Antifades and Other Tools for Fluorescence

Transcription

1 Molecular Probes Handbook A Guide to Fluorescent Probes and Labeling Technologies 11th Edition (2010) CHAPTER 23 Antifades and ther Tools for Fluorescence Molecular Probes Resources Molecular Probes Handbook (online version) Comprehensive guide to fl uorescent probes and labeling technologies thermofisher.com/handbook Molecular Probes Fluorescence SpectraViewer Identify compatible sets of fl uorescent dyes and cell structure probes thermofisher.com/spectraviewer BioProbes Journal of Cell Biology Applications Award-winning magazine highlighting cell biology products and applications thermofisher.com/bioprobes Access all Molecular Probes educational resources at

2 CHAPTER 23 Antifades and ther Tools for Fluorescence 23.1 Fluorescence Microscopy Accessories and Reference Standards Antifade Reagents and Mounting Media ProLong Gold Antifade Reagent ProLong Antifade Kit SlowFade Antifade Kit SlowFade Gold Antifade Reagent Qmount Qdot Mounting Media Image-iT FX Signal Enhancer FluoCells Prepared Microscope Slides FocalCheck Fluorescent Microspheres FocalCheck Ring-Stained Fluorescent Microspheres FocalCheck Thin-Ring Fluorescent Microspheres FocalCheck Microspheres Pre-Mounted on Microscope Slides FocalCheck Fluorescence Microscope Test Slides FocalCheck Fluorescent Microspheres for Spectral Unmixing MultiSpeck, TetraSpeck and Constellation Fluorescent Microspheres MultiSpeck Multispectral Fluorescence Microscopy Standards Kit TetraSpeck Fluorescent Microspheres and Sampler Kits Constellation Microspheres for Imaging PS-Speck Microscope Point Source Kit InSpeck Microscopy Image Intensity Calibration Kits Fluorescence Reference Standards Fluorescein NIST-Traceable Standard Reference Dye Sampler Kit Sample Chambers, Slides and Coverslips CultureWell Cell Culture Systems CultureWell Chambered Coverslips CultureWell Chambered Coverglasses CoverWell Imaging Chamber Gaskets CoverWell Perfusion Chamber Gaskets CoverWell Incubation Chamber Gaskets Press-to-Seal Silicone Isolators and Secure-Seal Adhesive Spacers HybriSlip Hybridization Covers HybriWell Hybridization Sealing Systems Secure-Seal Hybridization Chambers NCYTE MultiWells with Slide and Matching Gasket ProPlate Multi-Array System Attofluor Cell Chamber Coverslip Mini-Rack and Coverslip Maxi-Rack Product List 23.1 Fluorescence Microscopy Accessories and Reference Standards TWENTY-THREE The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPRTANT NTICE page : The 971 products and Master described Product List in on this page manual 975. Products are covered are For by Research one or more Use nly. Limited Not intended Use Label for License(s). any animal or Please human refer therapeutic to the Appendix or diagnostic on use. thermofi sher.com/probes 939

3 23.2 Flow Cytometry Reference Standards AlignFlow and AlignFlow Plus Flow Cytometry Alignment Beads LinearFlow Flow Cytometry Intensity Calibration Kits PeakFlow Flow Cytometry Reference Beads AbC and ArC Bead Kits for Flow Cytometry Compensation AbC Anti-Mouse and AbC Anti-Rat/Hamster Bead Kits ArC Amine-Reactive Compensation Bead Kit Flow Cytometry Size Calibration Kit CountBright Absolute Counting Beads Product List 23.2 Flow Cytometry Reference Standards The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT NTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page IMPRTANT 971 and Master NTICE Product : The List on products page 975. described Products in are this For Research manual are Use covered nly. Not by intended one or for more any Limited animal or Use human Label therapeutic License(s). or diagnostic Please refer use. to the Appendix on

4 23.1 Fluorescence Microscopy Accessories and Reference Standards To obtain accurate and reproducible results from fluorescence imaging applications, it is essential to maximize the intensity and stability of the fluorescence signal in the experimental sample. We have developed several effective antifade reagents that minimize photobleaching of fluorescently labeled specimens. The Image-iT FX signal enhancer (I36933) blocks nonspecific binding of dye-labeled antibodies, resulting in improved signal:background characteristics in images of immunolabeled cells and tissues. ur FluoCells prepared microscope slides provide ready-to-use, multicolor-labeled cell or tissue preparations for educational and commercial fluorescence microscopy demonstrations. In collaboration with Grace Bio-Labs, we also offer a wide selection of microscopy accessories, including sample chambers, slides and coverslips. Likewise, accurate and reproducible results depend on optimal performance of the optical system. The spectral compatibility of dyes and probes with excitation light sources (Table 23.1) and emission wavelength filters must be carefully evaluated. Downloadable reference spectra are available through our online Fluorescence SpectraViewer tool ( Using the Fluorescence SpectraViewer Note 23.1), and guidance in choosing optical filters can be found in Selecting ptical Filters for Fluorescence Microscopy Note Careful calibration and instrumentation adjustment are also required for high-precision imaging of fluorescent probes, particularly in multicolor applications that involve multiple exposures, repetitive scans or three-dimensional sectioning. We offer a variety of microsphere reference standards designed to facilitate adjustment and calibration of both conventional fluorescence microscopes and confocal laser-scanning microscopes. In addition, the Reference Dye Sampler Kit (R14782) provides ready-made stock solutions of five extensively characterized fluorescence standards for use in spectrofluorometers and fluorescence microplate readers. We are also the source of the NIST-traceable fluorescein standard (F36915), which is directly traceable to the fluorescein standard adopted and maintained by the National Institute of Standards and Technology. Antifade Reagents and Mounting Media Loss of fluorescence through irreversible photobleaching processes can lead to a significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration. 1 3 To minimize photobleaching of experimental samples, we have developed the ProLong, ProLong Gold, SlowFade and SlowFade Gold Antifade Kits and reagents, which have been shown to increase the photostability of many of our fluorophores in fixed cells, fixed tissues and cell-free preparations. The primary function of any antifade reagent is to sustain dye fluorescence, usually by inhibiting the generation and diffusion of reactive oxygen species. ther strategies for avoiding photobleaching include reducing the excitation light intensity by using high numerical aperture objectives and low magnification as well as hardware control of the excitation light s spatial 3 and temporal 4 distribution. Loss of fluorescence signal due to attenuated excitation can be compensated to some extent by use of high-quality optical filters and high-efficiency photodetectors. ProLong Gold Antifade Reagent ProLong Gold antifade reagent is an improved version of the ProLong antifade reagent, a component of the ProLong Antifade Kit described below. The ProLong Gold antifade reagent causes little or no quenching of the fluorescent signal while protecting the sample from A B Average fluorescence intensity Time (seconds) Figure A comparison of the performance of Molecular Probes antifade reagents. Bovine pulmonary arterial endothelial cells were labeled with fluorescein phalloidin and mounted using A) PBS, ProLong Gold antifade reagent (P36930, P36934) or ProLong Antifade Kit (P7481); B) PBS, SlowFade Gold antifade reagent (SS36936, S36937) or SlowFade Antifade Kit (S2828). Samples were imaged using a 1.3 NA 40 oil immersion lens, mega XF100-2 filter set and frame-capture rate of 1 image/second. Images were acquired with a Hamamatsu rca ER camera using the same exposure time for all samples. Y-axis values represent averages of the top 10% of the intensity-binned pixel values. Table 23.1 Fluorescence excitation sources. SlowFade Gold PBS SlowFade Source Principal Lines (nm) Mercury-arc lamp 366, 405, 436, 546, 578 * Xenon-arc lamp * Tungsten halogen lamp * Violet diode laser 405 Helium cadmium laser 325, 442 Argon-ion laser 457, 488, 514 Nd:YAG laser 532 Helium neon laser 543, 594, 633 Yellow diode laser 561 Krypton-ion laser 568, 647 Red diode laser 635 * Continuous white-light source. Frequency-doubled principal line output. The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by covered one or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to the Appendix Appendix on on 941

5 NTE 23.1 Using the Fluorescence SpectraViewer The Fluorescence SpectraViewer ( is an online tool that allows researchers to assess the spectral compatibility of dyes and probes in the course of designing experiments that utilize fluorescence detection techniques. This note outlines the functionality of the SpectraViewer (Figure 1A) and examples of its utility in the experimental design process (Figure 1B, Figure 1C). A B C Figure 1A SpectraViewer features: A. Fluorophore selection menu. Up to 5 fluorophore data sets may be displayed simultaneously. pen the drop-down menu (.) and select from the list of available excitation/emission data sets. The number of available data sets (as of July 2010) is 300, encompassing organic dyes, fluorescent proteins and Qdot nanocrystals. Note that data sets may be added, deleted or modified at our editorial discretion and without notice, although in practice we aim to keep such changes to a minimum to maintain stability of the database. These data sets may be downloaded from our website as 4-column text files for importing into other plotting and calculation utilities. For each selected fluorophore data set, a legend containing sample context information is displayed on the panel to the right of the plot. B. For each fluorophore data set, excitation (ex) and emission (em) data may be displayed or hidden by checking or unchecking the respective boxes. C. Y-axis scaling for excitation and emission spectra is in terms of percentage of peak intensity value. For Qdot nanocrystals, which exhibit quasi-continuous excitation profiles, the 100% intensity value has been arbitrarily defined as that at 300 nm. X-axis values on all plots are wavelengths in nanometers (nm). D. Excitation source spectral characteristics may be superimposed on the plot in the form of laser lines selected from a drop-down menu or filter characteristics input as numeric center wavelength (CWL) and bandpass (BP) values in nm in the boxes provided. In this example, laser excitation at 488 nm is indicated. E. Emission filter spectral characteristics may be superimposed on the plot in the form of numeric CWL and BP values in nm entered in the boxes provided. In this example, a typical FITC emission filter with CWL = 535 nm and BP = 50 nm is indicated. The transmission window of the filter is shown on the plot as a green-shaded rectangle. F. Mouse-controlled X,Y cursor. The crosshairs may be moved to any user-selected location within the plot window and are coupled to a numeric display of the corresponding (X,Y) values. Figure 1B Evaluating fluorophores for multiplex detection experiments: Excitation spectra. This overlay of the fluorescence excitation spectra of Alexa Fluor 488 and Alexa Fluor 568 dyes provides a useful initial assessment of their suitability for use in a multiplex detection experiment. It also serves to highlight scaling factors that are critical determinants of the final detected signal levels but that are excluded from the SpectraViewer comparison. The plot indicates that excitation of Alexa Fluor 568 at 488 nm is relatively inefficient (~5% of maximum indicated by the cursor). However, this consideration takes no account of the molar absorptivities (extinction coefficient, EC) of the fluorophores. The maximum EC values of Alexa Fluor 488 and Alexa Fluor 568 dyes are actually quite similar (75,000 cm 1 M 1 and 93,000 cm 1 M 1, respectively, as listed in The Molecular Probes Handbook data tables). An even more significant weighting factor is the relative abundances of the molecular targets of the fluorophores in the detector field of view; in general, cellular abundances of proteins vary over more orders of magnitude than the extinction coefficients of fluorophores. In the situation illustrated with excitation limited to 488 nm, the preferred course would be to use Alexa Fluor 568 to detect the more abundant of the two molecular targets and Alexa Fluor 488 to detect the less abundant target, thereby offsetting the absorptivity and target abundance factors. Figure 1C Evaluating fluorophores for multiplex detection experiments: Emission spectra. The fluorescence emission spectra of Alexa Fluor 488 and Alexa Fluor 568 dyes are shown with the spectral characteristics of typical emission filters superimposed. In this case, the main practical concern is the extent of overspill of Alexa Fluor 488 fluorescence into the Alexa Fluor 568 detection channel (CWL = 645 nm, BP = 75 nm), which can lead to false indications of molecular target colocalization in imaging applications. As in the excitation spectra comparison, this peaknormalized overlay provides an initial assessment of the suitability of fluorophore combinations for multiplex detection, but weighting of fluorescence signals by the relative abundances of molecular targets and other factors will heavily influence the final experimentally observed outcome. 942 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT NTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPRTANT NTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on

6 photobleaching (Figure , Figure ). Furthermore, unlike the reagents in the ProLong Antifade Kit, the ProLong Gold antifade reagent is premixed and ready to use just add a drop to the preparation and mount. As with our original ProLong antifade reagent, ProLong Gold reagent cures within 24 hours and the sample can be saved for months after mounting. ProLong Gold reagent offers excellent compatibility with a multitude of dyes and dye complexes, making it an especially valuable tool for multicolor applications 5 7 (Figure ). The ProLong Gold antifade reagent is available in a single 10 ml bottle (P36930), as well as in a set of five 2 ml bottles (P36934). As an added convenience, we also offer ProLong Gold antifade reagent containing DAPI, the popular nuclear and chromosome stain that emits blue fluorescence upon binding to DNA. The addition of DAPI in the mounting media eliminates the need for a separate counterstaining step. ProLong Gold antifade reagent with DAPI is available in a single 10 ml bottle (P36931), as well as in a set of five 2 ml bottles (P36935). ProLong Antifade Kit The ProLong Antifade Kit (P7481, Figure ) contains our original ProLong antifade reagent, which has proven to effectively enhance the resistance of many different fluorophores to photobleaching. Furthermore, specimens mounted using the ProLong Antifade Kit exhibit little or no quenching of the fluorescent signal of most dyes. Each ProLong Antifade Kit contains: ProLong antifade reagent powder ProLong mounting medium Detailed protocols for mounting samples 500 Figure A 20-second time series showing enhanced resistance to photobleaching afforded by ProLong Gold antifade reagent. Fixed bovine pulmonary artery endothelial cells were labeled with anti α-tubulin (A11126) and visualized with fluorescein goat anti mouse IgG (F2761). The samples were mounted in ProLong Gold antifade reagent (P36930; top) or phosphate-buffered saline (bottom). Images were acquired at 5-second intervals using a 40x/1.3 NA oil immersion objective with continuous illumination from a standard 100 watt mercury-arc lamp. Figure The relative photobleaching rates of fluorescein and Texas Red fluorophores with buffer alone (right series) or after treatment with the ProLong antifade reagent (left series). Bovine pulmonary artery endothelial cells were fixed, permeabilized and labeled with Texas Red -X phalloidin (T7471), which labels F-actin, and with a mouse monoclonal anti ß-tubulin antibody and fluorescein goat anti mouse IgG antibody (F2761), which label microtubules. Images were acquired at appropriate wavelengths using a cooled CCD camera. The numbers on the left of each pair of frames represent the duration of continuous excitation in seconds. Figure HeLa cell transfected with pshooter pcmv/myc/mito/gfp, then fixed and permeabilized. Green Fluorescent Protein (GFP) localized in the mitochondria was labeled with anti-gfp mouse IgG 2a (A11120) and detected with orange-fluorescent Alexa Fluor 555 goat anti mouse IgG (A21422), which colocalized with the dim GFP fluorescence. F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (A12379), and the nucleus was stained with blue-fluorescent DAPI (D1306, D3571, D21490). The sample was mounted using ProLong Gold antifade reagent (P36930). Some GFP fluorescence is retained in the mitochondria after fixation (left), but immunolabeling and detection greatly improve visualization (right). The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in described this manual in this are manual covered are by covered one or by more one Limited or more Limited Use Label Use License(s). Label License(s). Please Please refer refer to the to the Appendix on on thermofi sher.com/probes 943

7 NTE 23.2 Selecting ptical Filters for Fluorescence Microscopy Sensitive and versatile fluorescence detection techniques are of ever-increasing importance and popularity in biological research microscopy. In the now-standard epi-illuminated microscope configuration, the optical filter set performs a critical function in separating the fluorescence emission photons that will form the final image from the more-intense excitation light field. For practical purposes, it is necessary to reduce the excitation light intensity in the detection path by a factor of This design objective has to be achieved in parallel with capturing as many of the available fluorescence photons as possible. High capture efficiency allows compensating reductions in overall excitation light levels, with accompanying reductions in dye photobleaching and cellular phototoxicity. This technical note provides some basic fluorescence microscopy information, including discussions of: The ptical Filter Set The Trade-ff in ptical Filter Design Selecting an ptical Filter Set The Fluorescence SpectraViewer ( spectraviewer) is an online tool that allows researchers to assess the spectral compatibility of dyes, probes and optical filters in the course of designing experiments (Using the Fluorescence SpectraViewer Note 23.1). EM λ 4 DETECTR The ptical Filter Set A set of optical filters for selective excitation and detection of fluorescence typically consists of a minimum of three components: an excitation filter, a dichroic beamsplitter ( dichroic mirror ) and an emission filter ( barrier filter ) (Figure 1). The excitation filter selectively transmits a portion of the spectral output from the light source (Table 23.1). The dichroic beamsplitter then reflects the selected light, directing it to the sample. Fluorescence emission photons traveling from the sample towards the detector are transmitted by the dichroic beamsplitter, while excitation light reflected back from the sample is diverted out of the detection light path. The emission filter blocks unwanted spectral components of the emitted fluorescence (e.g., sample autofluorescence) as well as any residual excitation light. An interactive Java tutorial demonstrating these functions is available online at the Molecular Expressions website of Florida State University ( filtersetprofiles/index.html). The Trade-ff in ptical Filter Set Design For optimal fluorescence detection when using a single dye, the excitation and emission filters should be centered on the dye s absorption and emission peaks. To maximize the signal, one can choose excitation and emission filters with wide bandwidths. However, this strategy may result in unacceptable overlap of the emission signal with the excitation signal, resulting in poor resolution. To minimize spectral overlap, one can instead choose excitation and emission filters that are narrow in bandwidth and are spectrally well separated to increase signal isolation. This approach will reduce optical noise but may also reduce the signal strength to unacceptable levels. When overlapping signals from multiple fluorophores in the same sample are being differentiated, both the spectra of the dyes and their expected intensities must be considered before choosing an LAMP λ 1 EX DB λ 2 λ 2 λ 5 λ 3 SAMPLE Figure 1 Functions of fluorescence microscope filter set components. The desired excitation wavelength (λ 2 ) is selected from the spectral output of the lamp by the excitation filter (EX) and directed to the sample via the dichroic beamsplitter (DB). The beamsplitter separates emitted fluorescence ( ) from scattered excitation light ( ). The emission filter (EM) selectively transmits a portion of the sample s fluorescence emission (λ 4 ) for detection and blocks other emission components (λ 5 ). Figure 2 Potential-dependent staining of mitochondria in CCL64 fibroblasts by JC-1 (T3168). The mitochondria were visualized by epifluorescence microscopy using a 520 nm longpass optical filter. Regions of high mitochondrial polarization are indicated by red fluorescence due to J-aggregate formation by the concentrated dye. Depolarized regions are indicated by the green fluorescence of the JC-1 monomers. The image was contributed by Lan Bo Chen, Dana Farber Cancer Institute, Harvard Medical School. 944 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT IMPRTANT NTICE: NTICE The products : The products described described in this manual in this are manual covered are by covered one or more by one Limited or more Use Label Limited License(s). Use Label Please License(s). refer to the Please Appendix refer on to the Appendix on

8 optical filter. Complete spectral data for Molecular Probes fluorophores can be found at using our interactive Fluorescence SpectraViewer utility (Using the Fluorescence SpectraViewer Note 23.1). An interactive Java tutorial illustrating the trade-off among these parameters is available online at the Molecular Expressions website of Florida State University ( edu/primer/java/fluorescence/fluorocubes/index.html). Selecting an ptical Filter Set Filter set selection may involve a straightforward recommendation or a complex analysis of the spectral relationships of dyes and optical filters. 1 Emission filters are available with either longpass or bandpass wavelength transmission profiles. A typical longpass emission filter might transmit all wavelengths 530 nm, whereas a typical bandpass filter might transmit only wavelengths between 515 and 545 nm. Longpass filters should be used when the application requires maximum emission collection and when spectral discrimination is not desirable or necessary, which is generally the case for probes that generate a single emitting species in specimens with relatively low levels of background autofluorescence. Longpass filters are also useful for simultaneous detection of spectrally distinct dual emissions such as the monomer and J-aggregate forms of JC-1 (T3168, Section 12.2, Figure 2) and the monomer and excimer forms of BDIPY FL C 5 -ceramide (D3521, B22650; Section 12.4; Figure 3). Bandpass filters are designed to maximize the signal-to-noise ratio for applications where discrimination of signal components is more important than overall image brightness. The spectral sensitivity of the detection system should also be considered in order to achieve optimum detector signal-to-noise or accurate color rendition. Some applications, such as confocal laser-scanning microscopy, may require the use of sensitive photomultiplier (PMT) detectors. Alternatively, an electron-multiplying charge-coupled device (EMCCD) may be employed for quantitative imaging or microspectrofluorometry. Dual-, triple- and quadruple-band filter sets enable microscopists to excite and detect two, three or four fluorophores simultaneously instead of performing sequential image acquisitions with intervening filter changes (Figure 4). Selecting optimal filter sets for fluorescence microscopy applications requires matching optical filter specifications to the spectral characteristics of dyes. Comparisons should be made with care because some dyes have significantly different spectral properties in a particular application than those reported for the dye in solution. For example, the spectral characteristics of many nucleic acid stains depend on whether the dyes are in aqueous solution or bound to DNA or RNA. Similarly, styryl dyes such as FM 1-43 (T3163, T35356; Section 14.4, Section 16.1) and di-4-anepps (D1199, Section 22.2) have emission maxima that depend on whether they are dissolved in solvent or associated with membranes. To provide selection guidelines, we have compiled excitation and emission spectra for many of the most widely used probes in fluorescence microscopy in an online tool, the Fluorescence SpectraViewer ( Technical Support We invite customers to call our Technical Assistance Department for help in selecting the correct optical filter for a specific application. When calling, please be prepared to describe the dye(s), instrumentation and method of detection being used. A technical support scientist will then offer advice on the most effective filter configuration for the specified purposes. Alternatively, we recommend contacting Chroma Technology Corp., mega ptical, Inc. or Semrock, Inc. or the microscope manufacturer for this information. Chroma Technology, mega ptical and Semrock provide complete transmission curves and information on their specialty filters for ratio imaging, uncaging, multiphoton and other applications at their respective web sites ( Biophotonics Int (1999) 6: % Transmission Wavelength (nm) 750 Figure 3 Selective staining of the Golgi apparatus using the green-fluorescent BDIPY FL C 5 -ceramide (D3521) (right panel). At high concentrations, the BDIPY FL fluorophore forms excimers that can be visualized using a red longpass optical filter (left panel). The BDIPY FL C 5 -ceramide accumulation in the trans-golgi is sufficient for excimer formation (J Cell Biol (1991) 113:1267). Images contributed by Richard Pagano, Mayo Foundation. Figure 4 ptical transmission characteristics of a triple-band filter set (XF63, mega ptical Inc.) designed for simultaneous imaging of DAPI, fluorescein and Texas Red dyes. Transmission curves for the individual filter set components are shown in blue (excitation filter), black (dichroic beamsplitter) and red (emission filter). Graphic supplied by and used with permission of mega ptical Inc., Brattleboro, VT. The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by covered one or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to Appendix the Appendix on on 945

9 Bovine pulmonary arterial epithelial cells (BPAEC) labeled with fluorescein phalloidin (F432) photobleached to about 12% of the initial value in 30 seconds in PBS, while staying at the initial value under the same illumination conditions when mounted using the ProLong Antifade Kit (Figure ). As shown in Figure , the ProLong Antifade Kit provides more fluorescence output than a popular p- phenylenediamine containing antifade reagent 8 when used to mount fluorescein-stained HEp-2 cells. The ProLong antifade reagent also inhibits the fading of tetramethylrhodamine, as well as the fading of DNA-bound nucleic acid stains such as DAPI, propidium iodide and YY -1 (Section 8.1), again without significantly quenching the fluorescence of these dyes. 9 The compatibility of the ProLong antifade reagent with a multitude of dyes and dye complexes makes it an especially valuable tool for multicolor analysis procedures such as multiplexed fluorescence in situ hybridization. 10 Figure Bovine pulmonary artery endothelial cells were labeled with fluorescein phalloidin (F432), which labels filamentous actin, and placed under constant illumination on the microscope with a FITC filter set using a 60 objective. Images were acquired at 1-second intervals for 30 seconds. Under these illumination conditions, fluorescein photobleached to about 12% of its initial value in 30 seconds in PBS (left), but stayed at the initial value under the same illumination conditions when mounted using the reagents in the ProLong Antifade Kit (right, P7481). SlowFade Antifade Kit ur original SlowFade antifade formulation (S2828) was designed to reduce the fading rate of fluorescein to almost zero. Because it provides nearly constant emission intensity from fluorescein, this SlowFade antifade reagent is especially useful for quantitative measurements and applications that employ confocal laser-scanning microscopy. 11 However, this original SlowFade formulation substantially quenches the fluorescence of fluorescein and almost completely quenches that of the Alexa Fluor 350, Alexa Fluor 405 and Cascade Blue fluorophores. Each SlowFade Antifade Kit contains: SlowFade antifade reagent in 50% (v/v) glycerol, ready to use and sufficient for at least 200 coverslip-size experiments Concentrated SlowFade antifade reagent solution, provided for those applications in which glycerol may not be compatible Equilibration buffer, which raises the ph of the sample, increasing the protection afforded by the SlowFade antifade formulation Detailed protocols for mounting samples SlowFade Gold Antifade Reagent To overcome the limitations of the original SlowFade antifade reagent, especially with blue fluorophores, we have developed the SlowFade Gold antifade reagent. The SlowFade Gold antifade formulation slows fluorescein s fading rate by about fivefold without significantly reducing its initial fluorescence intensity, thereby dramatically increasing the signal-to-noise ratio in photomicro scopy. Moreover, quenching of the Alexa Fluor 350, Alexa Fluor 405, Cascade Blue, tetramethylrhodamine and Texas Red dyes is minimal. In fact, the SlowFade Gold antifade reagent reduces the fading rate of the Cascade Blue fluorophore to almost zero, while decreasing its emission intensity by only about 30%. The SlowFade Gold antifade reagent is available in a single 10 ml bottle (S36936), as well as in a set of five 2 ml bottles (S36937). As with the ProLong Gold antifade reagents, we also offer SlowFade Gold antifade reagent containing the blue-fluorescent nuclear counterstain DAPI. SlowFade Gold antifade reagent with DAPI is available A Normalized fluorescene emission ProLong Product X Illumination time (sec) No antifade B Illumination time (sec) Figure Bleaching profiles of A) fluorescein and B) Texas Red dye conjugates in cell samples. In these photobleaching experiments, human epithelial (HEp-2) cells were probed with human anti-nuclear antibodies and then developed for visualization with fluorophore-labeled secondary reagents. Identical samples were mounted in ProLong antifade reagent (n), Product X (+) or medium containing no antifade reagent (s). Although these data were normalized, we observed little or no quenching of samples mounted with the ProLong mounting medium. Normalized fluorescene emission ProLong No antifade Figure Human carcinoma (HeLa) cell labeled with Qdot nanocrystals and mounted with Qmount media. Mitochondria were detected with anti xphos complex V inhibitor protein IgG (A21355) and labeled with Qdot 625 goat F(ab ) 2 anti mouse IgG antibody (A10195, red fluorescence); the Golgi apparatus was detected with rabbit anti-giantin antibody and labeled with Qdot 585 goat F(ab ) 2 anti rabbit IgG antibody (Q11411MP, yellow fluorescence); tubulin was detected with rat anti-tubulin antibody and labeled with DSB-X biotin goat anti rat IgG antibody (D20697) and Qdot 525 streptavidin (Q10141MP, green fluorescence). The nucleus was labeled with Qnuclear Deep Red Stain (Q10363, purple fluorescence), and the slide was mounted with Qmount Qdot mounting media (Q10336). 946 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT NTICE: The products described in this manual are covered by one or more Limited Use Label License(s). 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10 in a single 10 ml bottle (S36938), as well as in a set of five 2 ml bottles (S36939). These reagents permit simultaneous nuclear staining and protection of the stained sample from photobleaching. 12 Unlike the ProLong and ProLong Gold antifade reagents, the SlowFade and SlowFade Gold antifade reagents do not cure over time so samples can be viewed immediately; however, SlowFade Gold reagents are intended for short-term use (3 4 weeks) only and mounted samples may degrade over longer time periods. Secondary sealing of coverslips with wax or nail polish is recommended when working with high magnification objectives or preparing specimens for storage and subsequent imaging. Qmount Qdot Mounting Media The unique physical properties of Qdot nanocrystals (Section 6.6) make them largely incompatible with ProLong Gold, SlowFade Gold and other mounting media designed primarily for use with organic dyes. Qmount Qdot mounting media (Q10336) is a specialized mountant that preserves the fluorescence signal of Qdot nanocrystals with little to no quenching of the signal s initial intensity. The formulation cures within 12 hours and is provided in a convenient and easy-to-use dropper bottle. This mounting medium offers excellent compatibility with all eight Qdot nanocrystal spectral types (Section 6.6) and their conjugates, as well as the nuclear counterstain Qnuclear Deep Red stain (Q10363, Section 12.5), making it an especially valuable tool for multicolor Qdot nanocrystal imaging applications (Figure ). Qmount Qdot mounting media is not recommended for use with Alexa Fluor dyes or fluorescent proteins. Image-iT FX Signal Enhancer By efficiently blocking nonspecific electrostatic interactions of anionic fluorescent dyes with cationic cell and tissue constituents, the Image-iT FX signal enhancer (I36933) dramatically improves the signal-to-noise ratio of immunolabeled cells and tissues, allowing clear visualization of targets that would normally be indistinguishable due to background fluorescence (Figure , Figure , Figure ). Background staining seen with fluorescent conjugates of streptavidin, goat anti mouse IgG antibody or goat anti rabbit IgG antibody is largely eliminated when Image-iT FX signal enhancer is applied to fixed and permeabilized cells prior to staining. FluoCells Prepared Microscope Slides Ideal for educators and instrument manufacturers, our popular FluoCells prepared microscope slides contain multilabeled cell preparations for observation by epifluorescence or confocal laser-scanning microscopy. The multicolor staining in these cell and tissue preparations can deliver publication-quality images and lasts through repeated viewings. These slides are especially useful for setting up microscopes and camera systems and for assessing the capabilities of optical filter sets When stored properly, these permanently mounted specimens will retain their bright and specific staining patterns for at least six months from the date of purchase. We currently offer five different FluoCells prepared microscope slides: Figure Reduced background staining afforded by Image-iT FX signal enhancer. Mouse brain cryosections were permeabilized and antigen retrieval was carried out. The sections were then treated for 30 minutes with Image-iT FX signal enhancer (I36933, left) or left untreated (right). Sections were labeled with the neural cell body selective antibody anti Hu C/D (A21271) and visualized using TSA Kit #2 (T20912) with the HRP conjugate of goat anti mouse IgG and Alexa Fluor 488 tyramide. Sections were mounted using the reagents in the ProLong Antifade Kit (P7481). Figure Increased label specificity and resolution provided by Image-iT FX signal enhancer. Fixed and permeabilized bovine pulmonary artery endothelial cells were treated with Image-iT FX signal enhancer (I36933, left) or left untreated (right) and then labeled with tetra methylrhodamine streptavidin (S870). Figure Golgi complexes in fixed and permeabilized HeLa cells labeled with anti golgin-97 anti body (A21270) and visualized with green-fluorescent Alexa Fluor 488 goat anti mouse IgG antibody (A11001). Actin was stained with red-fluorescent Alexa Fluor 594 phalloidin (A12381); nuclei were stained with blue-fluorescent DAPI (D1306, D3571, D21490). Treatment with Image-iT FX signal enhancer (I36933) largely eliminates nonspecific dye binding (bottom panel) as compared with untreated slide (top panel). The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPRTANT NTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on thermofi sher.com/probes 947

11 Figure FluoCells prepared slide #2 (F14781), which shows bovine pulmonary artery endothelial cells (BPAEC) that have been stained with an anti ß-tubulin mouse monoclonal antibody in conjunction with BDIPY FL goat anti mouse IgG (B2752) for labeling microtubules, Texas Red -X phalloidin (T7471) for labeling F-actin and DAPI (D1306, D3571, D21490) for labeling nuclei. This multiple-exposure image was acquired using bandpass optical filter sets appropriate for DAPI, fluorescein and Texas Red dye. FluoCells prepared slide #1 (F36924) shows bovine pulmonary artery endothelial cells (BPAEC) stained with MitoTracker Red CMXRos for labeling mitochondria, Alexa Fluor 488 phalloidin for labeling F-actin and DAPI for labeling the nucleus. FluoCells prepared slide #2 19 (F14781, Figure ) again contains BPAEC, but this time stained with Texas Red -X phalloidin for labeling F-actin, mouse monoclonal anti α-tubulin antibody in conjunction with BDIPY FL goat anti mouse IgG antibody for labeling microtubules and DAPI for labeling the nucleus. FluoCells prepared slide #3 (F24630; Figure , Figure , Figure ) contains a 16 µm cryostat section of a mouse kidney. Green-fluorescent Alexa Fluor 488 wheat germ agglutinin stains the glomeruli and convoluted tubules; red-fluorescent Alexa Fluor 568 phalloidin labels actin, which is especially prevalent in glomeruli and the brush border of proximal convoluted tubules; finally, DAPI stains the nuclei with blue fluorescence. FluoCells prepared slide #4 (F24631, Figure ) contains a 16 µm cryostat section of a mouse intestine. Alexa Fluor 350 wheat germ agglutinin labels the mucus of goblet cells with blue fluorescence; the red-fluorescent Alexa Fluor 568 phalloidin labels actin filaments, which are especially prevalent in the brush border of the intestinal mucosa; and SYTX Green nucleic acid stain labels nuclei with green fluorescence. FluoCells prepared slide #6 (F36925, Figure ) contains muntjac skin fibroblast cells stained with a combination of fluorescent stains. Green-fluorescent Alexa Fluor 488 phalloidin labels the prominent filamentous actin in these cells; a mouse monoclonal anti xphos Complex V inhibitor protein antibody in conjunction with the orange-fluorescent Alexa Fluor 555 goat anti mouse IgG antibody labels mitochondria; far-red fluorescent T-PR -3 nucleic acid stain labels nuclei. Because it contains no blue-fluorescent dyes, this slide is ideal for use with confocal laser-scanning microscopes that rely on non UV laser light sources. FocalCheck Fluorescent Microspheres FocalCheck Ring-Stained Fluorescent Microspheres FocalCheck fluorescent microspheres are specifically designed for examining the alignment, sensitivity and stability of confocal laser-scanning microscopes. 17,20 They are particularly useful for confirming the optical sectioning thickness (Z-resolution) in three-dimensional imaging applications. These polystyrene beads available in 6 µm and 15 µm diameters have been treated using a proprietary method in which a fluorescent dye is allowed to penetrate to only a limited depth within the microsphere. The resulting beads have a well-defined dye layer that, when viewed in Figure FluoCells prepared slide #3 (F24630) contains a mouse kidney section stained with a combination of fluorescent dyes. Alexa Fluor 488 wheat germ agglutinin (W11261) is a green-fluorescent lectin that was used to label elements of the glomeruli and convoluted tubules. The filamentous actin prevalent in glomeruli and the brush border were stained with red-orange fluorescent Alexa Fluor 568 phalloidin (A12380). Finally, the nuclei were stained with the blue-fluorescent DNA stain DAPI (D1306, D3571, D21490). This pseudocolored image was acquired on a Zeiss confocal microscope located at the Institute of Neuroscience, University of regon. Figure FluoCells prepared slide #3 (F24630) contains a section of mouse kidney stained with a combination of fluorescent dyes. Alexa Fluor 488 wheat germ agglutinin (W11261), a green-fluorescent lectin, was used to label elements of the glomeruli and convoluted tubules. The filamentous actin prevalent in glomeruli and the brush border were stained with red-fluorescent Alexa Fluor 568 phalloidin (A12380). Finally, the nuclei were counterstained with the blue-fluorescent DNA stain DAPI (D1306, D3571, D21490). This image is a composite of three micrographs acquired using filter sets appropriate for fluorescein, tetramethylrhodamine and DAPI. Figure FluoCells prepared slide #3 (F24630) containing a 16 µm cryostat section of mouse kidney stained with green-fluorescent Alexa Fluor 488 wheat germ agglutinin (W11261), red-orange fluorescent Alexa Fluor 568 phalloidin (A12380) and blue-fluorescent DAPI (D1306, D3571, D21490). The image represents an optical section obtained by simultaneous two-photon excitation of all three dyes at 797 nm using a Bio-Rad Radiance 2100 multiphoton microscope system. The image was acquired at the D Microscopy of Living Cells Course, University of British Columbia, Vancouver, Canada, by John Jordan, Bio- Rad Laboratories; and Iain Johnson, Life Technologies. 948 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT IMPRTANT NTICE: NTICE The products : The products described described in this manual in this are manual covered are by covered one or more by one Limited or more Use Label Limited License(s). Use Label Please License(s). refer to the Please Appendix refer on to the Appendix on

12 cross section in the confocal laser-scanning microscope, appears as a fluorescent ring of varying dimensions depending on the focal plane (Figure , Figure ). We refer to this proprietary staining procedure as ring staining to differentiate it from routine staining throughout the bead. FocalCheck microspheres are available in a variety of color configurations provided by five different fluorescent stains: Blue (365/430 nm) Green (505/515 nm) range (560/580 nm) Red (575/600 nm) Dark red (660/680 nm) The excitation/emission maxima of the different stains are well matched to the laser sources and optical filters commonly used in confocal laser-scanning microscopy and are especially useful in testing and aligning confocal laser-scanning microscopes with multiple laser lines and detection channels (Figure ). Moreover, because the dyes are localized within the bead and therefore protected from environmental factors, the FocalCheck microspheres are brighter and much more photostable than conventional surface-stained beads. A B C range Blue Alignment Red Alignment Figure FluoCells prepared slide #4 (F24631) contains a section of mouse intestine stained with a combination of fluorescent stains. Alexa Fluor 350 wheat germ agglutinin (W11263) is a blue-fluorescent lectin that was used to stain the mucus of goblet cells. The filamentous actin prevalent in the brush border was stained with red-orange fluorescent Alexa Fluor 568 phalloidin (A12380). Finally, the nuclei were stained with SYTX Green nucleic acid stain (S7020). This image is a composite of three digitized images obtained with filter sets appropriate for fluorescein, DAPI and tetramethylrhodamine. Figure FluoCells prepared slide #6 (F36925) showing a fixed, permeabilized and labeled muntjac skin fibroblast. Mitochondria were labeled with mouse anti xphos Complex V inhibitor protein antibody and visualized using orange-fluorescent Alexa Fluor 555 goat anti mouse IgG antibody (A21422). F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (A12379), and the nucleus was stained with T-PR -3 iodide (T3605, pseudocolored magenta). range Green Figure Confocal laser-scanning microscope optical cross-sectioning and alignment with FocalCheck microspheres. A) Serial optical sectioning from top to bottom along the z-axis of ring-stained microspheres reveals a continuous pattern of disc-to-ring-to-disc images. B) The diameter of the fluorescent ring (or disc) seen is dependent on the depth of the optical focal plane. C) In the confocal laser-scanning microscope, separate light paths exist for UV and visible wavelengths. Also, emitted fluorescence is detected by different photomultipliers. Proper optical alignment may be obtained with either of two types of FocalCheck microspheres. For example, the microspheres with an orange ring stain that are blue-fluorescent throughout the bead allow UV/visible wavelength alignment in three dimensions upon aligning the orange ring with the blue disc. Focal alignment is also possible simultaneously in three colors by aligning the green, orange and dark red rings of the FocalCheck microspheres containing fluorescent green/orange/dark red ring stains. Ar 351 Ar 488 Ar 514 He-Ne 543 Kr-Ar 568 Kr-Ar 647 He-He 633 Figure Images from confocal laser-scanning microscope optical cross-sectioning of our 15 µm FocalCheck microspheres that have a dark red fluorescent ring stain with a green-fluorescent stain throughout the bead (F7239). The left panel provides a clear visual representation of poor instrument alignment. Correct image registration has been achieved in the right panel, where the dark red ring is aligned with the green disk. The image was contributed by Paulette Brunner, Keck Imaging Center, University of Washington, and Yu-Zhong Zhang, Life Technologies Wavelength (nm) Figure Normalized excitation spectra of the dyes contained in the FocalCheck microspheres. Emission lines of several commonly used laser excitation sources are superimposed on the dyes excitation spectra to illustrate the wide range of usage of these beads as calibration references. Ar = Argon-ion laser. Kr-Ar = Krypton-argon laser. He-Ne = Helium-neon laser. The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by covered one or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to Appendix the Appendix on on 949

13 FocalCheck products are available in various different color configurations, including three suspensions that contain microspheres exhibiting ring stains of two or three different fluorescent colors: Blue-fluorescent and orange-fluorescent ring stains (15 µm, F7234) Green-fluorescent and dark red fluorescent ring stains (15 µm, F7240) Green-fluorescent, orange-fluorescent and dark red fluorescent ring stains (6 µm, F14806; 15 µm, F7235) We also supply four suspensions that contain microspheres exhibiting a ring stain of one fluorescent color combined with a stain of a second fluorescent color throughout the bead: Green-fluorescent ring stain with blue-fluorescent stain throughout (6 µm, F14808; 15 µm, F7237) Green-fluorescent ring stain with dark red fluorescent stain throughout (15 µm, F7238) range-fluorescent ring stain with blue-fluorescent stain throughout (15 µm, F7236) Dark red fluorescent ring stain with green-fluorescent stain throughout (6 µm, F14807; 15 µm, F7239, Figure ) The sharp ring stains exhibited by the FocalCheck microspheres produce a striking visual representation of instrument misalignment or other aberrations, making them ideal as reference standards for confocal laser-scanning microscopy. Correct image registration is indicated when the multiple ring images of the ring-stained FocalCheck beads (or the ring and disk images of the combination ring-stained and stained-throughout FocalCheck beads) are perfectly coincident in all dimensions (Figure ). FocalCheck Thin-Ring Fluorescent Microspheres ur FocalCheck Thin-Ring Fluorescent Microspheres Kit (F14791) contains smaller-dia meter microspheres that have spectral and physical features similar to those of our 6 µm and 15 µm FocalCheck microspheres. Because we prepare these 1.0 µm beads using fluorescent stains that are restricted to the surface only, they exhibit sharper and thinner fluorescent ring patterns when viewed in cross section with a confocal laser-scanning microscope. The FocalCheck Thin-Ring Fluorescent Microspheres Kit contains three 200 µl suspensions of 1.0 µm beads. Each suspension contains beads with a different color configuration: Green-fluorescent (495/515 nm) and red-fluorescent (575/600 nm) ring stains Green-fluorescent (495/515 nm) ring stain with dark red fluorescent (660/680 nm) stain throughout the bead Red-fluorescent (575/600 nm) ring stain with blue-fluorescent (365/430 nm) stain throughout the bead FocalCheck Microspheres Pre-Mounted on Microscope Slides In addition to the bead suspensions described above, we offer FocalCheck microspheres pre-mounted on microscope slides. The FocalCheck Fluorescent Microsphere Kits feature mounted samples of three different color configurations, in either the 6 µm (F24633) or the 15 µm (F24634) bead size: FocalCheck beads with green-fluorescent, orange-fluorescent and dark red fluorescent ring stains FocalCheck beads with green-fluorescent ring stain and bluefluorescent stain throughout the bead FocalCheck beads with dark red fluorescent ring stain and greenfluorescent stain throughout the bead FocalCheck Fluorescence Microscope Test Slides FocalCheck fluorescence microscope test slides #1, #2 and #3 are specifically designed for calibrating fluorescence microscope systems and evaluating system and filter performance: FocalCheck fluorescence microscope test slide #1 (F36909) is ideal for routine checking and calibration of confocal and widefield fluorescence microscopes 21 FocalCheck fluorescence microscope test slide #2 (F36913) provides a robust, reproducible method of evaluating the performance of spectral imaging systems, 22 as well as the ability to discriminate closely overlapping spectra. The slide consists of 6 µm diameter microspheres labeled with a series of spectrally overlapping dyes FocalCheck fluorescence microscope test slide #3 (F36914) is useful for basic evaluation of filter performance and as a general practice slide for fluorescence microscopy and digital imaging The slides each contain 10 sample areas arranged in two rows coated with proprietary fluorescent microspheres designed specifically for microscopy applications. The microspheres are mounted in optical cement (refractive index ~1.52) for maximal stability. The optical thickness of the mounted bead specimens may result in spherical aberration or inability to focus when using certain types of objectives. Users who encounter these problems are encouraged to contact our Technical Assistance Department. Figure A double-labeled microsphere from the FocalCheck DoubleGreen Fluorescent Microsphere Kit (F36905). The bead was imaged as a z-series using a Carl Zeiss LSM 510 META system. The two green-fluorescent dyes were separated by spectral unmixing, and one of the dyes was pseudocolored red. In this composite image, the complete z-series is shown prior to software rendering. Rendering fills in the missing information between the slices by interpolation to create a solid object. FocalCheck Fluorescent Microspheres for Spectral Unmixing We have prepared two FocalCheck Fluorescent Microspheres Kits for testing spectral separation on spectral imaging systems. 23 These microspheres are stained with two different fluorescent dyes that appear 950 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT NTICE: NTICE The products : The products described described in this manual in this are manual covered are by covered one or more by one Limited or more Use Label Limited License(s). Use Label Please License(s). refer to the Please Appendix refer on to the Appendix on

14 similar in color by eye but are sufficiently different to be resolved by linear-unmixing techniques. When linear-unmixing data-processing algorithms are applied, the dyes are shown to be spectrally distinct and spatially separated one appears only within the outer ring and the other appears throughout the microsphere (Figure ). These 6 µm, dual-stained microspheres are provided mounted on a microscope slide in each of the following kits: FocalCheck DoubleGreen Fluorescent Microspheres Kit (F36905), with a green-fluorescent (500/512 nm) ring dye and a slightly longer-wavelength green-fluorescent (512/525 nm) core dye FocalCheck Doublerange Fluorescent Microspheres Kit (F36906), with an orange-fluorescent (532/552 nm) ring dye and a slightly longer-wavelength orange-fluorescent (545/565 nm) core dye For generating reference spectra, these kits also contain two additional slides containing micro spheres stained uniformly with each of the individual dyes. Downloadable reference spectra are also available through our online Fluorescence SpectraViewer tool ( Using the Fluorescence SpectraViewer Note 23.1). MultiSpeck, TetraSpeck and Constellation Fluorescent Microspheres ur MultiSpeck and TetraSpeck fluorescent microspheres greatly facilitate the adjustment and calibration of conventional fluorescence microscopes, confocal laser-scanning microscopes and associated image-processing equipment for multicolor applications. These uniform, multiply stained microspheres are useful for colocalizing and focusing different wavelengths of light in the same optical plane, as well as for checking multicolor image resolution, magnification and sensitivity. MultiSpeck Multispectral Fluorescence Microscopy Standards Kit The 4 µm MultiSpeck microspheres in our MultiSpeck Multispectral Fluorescence Microscopy Standards Kit (M7901) exhibit three relatively distinct emission bands blue, green and red throughout every particle (Figure ). The spectral characteristics (excitation/ emission peaks at 365/405 nm, 520/525 nm and 580/600 nm) are compatible with optical filter sets designed for commonly used blue, green and red fluorophores (e.g., DAPI, fluorescein and rhodamine or Texas Red dyes or their Alexa Fluor dye counterparts). The MultiSpeck beads can be used as external references for assessing image registration in two and three dimensions, allowing the researcher to accurately determine the spatial relationships of different labels in multiparameter experiments. The MultiSpeck Multispectral Fluorescence Microscopy Standards Kit contains: Figure Multiple exposures of a prepared slide from our MultiSpeck Multispectral Fluorescence Microscopy Standards Kit (M7901). This kit provides fluorescent MultiSpeck microspheres that exhibit three relatively distinct excitation/emission bands blue, green and red all in the same particle. Thus, the same microsphere can appear a different color depending on the optical filter set used. This photograph was taken through bandpass optical filter sets appropriate for DAPI, fluorescein and Texas Red dyes, with the field of view shifted slightly between exposures. Figure Four separate exposures of three Tetra- Speck beads (T7283) photographed using optical filter sets appropriate for DAPI, fluorescein, tetramethylrhodamine and the Texas Red dye. The stage was shifted after each exposure. Note that the same beads appear blue, green, orange or red, depending on the filters used. Suspension of MultiSpeck multispectral microspheres Mixed suspension of separately stained blue-, green- and red-fluorescent microspheres, which exhibit the same three excitation/emission bands as the multispectral microspheres but in separate beads Mounting medium Slide-mounting protocol Both suspensions are provided at a ready-to-use density and can be mounted on slides or incorporated into an experimental sample. Each kit supplies a sufficient amount of material for ~50 slide preparations using either of the two bead suspensions provided. TetraSpeck Fluorescent Microspheres and Sampler Kits ur TetraSpeck fluorescent microspheres 17,19,20 expand the multispectral strategy introduced with the MultiSpeck beads in two important ways. First, the TetraSpeck beads have been stained throughout with a mixture of four different fluorescent dyes, yielding four well-separated excitation and emission peaks (Figure ). The excitation/emission maxima of the dyes are The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by one covered or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to Appendix the Appendix on on 951

15 365/430 nm (blue), 505/515 nm (green), 560/580 nm (orange) and 660/680 nm (dark red). Second, these microspheres are available in five nominal sizes (actual bead diameters are indicated on the product labels), spanning the range from subresolution to nearly cell-size particles: 0.1 µm (T7279) 0.2 µm (T7280) 0.5 µm (T7281) 1.0 µm (T7282) 4.0 µm (T7283) Figure Luminescent microsphere products provide an extensive range of sizes and fluorescent colors, illustrated by a sample of our Constellation microspheres for imaging (C14837). Figure Constellation microspheres (C14837) provide an assortment of sizes and colors for use in fluorescence microscopy demonstrations. Each of these products provides a 0.5 ml suspension sample of TetraSpeck microspheres that is sufficient for about 100 slides. We offer the TetraSpeck Fluorescent Microspheres Sampler Kit (T7284) and the TetraSpeck Fluorescent Microspheres Size Kit (T14792). The TetraSpeck Fluorescent Microspheres Sampler Kit consists of separate suspension samples of our 0.1 µm, 0.5 µm and 4.0 µm TetraSpeck beads, each sufficient for preparing about 20 slides. The TetraSpeck Fluorescent Microspheres Size Kit contains six microscope slides; five slides with a mounted sample of the 0.1 µm, 0.2 µm, 0.5 µm, 1.0 µm or 4.0 µm diameter TetraSpeck microspheres, and a sixth slide with a mixture of all five sizes. TetraSpeck microspheres have been used to calibrate the spatial distribution of illumination for high-content screening (HCS), 24,25 and as reference markers for image alignment in high-resolution immunofluorescence colocalization analysis. 24,26 In addition, various FluoSpheres and TetraSpeck beads have been used as reference standards for two-photon excitation microscopy, and in particular for the in situ determination of the two-photon excitation point-spread function (PSF) Constellation Microspheres for Imaging Constellation microspheres for imaging (C14837; Figure , Figure ) are 3 ml suspensions of assorted fluorescent microspheres with a variety of sizes and colors. Designed for use in laboratory tutorials and customer training sessions, they provide inexpensive and robust test samples for demonstrating filter switching, focus adjustment and other functional capabilities of fluorescence microscopes. The Constellation microspheres can be stored at room temperature, protected from light. PS-Speck Microscope Point Source Kit The fluorescent microspheres in the PS-Speck Microscope Point Source Kit (P7220) have a diameter of ± µm, making them ideal subresolution fluorescent sources for calibrating instrument optics. They are particularly useful for measuring the point-spread function (PSF) required for computational image deconvolution procedures 17,30 32 (Figure , Figure ). This kit s four ready-to-use suspensions contain bright monodisperse particles in the following fluorescent colors (and excitation/emission peaks): Blue (360/440 nm) Green (505/515 nm) range (540/560 nm) Deep red (633/660 nm) Figure ne PS-Speck green-fluorescent microsphere (P7220) used for point-spread function analysis. Shown are individual images, in pseudocolor, taken in x-y planes with 0.25 µm increments in the z-axis between planes. The image was contributed by Regina Armstrong, Uniformed Services University of the Health Sciences, Bethesda, Maryland. The kit also includes mounting medium and a mounting protocol for the user s convenience. Each suspension provides sufficient material to mount about 100 slides. PS-Speck microspheres are too large to represent point source objects for microscopes equipped with high numerical aperture (e.g., NA 1.4) oil immersion objectives. In such cases, we recommend our 0.1 µm TetraSpeck fluorescent microspheres (T7279) or 0.1 µm and 0.04 µm FluoSpheres carboxylate-modified microspheres (Section 6.5) for PSF determinations. InSpeck Microscopy Image Intensity Calibration Kits InSpeck Microscope Image Intensity Calibration Kits provide microsphere standards that generate a series of well-defined fluorescence intensity levels (Figure ) for constructing calibration curves and evaluating sample brightness. 6,25,33 InSpeck microspheres have been used to estimate the global background and signal response for high-content screening (HCS) The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT IMPRTANT NTICE: NTICE The products : The products described described in this manual in this are manual covered are by covered one or more by one Limited or more Use Label Limited License(s). Use Label Please License(s). refer to the Please Appendix refer on to the Appendix on

16 Most of the kits are offered in a choice of two different microsphere sizes (2.5 µm or 6 µm) and five different fluorescent colors: InSpeck Blue (350/440 nm) Kit, (2.5 µm, I7221) InSpeck Green (505/515 nm) Kits (2.5 µm, I7219; 6 µm, I14785) InSpeck range (540/560 nm) Kits (2.5 µm, I7223; 6 µm, I14786) InSpeck Red (580/605 nm) Kits (2.5 µm, I7224; 6 µm, I14787) InSpeck Deep Red (633/660 nm) Kit (2.5 µm, I7225) Each kit includes six separate suspensions of InSpeck fluorescent microspheres with relative fluorescence intensities of 100%, 30%, 10%, 3%, 1% and 0.3% (Figure ), covering the range of intensities commonly encountered in microscopy applications. Unstained control beads and mounting medium are also supplied. The aqueous suspensions of microspheres may be applied directly to the sample for calibrating fluorescence intensities or mounted separately in an adjacent well or on another slide. Each suspension provides sufficient material to prepare about 100 slides. Fluorescence Reference Standards Fluorescein NIST-Traceable Standard The National Institute of Standards and Technology (NIST) chose high-grade Molecular Probes fluorescein to create Standard Reference Material 1932 (SRM 1932), a certified fluorescein solution. We now offer a NIST-traceable fluorescein standard (F36915) that not only meets the stringent criteria established by NIST, but is also directly traceable to SRM We supply our NIST-traceable fluorescein standard as a calibrated 50 µm solution of fluorescein in 100 mm sodium borate buffer, ph 9.5; under these conditions, fluorescein is completely ionized 34 and is therefore in its most fluorescent form (Figure , Figure ), exhibiting an extremely high quantum yield of Academic researchers and industry scientists alike can use our NIST-traceable fluorescein standard to assess day-to-day or experiment-to-experiment variation in fluorescence-based instrumentation, as well as to determine the Molecules of Equivalent Soluble Fluorophore (MESF) value for an experimental solution. The MESF value is defined not as the actual number of dye molecules present, but rather as the number of fluorophores that would yield a fluorescence intensity equivalent to that of the experimental solution when analyzed on the same instrument under the same conditions Consequently, the MESF value is an important tool for characterizing the fluorescence intensity of a solution containing spectrally similar dye molecules attached to antibodies, nucleic acids, microspheres or other substrates that might enhance or diminish the fluorescence. When its ph is carefully matched with that of the experimental solution, our NISTtraceable fluorescein standard can be used for accurate MESF determinations of a wide range of green-fluorescent dye solutions and on an assortment of fluorescence-based instruments. _ dianion _ C H _ C monoanion Figure Ionization equilibria of fluorescein. H H neutral CH lactone H H cation CH + H Figure An orthogonal (x-z) display representing a point-spread function. The microsphere used is a component of the PS-Speck Microscope Point Source Kit (P7220). This pseudocolored image was generated electronically from a series of microsphere images taken in x-y planes. The image was contributed by Jennifer Kramer, Scanalytics. Number of particles Fluorescence intensity Forward scattering Figure Flow cytometric analysis of the beads in the 6 µm InSpeck Green Microscope Image Intensity Calibration Kit (I14785). The microspheres have nominal relative fluorescence intensities of 100%, 30%, 10%, 3%, 1%, 0.3%. For each lot, actual relative intensities are determined by flow cytometry and printed on the product labels. A Fluorescence emission Absorption B Figure The ph-dependent spectra of fluorescein (F1300): A) absorption spectra, B) emission spectra ph Wavelength (nm) ph Wavelength (nm) Ex = 488 nm 650 The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by covered one or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to the Appendix Appendix on on 953

17 Reference Dye Sampler Kit ur Reference Dye Sampler Kit (R14782) provides samples of five extensively characterized fluorescence standards with emission spectra covering the entire visible wavelength range. 39 All five fluorescent standards are supplied as 1 mm stock solutions in 1 ml units, sufficient to prepare approximately 500 diluted working samples for spectrofluorometry. The compositions of the stock solutions are as follows: Quinine sulfate in 0.1 M sulfuric acid (H 2 S 4 ) (Figure ) Fluorescein in dimethylsulfoxide (DMS) (Figure ) 5-Carboxytetramethylrhodamine in DMS (Figure ) Sulforhodamine 101 in DMS (Figure ) Nile blue perchlorate in DMS (Figure ) Spectroscopic data for the five standards are summarized in Table Reference spectra for all five fluorescent standard solutions are provided through our online Fluorescence SpectraViewer tool ( Sample Chambers, Slides and Coverslips In collaboration with Grace Bio-Labs, we offer a collection of accessories for imaging and microscopy. These accessories make slide preparation easy, facilitate sample perfusion and simplify sample manipulation during in situ hybridization and other procedures that involve multiple wash steps. CultureWell Cell Culture Systems The CultureWell cell culture systems provide an integrated set of tools for preparing cultured cells for staining and imaging. Each system uses medical-grade silicone gaskets preassembled with standard optical-quality coverslips into convenient inserts that fit into matching cell culture plates (Figure ). The entire system is provided sterile and ready to use. Cell culture, treatment and staining are performed on the coverslip, which adheres securely to the culture plate via a silicone backing. The samples can then be imaged with or without the silicone gaskets. Two types of systems are available in several configurations to suit a variety of needs (Table 23.3). The CultureWell multiwell cell culture systems use precut silicone gaskets to form convenient no-leak wells on mm coverslips. The wells are spaced for compatibility with microfluidic handling robots. Low numbers of wells are ideal for titering antibody dilutions or other staining conditions, whereas the higher numbers of wells facilitate high-throughput screening. Each insert includes four coverslips with gaskets, preassembled into a culture plate. Silicone dividers (C24770, Figure ) are also available for separating portions of the coverslip into leak-proof wells, for different treatment and washing conditions. The CultureWell multislip cell culture systems comprise multiple coverslips arrayed on a sheet of silicone and assembled into a convenient insert. The silicone backing adheres the coverslips to the tissue culture plate, preventing movement during plating, cell culture and washing steps. Each coverslip can be removed separately for individual Figure Absorption and fluorescence emission spectra of quinine sulfate, dihydrate in 0.5 M sulfuric acid. Figure Absorption and fluorescence emission spectra of fluorescein in 0.1 M sodium hydroxide. Figure Absorption and fluorescence emission spectra of 5-carboxytetramethylrhodamine (5-TAMRA) in methanol. Figure Absorption and fluorescence emission spectra of sulforhodamine 101 in ethanol. Figure Absorption and fluorescence emission spectra of nile blue in ethanol. Table 23.2 Spectroscopic data for components of the Reference Dye Sampler Kit. Component Solvent Abs (nm) * Em (nm) * QY Quinine sulfate Water Fluorescein Water CTMR ** Methanol Sulforhodamine Ethanol Nile blue Ethanol * Approximate absorbance (Abs) and fluorescence emission (Em) maxima. Fluorescence quantum yield at 22 C. Standard values in 0.5 M H 2 S 4. Standard values in 0.1 M NaH. ** CTMR = Carboxytetramethylrhodamine. 954 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT IMPRTANT NTICE: NTICE The products : The products described described in this manual in this are manual covered are by one covered or more by Limited one or more Use Label Limited License(s). Use Label Please License(s). refer to the Please Appendix refer on to the Appendix on

18 staining experiments. The CultureWell cell culture systems are provided in a set of 10 preassembled inserts in plates. CultureWell Chambered Coverslips The CultureWell chambered coverslips (Table 23.3, Figure ) are the same gasketed coverslips provided in our CultureWell cell culture systems, but they are not preassembled into inserts. The chambered coverslip can be placed in CultureWell plates (C24769) or other cell culture dishes for cell culture and staining. The silicone gasket can be easily removed and the coverslip placed on a slide for microscopy. The CultureWell chambered coverslips provide maximum versatility for designing smaller scale cell culture applications. They are provided in sets of five sterile pouches, with four chambered coverslips per pouch. CultureWell Chambered Coverglasses The CultureWell chambered coverglass provided sterile and ready to use contains 16 wells that can each hold up to 250 µl, allowing cells to be cultured in a number of different conditions on a single slide (Figure ). A silicone gasket forms a leakproof seal between the polystyrene upper structure and the coverglass. When the cells are ready to be imaged, coverglass removal is made easy by the use of a simple tool that separates the parts without the need for excessive force, eliminating the risk of coverglass breakage (Figure ). Removal of the Figure CultureWell chambered coverslips. Figure CultureWell cell culture system. Table 23.3 CultureWell cell culture systems and chambered coverslips. Figure CultureWell silicone dividers (C24770). Number of Coverslips per Insert Cat. No. Size of Coverslip (mm) Number of Wells per Coverslip Well Dimensions Depth Volume per Well CultureWell multiwell cell culture systems * C mm diameter 1 mm µl 4 C mm diameter 2 mm µl 4 C mm square 1 mm µl 4 C mm diameter 1 mm µl mm diameter 1 mm µl 4 C24766 C24767 C mm diameter 1 mm 3 10 µl 4 CultureWell chambered coverslips C mm diameter 1 mm µl NA C mm diameter 2 mm µl NA C mm diameter 1 mm µl NA C mm diameter 1 mm µl NA C mm diameter 1 mm µl NA C mm diameter 1 mm 3 10 µl NA CultureWell multislip cell culture systems * C NA NA NA NA 8 C NA NA NA NA 15 * Each system is supplied in packages of 10 inserts, each preassembled in an 86 mm 128 mm plate. A trial size is also available (C24767) that includes two inserts four coverslips with eight 6 mm wells each in two plates. Chambered coverslips are supplied in sets, in five pouches of four coverslips each. Packs of 10 sterile plates are also available separately (C24769). NA = Not applicable. Figure The CultureWell removable chambered coverglass for cell culture (C37000). Figure Using the CultureWell removable chambered coverglass for cell culture (C37000). When the cells are ready to be imaged, coverglass removal is made easy by the use of a simple tool (included with the coverglass) that separates the parts without the need for excessive force, eliminating the risk of coverglass breakage. The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by one covered or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to Appendix the Appendix on on 955

19 silicone gasket leaves no residue. The components of the chambered coverglass are manufactured and assembled with special orientation features to allow easy location of a specific specimen after the coverglass is mounted. Frosted microscope slides are also provided for mounting. The CultureWell removable chambered coverglass has several important features: Figure CoverWell imaging chamber gaskets. Black silicone gasket reduces light scatter, enhancing fluorescence applications. Inert, non-cytotoxic silicone permits edge-to-edge growth of cells in the wells. Wells are spaced to allow the use of multichannel pipettes for fast and easy cell culturing. Silicone gasket remains attached to the coverglass after separation, allowing the wells to be used as reagent reservoirs. Gasket design is ideally suited for small-volume incubations, in situ hybridization and immunostaining. Chambered coverglass is provided sterile and ready to use. We offer the CultureWell chambered coverglass in a package containing eight chambered coverglasses and the removal tool (C37000), as well as in a sample size containing a pair of chambered coverglasses (C37005). Figure CoverWell perfusion chamber gaskets. CoverWell Imaging Chamber Gaskets CoverWell imaging chamber gaskets (Table 23.4, Figure ) incorporate a thin, optically clear plastic cover, making them ideal for light, epifluorescence and confocal laser-scanning microscopy. 40 By simply pressing an imaging chamber gasket to a microscope slide or coverslip, a sealed chamber is formed to contain mounting medium. The watertight chamber supports and stabilizes thick and free-floating specimens, permitting resolution of fine internal structures and analysis of markers without the compression or movement artifacts that affect observations made using an ordinary coverslip. CoverWell Perfusion Chamber Gaskets CoverWell perfusion chamber gaskets (Table 23.4, Figure ) are designed for livecell imaging and manipulation. With the same silicone gasket technology as the CoverWell imaging chamber gaskets, these gaskets form watertight press-to-seal chambers with dualaccess ports for addition and removal of perfusing media. The access ports can be covered using Figure CoverWell incubation chamber gaskets. Table 23.4 CoverWell chamber gaskets. Cat. No. Number of Wells Well Dimensions Depth Approximate Volume per Chamber Quantity per Package CoverWell imaging chamber gaskets C mm diameter 0.5 mm 180 µl 40 C mm diameter 1.0 mm 300 µl 40 C24726 * 1 20 mm diameter 0.5 mm 180 µl 40 C24727 * 1 20 mm diameter 1.0 mm 300 µl 40 CoverWell perfusion chamber gaskets C mm 19 mm 0.5 mm 350 µl 40 C mm 19 mm 1.0 mm 550 µl 40 C mm 6 mm 0.5 mm 70 µl 40 C mm diameter 1.0 mm 300 µl 40 C mm diameter 0.5 mm 35 µl 20 C mm diameter 1.0 mm 60 µl 20 C mm diameter 2.0 mm 100 µl 20 C mm diameter 2.5 mm 150 µl 20 CoverWell incubation chamber gaskets C mm 22 mm 0.2 mm 200 µl 25 C mm 22 mm 0.5 mm 500 µl 50 C mm diameter 0.2 mm 20 µl 25 C mm diameter 0.5 mm 20 µl 50 * With adhesive on one side. 956 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT IMPRTANT NTICE: NTICE The products : The products described described in this manual in this are manual covered are by one covered or more by one Limited or more Use Label Limited License(s). Use Label Please License(s). refer to the Please Appendix refer on to the Appendix on

20 adhesive seal tabs (A18211), which are available separately. The heatresistant gaskets can be sterilized and used for direct culturing of cells and tissues. CoverWell perfusion chamber gaskets are available in single- or multiwell configurations, allowing multiple experiments to be performed on a single microscope slide or coverslip. CoverWell Incubation Chamber Gaskets CoverWell incubation chamber gaskets (Table 23.4, Figure ) are silicone gaskets with a clear plastic cover that are expressly designed for immunocytochemistry and in situ hybridization. 41,42 The gasket is simply pressed onto a wet or dry microscope slide to form a watertight chamber that holds reactants in place and prevents evaporation. The chambers improve the uniformity and sensitivity of staining by enclosing a large sample area while minimizing the reagent volume required. The incubation chamber gaskets are easily removed and reapplied for multiple-step procedures. These chamber gaskets are heat resistant, autoclavable and nuclease free. Press-to-Seal Silicone Isolators and Secure-Seal Adhesive Spacers For the ultimate in utility and flexibility, Press-to-Seal silicone isolators (Table 23.5, Figure ) are removable hydrophobic barriers that can be customized to meet specific experimental requirements. They may be used to isolate cells grown in culture dishes or to separate specimens on microscopy slides during staining procedures. The silicone material can be autoclaved and adheres to any smooth surface. Isolators without adhesive can be easily removed and reapplied for multiple incubation steps. Isolators are also available with adhesive on one side for added security or permanent mounting. In addition, we offer uncut silicone sheets that can easily be trimmed to prepare customized enclosures. Similar to the Press-to-Seal silicone isolators, Secure-Seal adhesive spacers are ultra-thin gaskets with adhesive that can be stacked to any depth desired. For high-resolution microscopy, the spacer and specimen can be sandwiched between two No. 0 glass coverslips. The spacers are available in several configurations (Table 23.5). Table 23.5 Press-to-Seal gaskets and Secure-Seal spacers. Cat. No. Number of Wells Well Dimensions Depth Quantity per Package Press-to-Seal silicone isolators P mm diameter 0.5 mm 50 P mm diameter 1.0 mm 50 P24740 * 1 20 mm diameter 0.5 mm 50 P24741 * 1 20 mm diameter 1.0 mm 50 P24742 * mm diameter 2.0 mm 25 P24743 * 8 9 mm diameter 0.5 mm 25 P24744 * 8 9 mm diameter 1.0 mm 25 Secure-Seal adhesive spacers S24735 * 1 13 mm diameter 0.12 mm 100 S24736 * 1 20 mm diameter 0.12 mm 100 S24737 * 8 9 mm diameter 0.12 mm 100 Press-to-Seal silicone sheets (13 cm 18 cm) P18178 NA NA 0.5 mm 5 P18179 NA NA 1.0 mm 5 P24745 * NA NA 0.5 mm 5 * With adhesive on one side. NA = Not applicable. HybriSlip Hybridization Covers HybriSlip hybridization covers (Table 23.6, Figure ) are nuclease free, ready-to-use and designed specifically for in situ hybridization. 43 These hydrophobic coverslips do not require lengthy preparation or blocking procedures to prevent probe trapping or binding. They are heat resistant and do not curl, even at high temperatures, making them ideal for denaturation steps or in situ PCR incubations. HybriSlip covers are available in three sizes. Figure Press-to-Seal silicone isolators. Figure HybriSlip hybridization covers. Table 23.6 Tools for hybridization experiments. Quantity Cat. No. Chamber Dimensions Depth Usable Volume per Package HybriWell hybridization sealing system H mm diameter 0.25 mm 30 µl 100 H mm diameter 0.15 mm 30 µl 100 H mm 22 mm 0.15 mm µl 100 H mm 21 mm 0.15 mm µl 100 H mm 22 mm 0.25 mm µl 100 Secure-Seal hybridization chambers S mm 22 mm 0.8 mm 250 µl 50 S mm diameter 0.8 mm 200 µl 40 S mm diameter 1.3 mm 280 µl 40 S mm diameter 0.8 mm 20 µl 20 S mm diameter 1.3 mm 40 µl 20 HybriSlip hybridization covers H mm 22 mm NA NA 500 H mm 22 mm NA NA 500 H mm 22 mm NA NA 500 Seal tabs A18211 Adhesive seal tab NA NA 400 NA = Not applicable. The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by covered one or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to Appendix the Appendix on on 957

21 Figure HybriWell hybridization sealing systems. HybriWell Hybridization Sealing Systems HybriWell hybridization sealing systems (Table 23.6, Figure ) are coverslip seal combinations that attach to microscope slides to form microwells optimized for carrying out in situ hybridization procedures. These ready-to-use hybridization gaskets have a special adhesive that bonds to glass slides in seconds, creating a water-tight seal that is temperature resistant but can also be removed cleanly and easily after hybridization. Solutions are easily added or removed through dual-access ports without disrupting the specimen. The hydrophobic coverslips are nuclease free and will not trap or bind probes, allowing uniform distribution of the reagent over the specimen. The HybriWell sealing systems also include a quick-seal tool to secure the hydrophobic cover to the microscope slide, as well as nuclease-free adhesive seal tabs to cover the access ports. Adhesive seal tabs (A18211) are also available separately in sets of 400. Secure-Seal Hybridization Chambers Like the HybriWell hybridization sealing systems, Secure-Seal hybridization chambers (Table 23.6, Figure ) are designed to isolate single or multiple specimens on a slide during in situ hybridization procedures. Access ports in the chamber surface allow for the addition or removal of solutions and are easily sealed using adhesive seal tabs (A18211), available separately. Because they are deeper than the HybriWell chambers, the Secure-Seal chambers provide optimum surface-to-volume fluid dynamics, which facilitate more uniform hybridization. However, the shallower chambers created by the HybriWell sealing systems hold a smaller reagent volume, minimizing the amount of probe required. Figure Secure-Seal hybridization chambers. Figure NCYTE MultiWells. NCYTE MultiWells with Slide and Matching Gasket The versatile NCYTE MultiWells (Figure ) consist of a two-piece set that includes a slide printed with nitrocellulose circles and a matching removable gasket to enclose and isolate each sample. The nitrocellulose coating on the slide is specially formulated for fluorescence imaging. This ultra-thin microporous coating ensures uniform binding of tissue prints, cells or macromolecules and becomes transparent in a variety of mounting media. The matching pressto-seal silicone gaskets adhere easily to the surface of the slide to isolate specimens and reagents and prevent cross contamination. A coverslip can be added to create enclosed chambers for long incubations. Gaskets can be removed and cleaned simply by peeling them off. NCYTE MultiWells are available in two configurations: a set of 20 slides and gaskets, each with 12 wells, 5 mm in diameter (24750), or a set of 20 slides and gaskets, each with a single well, 13 mm in diameter (24751). ProPlate Multi-Array System The ProPlate multi-array system (P37004) from Grace Bio-Labs allows integration of microscope array technology with automated microplate processing (Figure ). Individual modules (P37001), covered with seal strips (P37002), fit into a tray (P37003), producing a modular plate with a standard microplate footprint and well spacing. The modular design allows loading of 1 4 slides per tray for plate washing and reading. Individual ProPlate modules may also be processed by hand without the use of the tray. The large well volumes and isolation of 16 individual arrays (2 8) on a single microscope slide is particularly well suited for proteomics applications, including protein expression analysis, protein-protein interactions, antibody profiling and high-throughput automated analysis of multiple proteins. The ProPlate multi-array system can also be used to process cdna or oligonucleotide arrays. 44 Figure ProPlate multi-array system (P37004). Individual modules (P37001), covered with seal strips (P37002), fit into a tray (P37003), producing a modular plate with a standard microplate footprint and well spacing. 958 The Molecular Probes Handbook: A Guide to to Fluorescent Probes and Labeling Technologies IMPRTANT NTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPRTANT NTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on

22 Attofluor Cell Chamber The Attofluor cell chamber (A7816, Figure ) is a durable and practical coverslip holder designed for viewing live-cell specimens on upright or inverted microscopes; spare -rings for the Attofluor cell chamber are available in sets of 10 (14804). Features of the Attofluor cell chamber include: Surgical stainless steel construction Autoclavable, allowing cells to be grown directly in the chamber -ring seal design that prevents sample contamination by oil and leakage of media from the coverslip Accepts 25 mm diameter round coverslips and mounts in a standard 35 mm diameter stage holder Thin 0.5 mm base, allowing clearance for the objective when focusing Coverslip Mini-Rack and Coverslip Maxi-Rack ur unique coverslip mini-rack (C14784, Figure ) is a miniature support designed to vertically hold eight standard round or square coverslips. The mini-rack fits easily into a standard 50 ml beaker and can accommodate a small stir bar beneath the rack. Use of the mini-rack eliminates the necessity for repeatedly moving coverslips between solutions with forceps. Because it is constructed of Teflon, the mini-rack does not adsorb biopolymers, withstands strong acids and bases, is not damaged by heat and may be sterilized by a variety of methods such as autoclaving, organic solvent treatment or ethylene oxide exposure. The mini-rack is easily disassembled for cleaning and storage. The mini-rack is particularly useful in immunocytochemical and in situ hybridization procedures involving sequential wash steps where thorough and consistent removal staining, fixation, permeabilization or blocking reagents from the coverslips is critical. The coverslip maxi-rack (C24784, Figure ) provides efficient support for the simultaneous staining and washing of up to 50 samples on 18 mm square or circular coverslips in a self-contained covered container. The maxi-rack includes a convenient handle to remove the rack from the staining solution. Figure Attofluor cell chamber (A7816). Figure Coverslip mini-rack (C14784). REFERENCES 1. J Phys Chem A (2007) 111:429; 2. J Am Chem Soc (2007) 129:4643; 3. Nat Biotechnol (2007) 25:249; 4. Nat Methods (2008) 5:197; 5. Methods Mol Biol (2010) 611:151; 6. Cancer Epidemiol Biomarkers Prev (2007) 16:1371; 7. J Neurosci Methods (2008) 171:239; 8. J Histochem Cytochem (1993) 41:1833; 9. Methods Mol Biol (2006) 315:363; 10. Cytometry (1998) 32:163; 11. Dev Dyn (2009) 238:944; 12. Nat Protoc (2006) 1:2110; 13. Mol Pharm (2009) 6:1170; 14. J Immunol (2009) 182:4056; 15. J Cell Sci (2007) 120:101; 16. Microsc Res Tech (2005) 68:307; 17. Cytometry A (2006) 69:659; 18. Cytometry A (2006) 69:677; 19. Methods (1999) 18:447; 20. Appl Immunohistochem Mol Morphol (1999) 7:156; 21. Environ Sci Technol (2009) 43:6844; 22. Cytometry A (2007) 71:174; 23. PLoS ne (2009) 4:e4418; 24. Nat Protoc (2008) 3:619; 25. Cytometry A (2008) 73:904; 26. J Neurosci Methods (2009) 176:78; 27. Biophys J (2007) 93:2519; 28. Nat Neurosci (2008) 11:713; 29. Chembiochem (2006) 7:268; 30. Biochemistry (2006) 45:12411; 31. Biophys J (2004) 86:2517; 32. Biotechniques (2001) 31:1076; 33. J Microsc (2005) 218:148; 34. J Fluorescence (1996) 6:147; 35. J Microsc (2007) 228:390; 36. Anal Biochem (2007) 364:180; 37. J Res Natl Inst Stand Technol (2001) 106:381; 38. J Res Natl Inst Stand Technol (2002) 107:83; 39. J Fluoresc (2004) 14:465; 40. Appl Environ Microbiol (2009) 75:5952; 41. PLoS ne (2009) 4:e7637; 42. Cell Vision (1995) 2:165; 43. CSH Protoc (2010) 2010:pdb.prot5382; 44. Methods Mol Biol (2010) 632:141. Figure Coverslip maxi-rack (C24784). The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by covered one or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to Appendix the Appendix on on 959

23 PRDUCT LIST 23.1 FLURESCENCE MICRSCPY ACCESSRIES AND REFERENCE STANDARDS Cat. No. Product Quantity A18211 Adhesive seal-tab, for HybriWell hybridization sealing system *set of 400* 1 set A7816 Attofluor cell chamber *for microscopy* each C14837 Constellation microspheres for imaging *mixture of assorted sizes and colors* 3 ml C24784 coverslip maxi-rack *for 50 coverslips* each C14784 coverslip mini-rack *for 8 coverslips* each C18160 CoverWell imaging chamber gasket, one chamber, 20 mm diameter, 0.5 mm deep *set of 40* 1 set C18161 CoverWell imaging chamber gasket, one chamber, 20 mm diameter, 1.0 mm deep *set of 40* 1 set C24726 CoverWell imaging chamber gasket with adhesive, one chamber, 20 mm diameter, 0.5 mm deep *set of 40* 1 set C24727 CoverWell imaging chamber gasket with adhesive, one chamber, 20 mm diameter, 1.0 mm deep *set of 40* 1 set C18155 CoverWell incubation chamber gasket, one chamber, 13 mm diameter, 0.2 mm deep *set of 25* 1 set C18156 CoverWell incubation chamber gasket, one chamber, 13 mm diameter, 0.5 mm deep *set of 50* 1 set C18150 CoverWell incubation chamber gasket, one chamber, 40 mm x 22 mm, 0.2 mm deep *set of 25* 1 set C18151 CoverWell incubation chamber gasket, one chamber, 40 mm x 22 mm, 0.5 mm deep *set of 50* 1 set C18139 CoverWell perfusion chamber gasket, eight chambers, 9 mm diameter, 0.5 mm deep *set of 20* 1 set C18140 CoverWell perfusion chamber gasket, eight chambers, 9 mm diameter, 1.0 mm deep *set of 20* 1 set C18141 CoverWell perfusion chamber gasket, eight chambers, 9 mm diameter, 2.0 mm deep *set of 20* 1 set C18142 CoverWell perfusion chamber gasket, eight chambers, 9 mm diameter, 2.5 mm deep *set of 20* 1 set C18128 CoverWell perfusion chamber gasket, four chambers, 19 mm x 6 mm, 0.5 mm deep *set of 40* 1 set C18136 CoverWell perfusion chamber gasket, one chamber, 20 mm diameter, 1.0 mm deep *set of 40* 1 set C18120 CoverWell perfusion chamber gasket, one chamber, 32 mm x 19 mm, 0.5 mm deep *set of 40* 1 set C18121 CoverWell perfusion chamber gasket, one chamber, 32 mm x 19 mm, 1.0 mm deep *set of 40* 1 set C24769 CultureWell cell culture plate *set of 10* 1 set C37005 CultureWell chambered coverglass for cell culture *sixteen wells per coverglass* *set of 2* 1 pack C37000 CultureWell chambered coverglass for cell culture *sixteen wells per coverglass* *set of 8* 1 set C24770 CultureWell coverslip divider *set of 4* 1 set C24760 CultureWell multislip cell culture system MSI-12 *plate and insert, fifteen 12 mm coverslips per insert* *set of 10* 1 set C24761 CultureWell multislip cell culture system MSI-18 *plate and insert, eight 18 mm coverslips per insert* *set of 10* 1 set C24762 CultureWell multiwell cell culture system CWI 2R-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, two 1 mm-deep wells per coverslip* *set of 10* 1 set C24763 CultureWell multiwell cell culture system CWI 2R-2.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, two 2 mm-deep wells per coverslip* *set of 10* 1 set C24764 CultureWell multiwell cell culture system CWI 3S-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, three 1 mm-deep wells per coverslip* *set of 10* 1 set C24765 CultureWell multiwell cell culture system CWI 4R-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, four 1 mm-deep wells per coverslip* *set of 10* 1 set C24766 CultureWell multiwell cell culture system CWI 8R-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, eight 1 mm-deep wells per coverslip* *set of 10* 1 set C24767 CultureWell multiwell cell culture system CWI 8R-1.0 TS *plate and insert, four 24 mm x 50 mm coverslips per insert, eight 1 mm-deep wells per coverslip* *set of 2* 1 set C24768 CultureWell multiwell cell culture system CWI 50R-1.0 *plate and insert, four 24 mm x 50 mm coverslips per insert, fifty 1 mm-deep wells per coverslip* *set of 10* 1 set C24775 CultureWell multiwell chambered coverslip CWCS 2R-1.0 *24 mm x 50 mm coverslips, two 1 mm-deep wells per coverslip* *set of 20* 1 set C24776 CultureWell multiwell chambered coverslip CWCS 2R-2.0 *24 mm x 50 mm coverslips, two 2 mm-deep wells per coverslip* *set of 20* 1 set C24777 CultureWell multiwell chambered coverslip CWCS 3S-1.0 *24 mm x 50 mm coverslips, three 1 mm-deep wells per coverslip* *set of 20* 1 set C24778 CultureWell multiwell chambered coverslip CWCS 4R-1.0 *24 mm x 50 mm coverslips, four 1 mm-deep wells per coverslip* *set of 20* 1 set C24779 CultureWell multiwell chambered coverslip CWCS 8R-1.0 *24 mm x 50 mm coverslips, eight 1 mm-deep wells per coverslip* *set of 20* 1 set C24780 CultureWell multiwell chambered coverslip CWCS 50R-1.0 *24 mm x 50 mm coverslips, fifty 1 mm-deep wells per coverslip* *set of 20* 1 set F36924 FluoCells prepared slide #1 *BPAE cells with MitoTracker Red CMXRos, Alexa Fluor 488 phalloidin, DAPI* each F14781 FluoCells prepared slide #2 *BPAE cells with mouse anti-α-tubulin, BDIPY FL goat anti-mouse IgG, Texas Red -X phalloidin, DAPI* each F24630 FluoCells prepared slide #3 *mouse kidney section with Alexa Fluor 488 WGA, Alexa Fluor 568 phalloidin, DAPI* each F24631 FluoCells prepared slide #4 *mouse intestine section with Alexa Fluor 350 WGA, Alexa Fluor 568 phalloidin, SYTX Green* each F36925 FluoCells prepared slide #6 *muntjac cells with mouse anti-xphos Complex V inhibitor protein, Alexa Fluor 555 goat anti-mouse IgG, Alexa Fluor 488 each phalloidin, T-PR -3* F36915 fluorescein *NIST-traceable standard* *nominal concentration 50 µm* *special packaging* 5 x 1 ml F36905 FocalCheck DoubleGreen Fluorescent Microspheres Kit, 6 µm *mounted on slides* 1 kit F36906 FocalCheck Doublerange Fluorescent Microspheres Kit, 6 µm *mounted on slides* 1 kit F36909 FocalCheck fluorescence microscope test slide #1 *for alignment, intensity, and calibration* each F36913 FocalCheck fluorescence microscope test slide #2 *for spectral imaging systems* each F36914 FocalCheck fluorescence microscope test slide #3 *5 colors, high and low intensities* each F24633 FocalCheck Fluorescent Microspheres Kit, 6 µm *mounted on slides* 1 kit F24634 FocalCheck Fluorescent Microspheres Kit, 15 µm *mounted on slides* 1 kit F14807 FocalCheck microspheres, 6 µm, fluorescent dark-red ring stain/green throughout 0.5 ml F14808 FocalCheck microspheres, 6 µm, fluorescent green ring stain/blue throughout 0.5 ml F14806 FocalCheck microspheres, 6 µm, fluorescent green/orange/dark-red ring stains 0.5 ml F7234 FocalCheck microspheres, 15 µm, fluorescent blue/orange ring stains 0.5 ml F7239 FocalCheck microspheres, 15 µm, fluorescent dark-red ring stain/green throughout 0.5 ml 960 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT IMPRTANT NTICE: NTICE The products : The products described described in this manual in this are manual covered are by covered one or more by one Limited or more Use Label Limited License(s). 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24 PRDUCT LIST 23.1 FLURESCENCE MICRSCPY ACCESSRIES AND REFERENCE STANDARDS continued Cat. No. Product Quantity F7237 FocalCheck microspheres, 15 µm, fluorescent green ring stain/blue throughout 0.5 ml F7238 FocalCheck microspheres, 15 µm, fluorescent green ring stain/dark red throughout 0.5 ml F7240 FocalCheck microspheres, 15 µm, fluorescent green/dark-red ring stains 0.5 ml F7235 FocalCheck microspheres, 15 µm, fluorescent green/orange/dark-red ring stains 0.5 ml F7236 FocalCheck microspheres, 15 µm, fluorescent orange ring stain/blue throughout 0.5 ml F14791 FocalCheck Thin-Ring Fluorescent Microspheres Kit, 1.0 µm *three suspensions* 1 kit H18200 HybriSlip hybridization cover, 22 mm x 22 mm *RNase free* *set of 500* 1 set H18201 HybriSlip hybridization cover, 40 mm x 22 mm *RNase free* *set of 500* 1 set H18202 HybriSlip hybridization cover, 60 mm x 22 mm *RNase free* *set of 500* 1 set H24720 HybriWell hybridization sealing system, 13 mm diameter chamber, 0.25 mm deep *set of 100* 1 set H24721 HybriWell hybridization sealing system, 20 mm diameter chamber, 0.15 mm deep *set of 100* 1 set H24723 HybriWell hybridization sealing system, 22 mm x 22 mm chamber, 0.15 mm deep *set of 100* 1 set H18210 HybriWell hybridization sealing system, 40 mm x 21 mm chamber, 0.15 mm deep *set of 100* 1 set H24722 HybriWell hybridization sealing system, 40 mm x 22 mm chamber, 0.25 mm deep *set of 100* 1 set I36933 Image-iT FX signal enhancer 10 ml I7221 InSpeck Blue (350/440) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I7225 InSpeck Deep Red (633/660) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I7219 InSpeck Green (505/515) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I14785 InSpeck Green (505/515) Microscope Image Intensity Calibration Kit, 6 µm 1 kit I7223 InSpeck range (540/560) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I14786 InSpeck range (540/560) Microscope Image Intensity Calibration Kit, 6 µm 1 kit I7224 InSpeck Red (580/605) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I14787 InSpeck Red (580/605) Microscope Image Intensity Calibration Kit, 6 µm 1 kit M7901 MultiSpeck Multispectral Fluorescence Microscopy Standards Kit *in suspension* 1 kit NCYTE MultiWells, one well, 13 mm diameter, with slide and matching gasket *set of 20* 1 set NCYTE MultiWells, 12 wells, 5 mm diameter, with slide and matching gasket *set of 20* 1 set rings for Attofluor cell chamber, set of 10 each P18174 Press-to-Seal silicone isolator, one well, 20 mm diameter, 0.5 mm deep *set of 50* 1 set P18175 Press-to-Seal silicone isolator, one well, 20 mm diameter, 1.0 mm deep *set of 50* 1 set P24743 Press-to-Seal silicone isolator with adhesive, eight wells, 9 mm diameter, 0.5 mm deep *set of 25* 1 set P24744 Press-to-Seal silicone isolator with adhesive, eight wells, 9 mm diameter, 1.0 mm deep *set of 25* 1 set P24740 Press-to-Seal silicone isolator with adhesive, one well, 20 mm diameter, 0.5 mm deep *set of 50* 1 set P24741 Press-to-Seal silicone isolator with adhesive, one well, 20 mm diameter, 1.0 mm deep *set of 50* 1 set P24742 Press-to-Seal silicone isolator with adhesive, 24 wells, 2.5 mm diameter, 2.0 mm deep *set of 25* 1 set P24745 Press-to-Seal silicone sheet with adhesive, 13 cm x 18 cm, 0.5 mm thick *set of 5* 1 set P18178 Press-to-Seal silicone sheet, 13 cm x 18 cm, 0.5 mm thick *set of 5* 1 set P18179 Press-to-Seal silicone sheet, 13 cm x 18 cm, 1.0 mm thick *set of 5* 1 set P7481 ProLong Antifade Kit 1 kit P36930 ProLong Gold antifade reagent 10 ml P36934 ProLong Gold antifade reagent *special packaging* 5 x 2 ml P36931 ProLong Gold antifade reagent with DAPI 10 ml P36935 ProLong Gold antifade reagent with DAPI *special packaging* 5 x 2 ml P37002 ProPlate adhesive seal-strips *set of 50 seal-strips and one applicator* 1 set P37001 ProPlate multi-array slide module *set of 2* 1 set P37004 ProPlate multi-array system *includes four 16-well slide modules, one tray and cover, ten seal-strips and one applicator* 1 set P37003 ProPlate tray and cover *includes one tray and cover* 1 set P7220 PS-Speck Microscope Point Source Kit *blue, green, orange and deep red fluorescent beads* 1 kit Q10336 Qmount Qdot mounting media 3 x 2 ml R14782 Reference Dye Sampler Kit *five 1 mm solutions, 1 ml each* 1 kit S24732 Secure-Seal hybridization chamber gasket, eight chambers, 9 mm diameter, 0.8 mm deep *set of 20* 1 set S24733 Secure-Seal hybridization chamber gasket, eight chambers, 9 mm diameter, 1.3 mm deep *set of 20* 1 set S24730 Secure-Seal hybridization chamber gasket, one chamber, 20 mm diameter, 0.8 mm deep *set of 40* 1 set S24731 Secure-Seal hybridization chamber gasket, one chamber, 20 mm diameter, 1.3 mm deep *set of 40* 1 set S24734 Secure-Seal hybridization chamber gasket, one chamber, 22 mm x 22 mm, 0.8 mm deep *set of 50* 1 set S24737 Secure-Seal spacer, eight wells, 9 mm diameter, 0.12 mm deep *set of 100* 1 set The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The 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25 Section 23.2 Flow Cytometry Reference Standards 23.2 Flow Cytometry Reference Standards Flow cytometers are designed to perform quantitative measurements on individual cells and other particles with speed, accuracy and precision. As with all high-performance instrumentation, flow cytometers must be calibrated frequently to ensure accuracy and reliability. The stability, uniformity and reproducibility of our fluorescent microsphere products make them ideal reference standards for flow cytometry. However, because of the high variability in quantum yields of bound dyes and the heterogeneity of protein labeling, as well as problems with stoichiometry and accessibility in binding to targets, bead standards containing a known number of fluorophores per bead do not necessarily provide accurate information about the number of ligands bound to a cell. 1 4 AlignFlow and AlignFlow Plus Flow Cytometry Alignment Beads In order to ensure accurate and reproducible quantitative results, flow cytometers should be checked at least daily for proper performance. AlignFlow and AlignFlow Plus flow cytometry alignment beads permit the calibration of a flow cytometer s laser(s), optics and stream flow without wasting valuable and sensitive experimental material. 5 7 These fluorescently stained polystyrene microspheres are highly uniform with respect to both size and fluorescence intensity, and they are designed to approximately replicate the size, emission wavelength and intensity of biological samples. Because the dyes are contained inside the microsphere s matrix instead of on the surface, AlignFlow beads have excellent photochemical and physical stability, providing reliable reference signals for aligning, focusing and calibrating flow cytometers. The fluorescent dyes have been carefully selected for optimal excitation by laser sources commonly used in flow cytometry. The 2.5 µm AlignFlow flow cytometry alignment beads are available in four versions: for UV ( nm) excitation (A7304), for 488 nm excitation (A7302), for 633 nm excitation (A7312) and for nm excitation (A14835); the 6 µm AlignFlow Plus beads are available for the same four excitation-wavelength ranges: for UV ( nm) excitation (A7305), for 488 nm excitation (A7303), for 633 nm excitation (A7313) Number of particles Green range Red Fluorescence intensity Figure AlignFlow Plus (A7303) flow cytometry alignment beads excited at 488 nm by an argon-ion laser and monitored in three emission channels. The broad fluorescence emission is detected in all three channels. Note the exceptionally small variation of fluorescence intensity of the beads. Contributed by Carleton Stewart, Roswell Park Cancer Institute. and for nm excitation (A14836). The UV light excitable beads emit from 400 nm to 470 nm, the 488 nm light excitable beads emit broadly from 515 nm to 660 nm (Figure ), the 633 nm light excitable beads emit from 645 nm to 680 nm, and the nm light excitable beads emit from 670 nm to 720 nm. The AlignFlow and AlignFlow Plus flow cytometry alignment beads are supplied as suspensions packaged in dropper vials for convenient dispensation. LinearFlow Flow Cytometry Intensity Calibration Kits LinearFlow Flow Cytometry Intensity Calibration Kits provide flow cytometer operators with intensity references for generating calibration curves, establishing photomultiplier settings and evaluating sample brightness Each kit contains fluorescent microspheres in which the degree of staining has been carefully controlled to provide precisely determined intensity levels when excited in a flow cytometer (Figure ). The microspheres are supplied as suspensions packaged in dropper vials for convenient dispensation. The LinearFlow Flow Cytometry Intensity Calibration Kits are available in two different bead sizes (2.5 µm or 6 µm) and five different fluorescent colors covering the spectral ranges commonly encountered in flow cytometry: Blue (for UV excitation/430 nm emission). Available in 2.5 µm (L14812) and 6 µm (L14813) sizes; both kits contain microspheres stained at 100%, 20%, 4.0% and 0.8% relative fluorescence intensity levels. Green (for 488 nm excitation/515 emission). Available in 2.5 µm (L14821) and 6 µm (L14822) sizes; both kits contain microspheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative fluorescence intensity levels. The LinearFlow Green Flow Cytometry Low-Intensity Calibration Kits contain 2.5 µm (L14823) or 6 µm (L14824) diameter beads stained at 0.1%, 0.02%, 0.004% and 0.001% relative fluorescence intensity levels. The fluorescence from the beads with the lowest intensity level is approximately half that of the autofluorescence typically observed from unstained cells. range (for 488 nm excitation/575 nm emission). Available in 2.5 µm (L14814) and 6 µm (L14815) sizes; both kits contain microspheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative fluorescence intensity levels. Carmine (for 488 nm excitation/620 nm emission). Available in 2.5 µm (L14816) and 6 µm (L14817) sizes; both kits contain microspheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative fluorescence intensity levels. Deep Red (for 633 nm excitation/660 nm emission). Available in 2.5 µm (L14818) and 6 µm (L14819, Figure ) sizes; both kits contain microspheres stained at 100%, 20%, 4.0%, 0.8%, 0.2% and 0.04% relative fluorescence intensity levels. The 365/430 nm fluorescence excitation/emission maxima of the microspheres in the LinearFlow Blue Kits provide a close spectral match to samples stained with DAPI, Hoechst or Hoechst nucleic acid stains. These kits are ideal for intensity calibration of flow cytometers equipped with UV laser excitation. The microspheres in the 962 The The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT NTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPRTANT NTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on

26 Section 23.2 Flow Cytometry Reference Standards LinearFlow Green Kits are designed for calibrating the green (FL1) detection channel. Although the microspheres actually have an excitation maximum of ~505 nm, they are effectively excited by the 488 nm spectral line of the argon-ion laser. Their emission maximum of ~515 nm closely matches that of samples labeled with fluorescein, regon Green 488 or Alexa Fluor 488 dyes or with SYTX Green nucleic acid stain. Microspheres in the LinearFlow range Kit are spectrally similar to phycoerythrin and tetramethylrhodamine conjugates, making this kit useful for calibrating the orange (FL2) channel. Although these microspheres actually have an excitation maximum of ~570 nm, they are effectively excited by the 488 nm spectral line of the argon-ion laser. Microspheres in the LinearFlow Carmine Kit exhibit excitation and emission spectra similar to the spectra of the propidium iodide complex with DNA or the spectra of Texas Red or Alexa Fluor 594 dyes and are suitable for calibrating the red (FL3) channel. The microspheres in the LinearFlow Carmine Kit have an excitation maximum of ~580 nm, but they can also be excited by the 488 nm spectral line of the argon-ion laser. The microspheres in the LinearFlow Deep Red Kit have maximal emission at ~660 nm, closely matching that of Alexa Fluor 647 dye, Cy 5 dye and allophycocyanin, and they are useful for calibrating flow cytometers equipped with 633 nm He-Ne laser excitation. Although primarily intended for 633 nm excitation, the LinearFlow Deep Red microspheres can be adequately excited at 488 nm and will provide accurate relative intensity readings with this excitation. Number of particles % 0.2% 0.8% Red fluorescence Figure Fluorescence intensity histogram of the six different 6 µm polystyrene bead samples supplied in the LinearFlow Deep Red Flow Cytometry Intensity Calibration Kit (L14819). Fluorescence measurements were performed with a flow cytometer using excitation at 633 nm. This histogram is a composite of two graphs; the same mixture of micro spheres was sampled and analyzed using two distinct PMT voltage settings in order to cover the full intensity range. 4% 20% 100% PeakFlow Flow Cytometry Reference Beads PeakFlow flow cytometry reference beads are stained with fluorescent dyes that have been carefully selected to produce emission peaks coincident with labeled cells used in typical flow cytometry applications. The emission profiles for these standards are intentionally narrow in comparison to fluorescein-labeled cells (Figure ). Consequently, PeakFlow beads serve as reference sources with emissions centered upon the expected fluorescence of the experimental sample. Because PeakFlow beads are highly uniform with respect to both size and fluorescence intensity, and because they approximate the size, emission wavelength and intensity of many biological samples, they can be used to calibrate a flow cytometer s laser source, optics, stream flow and cell sorting system without wasting valuable and sensitive experimental material. Furthermore, due to their narrow emission profiles, PeakFlow beads of two different fluorescent colors exhibit minimal spectral overlap, and little or no color compensation is needed when setting up for multicolor experiments. As with all of our flow cytometry standard microspheres, PeakFlow beads are stained internally rather than on the surface. The dyes are therefore insulated from environmental interactions that could cause variable fluorescence output, resulting in excellent signal stability. These fluorescent polystyrene microspheres are supplied as suspensions packaged in dropper vials for convenient dispensation, with a choice of seven fluorescent colors and, for most products, two different sizes (Table 23.7). Figure Normalized emission spectra of PeakFlow Green flow cytometry reference beads (P14827, solid line) and fluorescein-labeled cells (dashed line). The narrow emission spectrum of PeakFlow beads is approximately centered on the broader emission spectrum of fluorescein. Table 23.7 Spectral characteristics of PeakFlow flow cytometry reference beads. Cat. No. Size (µm) Nominal Color Abs * (nm) Em * (nm) Emission Matches Cells Stained with: P Blue DAPI, Hoechst dyes P P Green Fluorescein, Alexa Fluor 488 dye, regon Green 488 dye, DiC 18 (3) ( Di ) P P range R-phycoerythrin, tetramethylrhodamine, Alexa Fluor 568 dye, DiIC 18 (3) ( DiI ) P P Carmine Propidium iodide, Texas Red dye, Alexa Fluor 594 dye P P Claret TT -3, Alexa Fluor 647 dye, Cy 5 dye, DiIC 18 (5) ( DiD ) P Ultra red Alexa Fluor 660 dye, Cy 5.5 dye P Infrared Alexa Fluor 750 dye, Cy 7 dye, DiIC 18 (7) ( DiR ) * Approximate absorption and emission maxima for beads in suspension. Suitable for excitation by the UV ( nm) spectral line of argon-ion lasers. Suitable for excitation by the 488 nm spectral line of argon-ion lasers. Suitable for excitation by the 633 nm spectral line of He-Ne lasers. The Molecular Probes Handbook: A Guide A Guide to Fluorescent to Probes Probes and and Labeling Technologies IMPRTANT NTICE IMPRTANT : The products NTICE: described The products in this described manual in are this covered manual are by covered one or more by one Limited or more Use Limited Label Use License(s). Label License(s). Please Please refer refer to the to the Appendix Appendix on on 963

27 Events counted Events counted CD3 (FITC fluorescence) Chapter 23 Antifades and ther Tools for Fluorescence Section 23.2 Flow Cytometry Reference Standards AbC and ArC Bead Kits for Flow Cytometry Compensation AbC Anti-Mouse and AbC Anti-Rat/Hamster Bead Kits The AbC Anti-Mouse Bead Kit (A10344) provides a consistent, accurate and simple-to-use technique for the setting of flow cytometry compensation when using fluorophore-conjugated mouse antibodies. The kit contains two types of specially modified polystyrene microspheres, the AbC capture beads, that bind all isotypes of mouse immunoglobulin, and the negative beads that have no antibody binding capacity. After incubation with a fluorophore-conjugated mouse antibody, the two bead components provide distinct positive and negative populations of beads that can be used to set compensation (Figure ). Because of the consistent nature of bead scatter and high surface antibody binding capacity, more consistent and accurate compensation settings for any combination of fluorophore-labeled mouse antibodies can be achieved. The AbC Anti-Rat/Hamster Bead Kit (A10389) provides parallel functionality for protocols using fluorophore-labeled rat or hamster antibodies. The AbC capture beads and negative beads have a diameter of approximately 6 µm (actual size for each lot is listed on the component vial). The bead suspensions are supplied in dropper vials for convenient sample application. ArC Amine-Reactive Compensation Bead Kit The ArC Amine-Reactive Compensation Bead Kit (A10346) provides a consistent, accurate and simple-to-use technique for the setting of flow cytometry compensation when using any of the LIVE/ DEAD fixable dead cell stains. The LIVE/DEAD Fixable Dead Cell Stain Kits (Section 15.3) use an amine-reactive dye labeling method to evaluate the viability of mammalian cells by flow cytometry. 11 The ArC Amine-Reactive Compensation Bead Kit includes two types of specially modified polystyrene microspheres to allow easy compensation of the LIVE/DEAD fixable stains: the ArC reactive beads (Component A), which bind any of the amine-reactive dyes, and the ArC negative beads (Component B), which have no reactivity. After incubation with any amine-reactive dye, the two kit components provide distinct positive and negative populations of beads that can be used to set compensation. Flow Cytometry Size Calibration Kit The Flow Cytometry Size Calibration Kit (F13838) provides nonfluorescent particle-size calibration standards for use in forward light scattering measurements of cell size by flow cyto metry. 12,13 This kit contains suspensions of six different nonfluorescent microspheres packaged in convenient dropper vials. The individual standards contain highly uniform polystyrene microspheres with nominal diameters of 1.0 µm, 2.0 µm, 4.0 µm, 6 µm, 10 µm and 15 µm. CountBright Absolute Counting Beads Flow cytometry provides a rapid method for quantitating cell characteristics; however, most flow cytometers cannot directly provide the cell concentration or absolute count of cells in a sample. Absolute cell counts have been widely used in quantitating cell populations and disease progression and are generally obtained either by combining a separate cell concentration determination from a hematology analyzer with flow cytometry population data (multiple-platform testing) or by adding an internal microsphere counting standard to the flow cytometry sample (single-platform testing). The single-platform method is preferred as it is technically less complicated and more accurate than multiple-platform testing. 19 To facilitate this single-platform method, we offer CountBright absolute counting beads (C36950), a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths and contain a known concentration of microspheres. For absolute counts, a specific volume of the CountBright microsphere suspension is added to a specific volume of sample, such that the ratio of sample volume to microsphere volume is known. 20 The volume of sample analyzed can be calculated from the number of microsphere events and then used with cell events to determine cell concentration. In general, at least 1000 bead events should be acquired to assure a statistically significant determination of sample volume. Sufficient reagents are provided for 100 flow cytometry assays, each using 50 µl of counting beads per test. CountBright absolute counting beads are broadly fluorescent and can be used with either a fluorescence or scatter threshold. Fluorescence can be excited by wavelengths from UV to 635 nm; fluorescence A 250 B 250 C R-PE fluorescence FITC fluorescence CD56 (R-PE fluorescence) Figure Compensation using the AbC Anti-Mouse Bead Kit (A10344). (A) R-Phycoerythrin (R-PE) conjugated mouse anti human CD56 antibodies (MHCD56044) label the AbC capture beads for a positive signal, and negative beads provide a negative signal. (B) FITC-conjugated mouse anti human CD3 antibodies (MHCD03014) label the AbC capture beads for a positive signal, and negative beads provide a negative signal. (C) Dual-parameter plot showing gated human lymphocytes labeled with R-PE conjugated mouse anti human CD56 and FITC-conjugated mouse anti human CD3 antibodies using compensation settings obtained with the AbC Anti-Mouse Bead Kit. 964 The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPRTANT IMPRTANT NTICE: NTICE The products : The products described described in this manual in this are manual covered are by covered one or more by one Limited or more Use Label Limited License(s). Use Label Please License(s). refer to the Please Appendix refer on to the Appendix on