Accessorize Your Imaging. Ian Clements Invitrogen Corp

Transcription

1 Accessorize Your Imaging Ian Clements Invitrogen Corp

2 Imaging Tools and Accessories Antifade Reagents Prolong Gold SlowFade Gold Image-iT FX Signal Enhancer FocalCheck Microscope Test Slides Fluorescent Microspheres FocalCheck Microspheres Tetraspeck Beads PS Speck Point Spread Function Beads InSpeck Intensity Calibration Beads FluoCells Prepared Microscope Slides Sample Imaging and Staining Accessories

3 Antifades and Photobleaching Problem: Under the high intensity illumination of a microscope, fluorescent dyes are prone to photobleaching. Photobleaching is the irreversible destruction of a dye by free radicals produced during excitation. Photobleaching can be countered in part by, Enhancing the dyes inherent photostability. Addition of an antifade agent.

4 Photobleaching in Action So what does photobleaching look like? Mounted in Antifade Mounted in Buffer Only

5 Antifade Reagents ProLong Gold Antifade Reagent Optimized formulation-superior antifade with little or no impact on initial fluorescence intensity Ready to use - dropper bottle Cures to a firm gel

6 SlowFade Gold Antifade Reagents SlowFade Gold Prolong SlowFade SlowFade Light PBS Average intensity 00:00 00:04 00:09 00:13 00:17 00:22 00:26 00:30 Time (seconds)

7 Image-iT FX Enhancer on Cells No Block NGS Only Blocked with Image-IT FX Blocking Solution Avg. Intensity No Block 5% NGS only Blocked Autofluorescence Block Condition

8 Image-iT FX Enhancer on Tissue No Block Non-Specific White Matter Binding in Brain Sections: Blocked with Image-IT FX Blocking Solution 16um Mouse Hippocampus Section, No primary, AF488 GAM

9 Imaging Standards. Currently there are no established standards used in fluorescence imaging. MPI s FluoCell line of fluorescently labeled cells and tissue sections are widely used as standard slides by instrument vendors. Fluorescence Microspheres are generally better standards as they are more uniformly labeled and more durable. MPI offers a variety of microsphere products specifically targeted at imaging applications.

10 FocalCheck Fluorescence Microscope Calibration Slide #1 15 um 3 ring FocalCheck Beads Green (ex 488) Orange (ex 543) Far Red (ex 633) 6 um Blue-Green FocalCheck Beads Green (ex 488) Blue (ex 365) 4 um TetraSpeck Beads Blue (ex 365) Green (ex 488) Red (ex 543) Far Red (ex 633) 1 um TetraSpeck Beads Blue (ex 365) Green (ex 488) Red (ex 543) Far Red (ex 633) 0.5 um TetraSpeck Beads Blue (ex 365) Green (ex 488) Red (ex 543) Far Red (ex 633) Top Row Bottom Row 100% Relative Intensity 33% Relative Intensity 10% Relative Intensity 3% Relative Intensity 0.667% Relative Intensity For general alignment and calibration

11 FocalCheck Fluorescence Microscope Calibration Slide # um Green Beads 6 um Orange Beads 6 um Red Beads 6 um Far Red Beads A Bead 1 503/511 Bead 1 541/555 Bead 1 578/605 Bead 1 657/676 Mixture of all 8 Bead 2 511/524 Bead 2 545/565 Bead 2 589/613 Bead 2 671/692 Reference Beads B Ring 503/511 Ring 541/555 Ring 589/613 Ring 671/692 Mixture of all 4 Core 511/524 Core 545/565 Core 578/605 Core 657/676 Double- Stained Beads Spectral Unmixing Standards

12 FocalCheck Fluorescence Microscope Calibration Slide #3 Sample Position Wavelengths 350/ / / / /660 A 2.5 um Blue Bright 100% intensity 2.5 um Green Bright 100% intensity 2.5 um Orange Bright 100% intensity 2.5 um Red Bright 100% intensity 2.5 um Deep Red Bright 100% intensity B 2.5 um Blue Dim 1% intensity 2.5 um Green Dim 1% intensity 2.5 um Orange Dim 1% intensity 2.5 um Red Dim 1% intensity 2.5 um Deep Red Dim 1% intensity General Colors, Two Intensities

13 Fluorescent Microspheres Image registration, X,Y alignment Point spread function (PSF) Z-step calibration Relative intensity Spectral unmixing Color range * FocalCheck X X X B/G/O/R/ NIR Mounted on slides (optional) X MultiSpeck X B/G/R TetraSpeck X X (0.2 µm beads) B/G/O/NI R PS-Speck X B/G/O/NI R X InSpeck X B/G/O/R/ NIR

14 FocalCheck fluorescent microsphere Ring stain * Core stain Bead size (µm) Cat. no. Blue, orange None 15 F7234 Green, dark red None 15 F7240 Green, orange, dark red None 15 F7235 Green, orange, dark red None 6 F14806 Green Blue 15 F7237 Green Blue 6 F14808 Green Dark red 15 F7238 Orange Blue 15 F7236 Dark red Green 15 F7239 Dark red Green 6 F14807 * The approximate fluorescence excitation and emission maxima, in nm, of the ring and core stains are: blue, 365/430; green, 505/515; orange, 560/580; and dark red, 660/680. For current prices, please visit our website at probes.invitrogen.com.

15 MultiSpeck and TetraSpeck fluorescent microspheres Microsphere Nominal bead diameter (µm) Cat. no. Blue, green, red fluorescence * MultiSpeck 4 M7901 Blue, green, orange, dark red fluorescence * TetraSpeck 0.1 T7279 TetraSpeck 0.2 T7280 TetraSpeck 0.5 T7281 TetraSpeck 1.0 T7282 TetraSpeck 4.0 T7283 * Corresponding excitation and emission peaks are 365/430 nm (blue), 505/515 nm (green), 560/580 nm (orange), 580/600 nm (red), and 660/680 nm (dark red). Contains a suspension of MultiSpeck beads and a mixed suspension of beads singly labeled with each of the three dyes.

16 Applications of Fluorescent Microspheres Chromatic Aberration Color Misalignment Spherical Aberration Point Spread Function Analysis Z axis errors Field Illumination Intensity Calibration

17 Color Registration and Misalignment While chromatic aberration can be a serious issue, Color Registration is a more common problem in fluorescence imaging. Misregistration is caused most commonly by alignment differences between the optical filters. Misalignment in a confocal system caused by using different light path configurations between scans.

18 Useful tools for demonstration and training #5 NIR Nucleus #4 For Confocal #3 For Confocal #2 Mitochondria #1 Last for years We do consider customs

19 FluoCells Prepared Slides Product # Description Utility F36924 F14781 F24630 F24631 F36925 FluoCells prepared slide #1 *BPAE cells with MitoTracker Red CMXRos, Alexa Fluor 488 phalloidin, DAPI* FluoCells prepared slide #2 *BPAE cells with mouse anti-α-tubulin, BODIPY FL goat antimouse IgG, Texas Red -X phalloidin, DAPI* FluoCells prepared slide #3 *mouse kidney section with Alexa Fluor 488 WGA, Alexa Fluor 568 phalloidin, DAPI* FluoCells prepared slide #4 *mouse intestine section with Alexa Fluor 350 WGA, Alexa Fluor 568 phalloidin, SYTOX Green* FluoCells prepared slide #6 *muntjac cells with mouse anti-oxphos Complex V inhibitor protein, Alexa Fluor 555 goat anti-mouse IgG, Alexa Fluor 488 phalloidin, TO-PRO -3* General 3 color RGB fluorescence sample General 3 color RGB fluorescence sample Tissue section. Use for confocal and 3D imaging. Tissue section. Use for confocal and 3D imaging Use for confocal and 3D imaging without UV excitation capability

20 Sample Imaging and Staining Accessories Attofluor Cell Chamber Coverslip Staining Racks CultureWell Silicone Gasket Systems CoverWell Silicone Gasket Systems Press to Seal Gaskets HybriSlip and HybriWell Systems

21 Attofluor cell chamber A-7816 Surgical stainless steel construction Autoclavable, allowing cells to be grown directly in the chamber O-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

22 Coverslip Racks C14784 Coverslip Mini-Rack Holds 8 coverslips (18 mm x 18 mm) C24784 Coverslip maxi-rack, Holds staining of up to 50 samples

23 CultureWell Cell Culture Systems Product group Details Example CultureWell MultiWell Cell Culture System CultureWell multislip cell culture systems CultureWell chambered coverslips CultureWell chambered coverglass Precut silicone gaskets form no-leak wells on mm coverslips. Each insert includes four coverslips with gaskets, preassembled into a culture plate. Multiple coverslips arrayed on a sheet of silicone and assembled into a convenient insert. Each coverslip can be removed separately for individual staining experiments Precut silicone gaskets form no-leak wells on mm coverslips. Can be used in any culture dish desired. 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

24 CoverWell Imaging Chamber Gaskets Product group Details Example CoverWell imaging chamber gaskets CoverWell perfusion chamber gaskets CoverWell Incubation Chamber Gaskets Incorporate a thin, optically clear plastic cover, making them ideal for light, epifluorescence and confocal laserscanning microscopy. The watertight chamber supports and stabilizes thick and free-floating specimens. Designed for live-cell imaging and manipulation. Silicone gasket technology form watertight chambers with dual-access ports for addition and removal of perfusing media. Improve the uniformity and sensitivity of staining by enclosing a large sample area while minimizing the reagent volume required

25 Press-to-Seal Silicone Isolators and Secure-Seal Adhesive Spacers Press-to-Seal silicone isolators are Removable hydrophobic barriers Customizable to meet specific experimental requirements Material can be autoclaved and adheres to any smooth surface. Isolators are also available with adhesive on one side for added security or permanent mounting. Uncut silicone sheets are also available that can easily be trimmed to prepare customized enclosures. 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 glass coverslips.

26 HybriSlip and HybriWell Hybridization Systems HybriSlip hybridization covers are Nuclease free, Ready-to-use Designed specifically for in situ hybridization. HybriWell hybridization sealing systems are: Coverslip seal combinations Ready-to-use hybridization gaskets with adhesive. Solutions added or removed through dual-access ports without disrupting the specimen

27 THANK YOU! Questions?