Optical Filters for Clinical Fluorescence Microscopy
|
|
- Rosalind King
- 6 years ago
- Views:
Transcription
1 clpmag.com Optical Filters for Clinical Fluorescence Microscopy Introduction The use of fluorescence in clinical microscopy has become routine in fields such as microbiology and immunology, as well as in pathology testing labs. Clinical applications encompass a variety of antibody, viral, and genetic screening procedures (Table 1 below). These include direct and indirect labeling of antibodies and proteins, 1 and staining of cells in mycological specimens. 2-4 Typically, a fluorescent molecule, called a fluorophore or fluorochrome, is conjugated to a certain target of interest in the specimen. Many commercial kits and assays are readily available for preparing patient samples for clinical microscopy analysis. Recently, more advanced methods are emerging from the research world and receiving increasing clinical acceptance, such as Fluorescence In Situ Hybridization (FISH), a technique that enables clinical-scale genetic screening based on molecular diagnostics. FISH uses highly specific DNA probes to detect genetic markers with fluorescent molecules (direct method) or nonfluorescent molecules that are tagged by fluorescent antibodies (indirect method). 5 The probe DNA are labeled by specific fluorescence emission color and hybridized to DNA in either interphase or metaphase chromosomes after denaturing, so that the probe nucleotide sequences seek and bind to specific regions on target chromosomes. Direct visualization of the relative positions of the probes, and therefore, translocated genetic sequences, is possible using a fluorescence microscope equipped with colorcontrasting fluorescence optical filter sets for each given fluorophore probe. All of these examples use a fluorescence microscope that is equipped with an intense light source (usually a mercury arc lamp) and one or more sets of optical filters. Optical filters are essential for observing the labeled or stained specimens. Each fluorophore requires a dedicated set of filters optimized for imaging the particular color associated with the fluorophore. In addition to providing the best visual or optical performance for accurate identification and ergonomy, important factors that must be considered for optical filters in clinical microscopy are durability and cost. More durable optical filters do not burn out as a result of the intense illumination source, thus avoiding the need for replacement and downtime, and they may be cleaned like other optical components in a microscope to maintain high performance year after year. Furthermore, while the highest-performance filters might cost only a small fraction of a sophisticated research microscope (such as one with automation and digital imaging), the cost of such components can become prohibitive when outfitting a lab with a typical clinical microscope that has little or no automation and is viewed primarily by eye. In many cases, a clinical microscope is purchased with money from a hospital budget, rather than from a government-research or capital-equipment grant. Hence, it is critical to achieve the best possible performance with the microscope optics, including filters, optimized for reasonable cost. The long-term durability of the optics also leads to a lower total cost of ownership over time. Fortunately, due to recent advances in optical filter technology, 6 filters that are affordable, durable, and exhibit excellent optical performance (such as the Semrock Brightline ClinicalTM filter series) now make advanced clinical observation possible. 1/8
2 Category Fluorphore, Probe, or Stain Applications Examples Direct and Indirect Fluorescent Antibody tests and assays FITC detection of bacteria, fungus, virus, parasite, autoimmune disorders FTA-ABS, ANA, Esptein-Barr virus, Toxoplasma, Legionella, Pneumophila, Giardia Nonspecific Stains Calcofluor White binds to cellulose and chitin of cell walls detection of fungi, pneumocystis, microsporidia, acanthamoeba Auramine, Auramine- Rhodamine binds to mycolic acid detection of Mycobacterium tuberculosis and other acid-fast bacilli Acridine Orange intercalates into both native and denatured nucleic acids detection of bacteria and fungi in blood, CSF and buffy coat preparations FISH and Genetic Molecular Diagnostics DAPI, SepctrumGreen SpectrumOrange detect amplification of Her- 2/neu gene PathVysion (assay to aid in the selection of patients for Herceptin therapy) FITC, Rhodamine BCR/ABL translocation probes Chronic myelogenous leukemia Table 1. Some examples of fluorescence used in clinical applications. Optical Filters and Fluorescence Microscopes Figure 1 shows a fluorescence microscope with an expanded view of the epi-fluorescence illuminator attachment where fluorescence filter cubes holding the optical filters are installed within a removable filter housing turret. Further detail of the fluorescence filter cube shows the configuration and placement of the three optical filters (excitation filter, dichroic beam splitter, and emission or barrier filter). It should be noted that modern fluoresence microscopes allow for easy installation or interchange of filter cubes by the user. The optical filters are key components enabling the detection of fluorescence light from the sample, thereby enabling the functionality that lies at the heart of a fluorescence microscope. The critical role of fluorescence optical filters is often not appreciated, yet their proper use and specification is crucial to realizing optimal performance of a particular clinical fluorescence test or assay. The operation of a fluorescence microscope using optical filters is schematically shown in Figure 2. The excitation filter passes a portion of light from the intense light source, which is then reflected by the dichroic beam splitter (also referred to as a mirror) such that it passes through the microscope objective and fully illuminates the specimen on the sample slide that is on the microscope stage. Some of the excitation light is absorbed by the fluorophore in the specimen, which then instantaneously emits longer-wavelength fluorescence-emission light. A substantial portion of the fluorescence emission is captured by the microscope objective, and then it is transmitted through the dichroic 2/8
3 Figure 1. Olympus BX-51 upright microscope showing detail of the Epi-fluorescence illuminator attachment, fluorescence filter cube placement in the turret housing, and optical fluorescence filters and their arrangement within a cube. Images courtesy of Mike Davidson (Molecular Expressions and Olympus America). beam splitter and through the emission filter to the microscope eyepieces. In this so-called epi-fluorescence configuration, the set of three optical filters function together as a complete set to capture fluorescence-emission light and block contaminating excitation light and other potential sources of background light that would otherwise reduce sensitivity or even suppress the fluorescence from the fluorophore. This separation of the excitation and fluorescence-emission light paths both physically and spectrally is essential, since the fluorescence emission is very faint it is typically four or more orders of magnitude weaker than the excitation light (< 1/10,000th). Fluorescence Filter Functionality and Specification An understanding of the crucial role optical filters play in fluorescence microscopes requires an understanding of the fluorescence absorption and emission process. 7 A fluorophore absorbs incoming excitation light at wavelengths within its absorption-spectrum profile. It then re-emits longer-wavelength fluorescence-emission light at wavelengths given by its emission-spectrum profile. 3/8
4 A simple example that illustrates the specification of optical filters for fluorescein isothiocyanate ( FITC) is shown in Figure 3. The optical filter spectral profiles are shown superimposed on the FITC absorption and emission spectral profiles. The parameters that describe the placement of excitation and emission filters are the center wavelengths (CW) and bandwidths (BW). Here for simplicity, BW refers to the full-width-at-halfmaximum transmission bandwidth, but bandwidth may also be specified as the minimum spectral width over which high transmission is guaranteed. For the example in the diagram, the excitation filter has CW = 475 nm with BW = 33 nm. A bandpass emission filter in this case (CW = 529 nm and BW = 33 nm) is designed so that it specifically captures fluorescence emission primarily from a single fluorophore (such as green emission). Figure 2. Schematic of optical fluorescence filter functionality. Figure 3. Absorption and emission profiles for FITC fluorophore shown with transmission of Brightline Clinical FITC filters. The long pass emitter set is shown at left, and the bandpass emitter set is shown at right. Note how center wavelength, bandwidth, and edge wavelength are specified. Successful observation of fluorescence emission requires the excitation and emission filters to transmit as much light as possible at wavelengths within their respective bands. It is worth noting that while the spectra of bandpass excitation and emission filters appear symmetric when the transmission is plotted on a linear scale, suggesting that an excitation filter may be used as an emission filter, or vice versa, so long as the CW and BW are appropriate, in fact the blocking (visible only on a logarithmic scale) is typically not symmetric. Excitation filters are optimized to maximize blocking in the emission-filter transmission passband, and emission filters are optimized to maximize blocking in the corresponding excitation filter passband (that is, no overlap). The function of the excitation and emission filters is therefore complimentary, ensuring that excitation or other stray light does not spectrally leak, contaminate, and suppress the fluorescence-emission light. The CWs of the filters should be spectrally positioned so that overlap with the absorption and emission spectral profiles maximizes brightness. 6 Widening the BWs of the filters also increases brightness. Another important consideration for clinical samples is that a large amount of nonspecific fluorescence (resulting from the fluorophore of interest not bound to the desired target species) and autofluorescence (from all other 4/8
5 fluorescing substances) may be generated from endogenous tissues, fluids, and other organic matter, resulting in a high background light level that can obscure the fluorescent tagged specimen and thus limit contrast. Nonspecific fluorescence and autofluorescence can be minimized by careful sample preparation 8 but not totally eliminated. Narrowing the bandwidths of the optical filters (particularly the excitation filter) can further reduce the background and enhance contrast (at the expense of brightness), since the observed autofluorescence background roughly increases in proportion to the bandwidth. 6 A balance, therefore, has to be struck between the level of brightness versus background based on the desired observation criteria for a given application. Finally, it should be noted that optical filters need to be properly oriented with respect to excitation light to ensure that autofluorescence generation is minimal. Dichroic beam splitters and long pass excitation filters are usually specified by a cut-on edge wavelength (EW) above which the filter transmits light. A long pass emission filter may transmit fluorescence from all fluorophores that emit light at wavelengths longer than the edge wavelength (such as green and orange emission). This property enables the clinical observer to see possible counterstains or other fluorescence emission that can be used to enhance contrast between different cells and parts of a biological cell. Durability of Optical Fluorescence Filters Figure 4. Transmission versus wavelength comparison between burned-out soft-coated, and Semrock UV excitation filters for DAPI fluorophore. Semrock filters have been exposed to more than 1,000 hours of continuous high intensity HG arc-lamp illumination. For clinical applications, durability of all aspects of the microscope, including the filters, is critical. Optical filters must be impervious to intense light sources that generate ultraviolet (UV) light that could lead to burnout (also known as photodarkening or solarization), particularly of the exciter filter as it is subjected to the full intensity of the illumination source. Most filters used in clinical microscopy use older soft-coating technology that may carry explicit warnings for exposed coatings not to be handled or otherwise touched to prevent damage; in some cases, the coatings can even be wiped right off the substrate glass if not handled carefully. Absorptive or colored substrate glasses are also used in some exciter filters (particularly those optimized for Calcofluor White, DAPI, and other UV-excited fluorophores), and these filters are particularly prone to burnout since they contain impurities that lead to photodarkening. This can lead to a disastrous loss of transmission that may render the filter useless until the filter is replaced. Modern filters such as the Brightline Clinical filter series do not suffer from catastrophic photodarkening or burnout, nor spectral shifting or any other possible variations with usage. These filters are based on a thin-film 5/8
6 manufacturing process called Ion-Beam Sputtered (IBS), which enables filters with hard oxide thin-film coatings to be constructed using a single, low-impurity glass substrate the result is the highest possible transmission with exceptional durability and reliability. Such durability is ensured by regular testing and stringent compliance to the latest optical MIL and ISO standards. 9 A typical example of filter burnout is shown in Figure 4, where soft-coated UV excitation filters that exhibit severe degradation in transmission (down to 10% to 15%) are shown in comparison to modern, hard-coated filters that have experienced more than 1,000 hours of continuous, high-intensity illumination from a mercury-arc excitation lamp. Considerations for Fluorescence Observation It has been discussed that there are many potential fluorescence applications in clinical microscopy and even within a particular application, there can be major differences in the types of specimens and samples. A clinical microscopist may be faced with large ranges in brightness and contrast due to variations in the concentration of fluorescently tagged specimens, as well as background, in clinically prepared slide sample slides. Despite this wide variation, however, most visual observations tend to fall within two broad categories governed by basic imaging properties. Case 1. Maximize Contrast and Brightness This case applies to sample slides that exhibit relatively little nonspecific fluorescence and autofluorescence and therefore tend to have a dark background away from the fluorescence target specimen. In this case, visual perception tends to favor the ratio of brightness to background (which can be thought of as contrast) more heavily than either attribute individually. Optimal observation is therefore obtained by maximizing both the contrast and the brightness. Optimal optical filter performance for this case is achieved by providing high transmission for the filter that directly impacts brightness, and also carefully selecting the CWs of the filters to maximize the fluorescence throughput (brightness) while simultaneously limiting the BWs of the filters to limit the amount of autofluorescence observed. Achieving all of these considerations simultaneously is not trivial, but the benefits are clear and evident. Mike Nasello, supervisor, Clinical Microbiology Laboratories, Strong Hospital, Rochester, NY, states, Having high brightness while maintaining good contrast allows identification of fluorescent tagged specimens not just in high titer (concentration) dilution samples but also for difficult to diagnose low titer cases. Figure 5. Comparative brightness and contrast between hard-coated Brightline Clinical FITC-LP01 (left) and soft-coated (right) optical filter sets on Toxoplasma Gondii specimens tagged with FITC. Brightness (signal) and contrast fields used in comparison are indicated. Example images of a FITC-tagged sample are shown in Figure 5. Here an image photographed using a Brightline Clinical FITC-LP01 filter set is compared to one taken using a commonly used soft-coated filter set. Case 2. Minimize Background and Maximize Brightness 6/8
7 This case applies to sample slides that exhibit very high background due to autofluorescence. Of particular relevance are samples containing fluorophores that require UV or blue excitation since shorter-wavelength light generates greater autofluorescence. 10 With a very high autofluorescence background, visual observation does not perceive good contrast no matter the brightness. The tasks of minimizing the absolute value of background while retaining high brightness are mutually exclusive. The optimal filter set is obtained by choosing as narrow and specific an exciter filter as possible for the fluorophore while maximizing the transmission of all the filters (excitation, beam splitter, and emission or barrier) using advanced filter manufacturing techniques in order to generate good brightness. In this case, Susan Romansky, Clinical Specialist, Microbiology Laboratory, Rochester General Hospital, Rochester, NY, explains that, A darker background provides a crisper image from which minute yet critical features such as cell walls, septa, and hyphae in mycobacterial samples can be distinguished. Figure 6. Comparative brightness and contrast between Brightline Clinical CFW-LP01 (left) and wide band UVexciter (right) optical filter set on fungal yeast cells stained with Calcofluor White. Signal (brightness) and background fields used in the comparison are indicated. All images captured using a 40x Olympus BX-41 microscope and Pixelink 1776 camera with 500 millisecond exposure. In Figure 6, an example is shown of fungal yeast cells stained with Calcofluor White viewed using the Brightline Clinical CFW-LP01 set and a commonly used optical filter set containing a wide-band UV exciter and long-pass emitter. The competitor image shows a diffuse background or fluorescence halo that extends over the entire observational field due to auto- and nonspecific fluorescence that washes out detail despite the fact that the specimen yeast cell appears quite bright. The markedly better contrast of the Brightline Clinical CFW-LP01 filter set allows clear visualization of the septa in the fungal yeast cells due to the narrow excitation filter (CW = 387 nm, BW = 14 nm) that generates much less autofluorescence while still providing high optical transmission. Note that camera noise has been subtracted from both brightness and background fields in Figures 5 and 6 in the numerical comparisons to most accurately reflect what would be observed visually. Summary For clinical fluorescence microscopy, it is crucial that optical filters be chosen to give the best performance for a given application and fluorophore. The wide prevalence and variation of clinical fluorescence samples, as well as the increasing number of observations required of clinical microscopists, puts a premium on performance. Good performance involves selecting and optimizing spectral features of the filters (CW, BW, or EW) to maximize a combination of brightness and contrast. Such performance is critical when scanning fluorescent specimens for features at lower magnifications that may be difficult to discern before subsequent inspection at higher magnifications can provide a confirming diagnosis. Equally important is the consideration of choosing durable coatings particularly exciter filters that do not burn out and permit reliable observations and measurements time after time. Given the availability of modern optical filters such as the Semrock Brightline Clinical series based on hard coated technology (IBS) at a competitive cost, clinical microscopists should consider replacing any current soft-coated optical filters with hard-coated ones. One can then be confident that robust, statistically significant observations can be performed without worrying about whether optical filters installed in the microscope will degrade. A critical example is the necessity of maintaining 7/8
8 clinical patient slide samples over time and being able to confidently make comparisons between past, present, and future samples. Atul Pradhan, PhD, is the principal engineer for Semrock Inc, Rochester, NY. He can be reached For more information search for microscopy at the top of the page. References 1. Lehman D. Rapid diagnostic testing in microbiology-clinical issues. Med Lab Obs. 2003;35(5). 2. Darken M. Applications of fluorescence brightners in biological techniques. Science. 1961;133: Hageage G, Harrington B. Use of calcofluor white in clinical mycology. Lab Med. 1984;15: Monheit JE, Cowan DF, Moore DG. Rapid detection of fungi in tissues using calcofluor white and fluorescence microscopy. Arch Pathol Lab Med. 1984;108: Werner M, Wilkens L, Aubele M, Nolte M, Zitzelsberger H, Komminoth P. Interphase cytogenetics in pathology: Principles, methods, and applications of fluorescence in situ hybridization (FISH). Histochem Cell Biol. 1997;108(4): Erdogan T, Pradhan A, Mizrahi V.Optical filters impact fluorescence fidelity. Biophotonics International. 2003;10(10): Lichtman J, Conchello JA. Fluorescence microscopy. Nat Methods. 2005;2(12): Neumann M, Gabel D. Simple method for reduction of autofluorescence in fluorescence microscopy. J Histochem Cytochem. 2002;50(3): Filter reliability. Available at: Accessed December 28, Drezek R, Brookner C, Pavlova I, et al. Autofluorescence microscopy of fresh cervical-tissue sections reveals alterations in tissue biochemistry with dysplasia. Photochem Photobiol. 2001;73(6): /8
Dino-Lite knowledge & education. Fluorescence Microscopes
Dino-Lite knowledge & education Fluorescence Microscopes Dino-Lite Fluorescence models Smallest fluorescence microscope in the world Revolution to biomedical and educational applications Flexible Easy
More informationFluorescent in-situ Hybridization
Fluorescent in-situ Hybridization Presented for: Presented by: Date: 2 Definition In situ hybridization is the method of localizing/ detecting specific nucleotide sequences in morphologically preserved
More informationFluorescence Microscopy. Terms and concepts to know: 10/11/2011. Visible spectrum (of light) and energy
Fluorescence Microscopy Louisiana Tech University Ruston, Louisiana Microscopy Workshop Dr. Mark DeCoster Associate Professor Biomedical Engineering 1 Terms and concepts to know: Signal to Noise Excitation
More informationWidefield Microscopy Bleed-Through
In widefield microscopy the excitation wavelengths which illuminate the sample, and the emission wavelengths which reach the CCD camera are selected throughout a filter cube. A filter cube consists of
More informationFluorescence spectroscopy
Fluorescence spectroscopy The light: electromagnetic wave Zoltán Ujfalusi Biophysics seminar Dept. of Biophysics, University of Pécs 14-16 February 2011 Luminescence: light is not generated by high temperatures!!!
More informationZytoLight SPEC RET Dual Color Break Apart Probe
ZytoLight SPEC RET Dual Color Break Apart Probe Z-2148-200 Z-2148-50 20 (0.2 ml) 5 (0.05 ml) For the detection of translocations involving the RET gene at 10q11.21 by fluorescence in situ hybridization
More informationZytoLight SPEC BCR/ABL Dual Color Dual Fusion Probe
ZytoLight SPEC BCR/ABL Dual Color Dual Fusion Probe Z-2111-200 20 (0.2 ml) For the detection of the translocation t(9;22)(q34;q11) by fluorescence in situ hybridization (FISH).... In vitro diagnostic medical
More informationConfocal Microscopy & Imaging Technology. Yan Wu
Confocal Microscopy & Imaging Technology Yan Wu Dec. 05, 2014 Cells under the microscope What we use to see the details of the cell? Light and Electron Microscopy - Bright light / fluorescence microscopy
More informationZytoLight SPEC ABL2 Dual Color Break Apart Probe
ZytoLight SPEC ABL2 Dual Color Break Apart Probe Z-2200-200 20 (0.2 ml) For the detection of translocations involving the ABL2 gene at 1q25.2 by fluorescence in situ hybridization (FISH).... In vitro diagnostic
More informationIllumatool ΤΜ Tunable Light System: A Non-Destructive Light Source For Molecular And Cellular Biology Applications. John Fox, Lightools Research.
Illumatool ΤΜ Tunable Light System: A Non-Destructive Light Source For Molecular And Cellular Biology Applications. John Fox, Lightools Research. Fluorescent dyes and proteins are basic analytical tools
More informationFluorescence spectroscopy
Fluorescence spectroscopy The light: electromagnetic wave Tamás Huber Biophysics seminar Dept. of Biophysics, University of Pécs 05-07. February 2013. Luminescence: light emission of an excited system.
More informationCharacterizing Phenotypes of Bacteria by Staining Method
Experiment 3 Laboratory to Biology III Diversity of Microorganisms / Wintersemester / page 1 Experiment 3 Characterizing Phenotypes of Bacteria by Staining Method Advisor NN Reading Chapters in BBOM 9
More informationCharacterizing Phenotypes of Bacteria by Staining Method
Experiment 3 Laboratory to Biology III Diversity of Microorganisms / Wintersemester / page 1 Experiment Characterizing Phenotypes of Bacteria by Staining Method Advisor Reading NN Chapters 3.1, 3.7, 3.8,
More informationZytoLight SPEC FGFR3 Dual Color Break Apart Probe
ZytoLight SPEC FGFR3 Dual Color Break Apart Probe Z-2170-200 20 (0.2 ml) For the detection of translocations involving the FGFR3 gene at 4p16.3 by fluorescence in situ hybridization (FISH).... In vitro
More informationPrinciples of flow cytometry: overview of flow cytometry and its uses for cell analysis and sorting. Shoreline Community College BIOL 288
Principles of flow cytometry: overview of flow cytometry and its uses for cell analysis and sorting Shoreline Community College BIOL 288 Flow Cytometry What is Flow Cytometry? Measurement of cells or particles
More informationCompensation: Fundamental Principles
Flow Cytometry Seminar Series 2017 : Fundamental Principles Spillover correction in multicolor flow cytometry 28.02.2017 http://www.cytometry.uzh.ch Contents Fluorescence and its detection Absorption and
More informationFluorescence spectroscopy
Fluorescence spectroscopy The light: electromagnetic wave Tamás Huber Biophysics seminar Dept. of Biophysics, University of Pécs 05-06. February 2014. 1 Luminescence: light emission of an excited system.
More informationMicrobiology Chapter 2 Laboratory Equipment and Procedures 2:1 The Light Microscope MICROSCOPE: any tool with a lens to magnify and observe tiny
Microbiology Chapter 2 Laboratory Equipment and Procedures 2:1 The Light Microscope MICROSCOPE: any tool with a lens to magnify and observe tiny details of specimens Micro tiny, small Scope to see SIMPLE
More informationZytoDot. 2C SPEC EWSR1 Break Apart Probe
ZytoDot 2C SPEC EWSR1 Break Apart Probe C-3043-100 10 (0.1 ml) For the detection of translocations involving the EWSR1 gene at 22q12 by chromogenic in situ hybridization (CISH).... In vitro diagnostic
More informationFoundations in Microbiology Seventh Edition
Lecture PowerPoint to accompany Foundations in Microbiology Seventh Edition Talaro Chapter 3 Tools of the Laboratory: The Methods for Studying Microorganisms Copyright The McGraw-Hill Companies, Inc. Permission
More informationPartha Roy
Fluorescence microscopy http://micro.magnet.fsu.edu/primer/index.html Partha Roy 1 Lecture Outline Definition of fluorescence Common fluorescent reagents Construction ti of a fluorescence microscope Optical
More informationZytoLight SPEC MDM2/CEN 12 Dual Color Probe
ZytoLight SPEC MDM2/CEN 12 Dual Color Probe Z-2013-200 Z-2013-50 20 (0.2 ml) 5 (0.05 ml) For the detection of the chromosomal region of the human MDM2 gene and alpha-satellites of chromosome 12 by fluorescence
More informationZytoLight Dual Color Dual Fusion Probe
ZytoLight SPEC ETV6/RUNX1 Dual Color Dual Fusion Probe Z-2157-200 20 (0.2 ml) For the detection of the translocation t(12;21)(p13;q22) by fluorescence in situ hybridization (FISH).... In vitro diagnostic
More informationImaging Quantum Dots using FUJIFILM LAS 4000
Imaging Quantum Dots using FUJIFILM LAS 4000 Application Note John Pizzonia, Ph.D. 9-28-07 Quantum dots (also known as nanocrystals) are a special class of materials known as semiconductors, which are
More informationBiophotonics I W. Petrich
Biophotonics I W. Petrich Slides of lecture #6 November 20 th, 2017 http://www.kip.uni-heidelberg.de/biophotonik/teaching Lecture Biophotonics I will be credited with 2 CP subject to successfully passing
More informationFluorescence Light Microscopy for Cell Biology
Fluorescence Light Microscopy for Cell Biology Why use light microscopy? Traditional questions that light microscopy has addressed: Structure within a cell Locations of specific molecules within a cell
More informationSURFACE ENHANCED RAMAN SCATTERING NANOPARTICLES AS AN ALTERNATIVE TO FLUORESCENT PROBES AN EVALUATION
APPLICATION NOTE SURFACE ENHANCED RAMAN SCATTERING NANOPARTICLES AS AN ALTERNATIVE TO FLUORESCENT PROBES AN EVALUATION Summary: Interest in using nanoparticles specifically, Surface Enhanced Raman Scattering
More informationFlow Cytometry - The Essentials
Flow Cytometry - The Essentials Pocket Guide to Flow Cytometry: 1. Know your Cytometer 2. Understanding Fluorescence and Fluorophores 3. Gating Process 4. Controls 5. Optimization 6. Panel Building 7.
More informationSelected Topics in Electrical Engineering: Flow Cytometry Data Analysis
Selected Topics in Electrical Engineering: Flow Cytometry Data Analysis Bilge Karaçalı, PhD Department of Electrical and Electronics Engineering Izmir Institute of Technology Outline Experimental design
More informationZytoLight FISH-Tissue Implementation Kit
ZytoLight FISH-Tissue Implementation Kit Z-2028-20 20 Z-2028-5 5 For fluorescence in situ hybridization (FISH) using any ZytoLight FISH probe.... In vitro diagnostic medical device according to EU directive
More information11/19/2013. Janine Zankl FACS Core Facility 13. November Cellular Parameters. Cellular Parameters. Monocytes. Granulocytes.
DEPARTEMENT BIOZENTRUM Janine Zankl FACS Core Facility 13. November 2013 Cellular Parameters Granulocytes Monocytes Basophils Neutrophils Lymphocytes Eosinophils Cellular Parameters 1 What Is Flow Cytometry?
More informationPropidium Iodide. Catalog Number: Structure: Molecular Formula: C 27H 34I 2N 4. Molecular Weight: CAS #
Catalog Number: 195458 Propidium Iodide Structure: Molecular Formula: C 27H 34I 2N 4 Molecular Weight: 668.45 CAS # 25535-16-4 Physical Description: Dark red crystals Description: Reagent used for the
More informationVisualizing Cells Molecular Biology of the Cell - Chapter 9
Visualizing Cells Molecular Biology of the Cell - Chapter 9 Resolution, Detection Magnification Interaction of Light with matter: Absorbtion, Refraction, Reflection, Fluorescence Light Microscopy Absorbtion
More informationZytoLight SPEC HER2/TOP2A/CEN 17
ZytoLight SPEC HER2/TOP2A/CEN 17 Triple Color Probe Z-2093-200 Z-2093-50 20 (0.2 ml) 5 (0.05 ml) For the detection of the human HER2 gene, the human TOP2A gene, and alpha-satellites of chromosome 17 by
More informationEach question may have MULTIPLE correct answers. Select all that are correct.
Knowledge Assessment Flow Cytometry Workshop, Part 1 April 20, 2015 Each question may have MULTIPLE correct answers. Select all that are correct. 1. Tandem dyes are a. highly stable fluorophores after
More informationBASICS OF FLOW CYTOMETRY
BASICS OF FLOW CYTOMETRY AUTHOR: Ana Isabel Vieira APPROVAL: Henrique Veiga Fernandes Ana Sílvia Gonçalves SOP.UCF.002 03-09-2015 Pag. 1/9 Overview Flow: Fluid Cyto: Cell Metry: Measurement Flow cytometry
More informationTime-resolved Measurements Using the Agilent Cary Eclipse Fluorescence Spectrophotometer A Versatile Instrument for Accurate Measurements
Time-resolved Measurements Using the Agilent Cary Eclipse Fluorescence Spectrophotometer A Versatile Instrument for Accurate Measurements Technical Overview Authors Dr. Fabian Zieschang, Katherine MacNamara,
More informationCOPYRIGHTED MATERIAL. Tissue Preparation and Microscopy. General Concepts. Chemical Fixation CHAPTER 1
CHAPTER 1 Tissue Preparation and Microscopy General Concepts I. Biological tissues must undergo a series of treatments to be observed with light and electron microscopes. The process begins by stabilization
More informationMore on fluorescence
More on fluorescence Last class Fluorescence Absorption emission Jablonski diagrams This class More on fluorescence Common fluorophores Jablonski diagrams to spectra Properties of fluorophores Excitation
More informationHydroxystilbamidine Protocol
ab138870 Hydroxystilbamidine Protocol Instructions for Use Staining of DNA and RNA in cells. This product is for research use only and is not intended for diagnostic use. 1 Table of Contents 1. Biological
More informationCavity Filters. KIGRE, INC., 100 Marshland Road, Hilton Head, SC 29926, USA PH: FAX: Web:
Cavity Filters Kigre, Inc. s expertise in laser glass technology has led the way in the company s development of a wide range of cavity filter glass for solid-state lasers. Filters are used inside the
More informationAmerican Society of Cytopathology Core Curriculum in Molecular Biology
American Society of Cytopathology Core Curriculum in Molecular Biology American Society of Cytopathology Core Curriculum in Molecular Biology Chapter 3 Molecular Techniques Separation and Detection, Part
More informationZytoLight SPEC BRAF/CEN 7 Dual Color Probe
ZytoLight SPEC BRAF/CEN 7 Dual Color Probe Z-2191-200 20 (0.2 ml) For the detection of the human BRAF gene and alphasatellites of chromosome 7 by fluorescence in situ hybridization (FISH).... In vitro
More informationDiagnostic Microbiology
Diagnostic Microbiology Identification of Microbes Lecture: 1 Out lines What is expected out of this course??? At the end of this course, you will be able to apply Conventional/ Molecular diagnostic methods
More informationComparative Genomic Hybridization
Comparative Genomic Hybridization Srikesh G. Arunajadai Division of Biostatistics University of California Berkeley PH 296 Presentation Fall 2002 December 9 th 2002 OUTLINE CGH Introduction Methodology,
More informationColor-Rich Fluoro-Max Dyed Microparticles March 2008
Fluoro-Max Dyed Microparticles March 2008 Introduction Dyed Microparticles Thermo Scientific Seradyn dyed and fluorescent microparticles are monodisperse particles prepared by unique and proprietary emulsion
More informationIQFISH on Dako Omnis. Panel for Lung Cancer. Dako FAST RESULTS. ALK, ROS1, RET and MET IQFISH. Dako Omnis. Agilent Pathology Solutions
PRODUCT INFORMATION Dako Omnis ALK, ROS1, RET and MET IQFISH Dako Agilent Pathology Solutions IQFISH on Dako Omnis Panel for Lung Cancer FAST RESULTS Fast, high-quality FISH Integrated into your IHC workflow
More informationby Ken Tsukii *, Akihiro Murakami *, Toru Takahashi * and Jie Xu *
by Ken Tsukii *, Akihiro Murakami *, Toru Takahashi * and Jie Xu * As we enter the post-genome sequencing era, great importance attaches to ABSTRACT techniques for analyzing single-nucleotide polymorphisms
More informationChapter-IV OPTICAL PROPERTIES
Chapter-IV OPTICAL PROPERTIES 4.1 Ultraviolet/ Visible Spectroscopy UV- visible spectroscopy of wavelength is shorter than visible light, but longer than X- rays. UV-Violet color is the shortest wavelength
More informationBIO 315 Lab Exam I. Section #: Name:
Section #: Name: Also provide this information on the computer grid sheet given to you. (Section # in special code box) BIO 315 Lab Exam I 1. In labeling the parts of a standard compound light microscope
More informationInnovations To Meet Your Needs
Innovations To Meet Your Needs Cooled CCD Camera 1340 x 1037 pixel resolution for greatest image quality 12-bit precision provides 3 orders of linear dynamic range Windows and Power Macintosh Software
More informationConfocal Microscopy Analyzes Cells
Choosing Filters for Fluorescence A Laurin Publication Photonic Solutions for Biotechnology and Medicine November 2002 Confocal Microscopy Analyzes Cells Reprinted from the November 2002 issue of Biophotonics
More informationReal-Time PCR Principles and Applications
Real-Time PCR Principles and Applications Dr Esam Ibraheem Azhar (BSc, MSc, Ph.D Molecular Medical Virology) Asst. Prof. Medical Laboratory Technology Department Objectives Real-Time PCR Principles and
More informationZytoLight FISH-Cytology. Implementation Kit. For fluorescence in situ hybridization (FISH) on cytology specimens using any ZytoLight FISH probe
ZytoLight FISH-Cytology Implementation Kit Z-2099-20 20 For fluorescence in situ hybridization (FISH) on cytology specimens using any ZytoLight FISH probe.... In vitro diagnostic medical device according
More informationHow to perform-control immunostaining experiment - microscopist subjective point of view. Pawel Pasierbek
How to perform-control immunostaining experiment - microscopist subjective point of view. Pawel Pasierbek Immunolabeling and fluorescent detection became such a standard procedure in the biomedical research
More informationSYNGENE APPLICATION TRAINING GUIDE
SYNGENE APPLICATION TRAINING GUIDE Contents Page Lighting and Filter combinations 3 Examples.. 6 Chemiluminescence versus Fluorescence Western blot detection. 8 Film versus digital imaging Chemiluminescence
More informationZytoDot SPEC EGFR Probe
ZytoDot SPEC EGFR Probe C-3007-400 C-3007-100 40 (0.4 ml) 10 (0.1 ml) For the detection of the human EGFR gene by chromogenic in situ hybridization (CISH).... In vitro diagnostic medical device according
More informationAzure cseries. A new way to see the light. c600 c500 c400 c300 c200
Azure cseries A new way to see the light. c6 c5 c4 c3 c2 Infrared Laser Excitation for Quantitative Western Blot Imaging in the NIR Improve Your Data Quality Imaging with infrared dyes offers signal stability
More informationab CytoPainter ER Staining Kit Red Fluorescence
ab139482 CytoPainter ER Staining Kit Red Fluorescence Instructions for Use Designed to detect Human endoplasmic reticulum by microscopy. This product is for research use only and is not intended for diagnostic
More informationAssays for gene expression and protein production
Assays for gene expression and protein production Module 3, Lecture 5! 20.109 Spring 2011! Topics for Lecture 5 Measuring protein levels! Measuring transcript levels! Imaging assays! 2 Module overview:
More informationAPPLICATION OF MOLECULAR TECHNICS FOR DIAGNOSIS OF VIRAL INFECTIONS
APPLICATION OF MOLECULAR TECHNICS FOR DIAGNOSIS OF VIRAL INFECTIONS Hossein Keyvani Basic Diagnostic Methods in Virology Immunology and serology techniques (Antigen-Antibody Reactions) 1 ELISA ( Enzyme
More informationarc lamp is substituted. Before
CE update [cytology hematology generalist] The Principles of Flow Cytometry Antony C. Bakke, PhD From the Department of Pathology, Oregon Health Sciences University, Portland, OR On completion of this
More informationALP (alkaline phosphatase) calibrators were analyzed manually in microtiter plates to find the linearity range by following this protocol:
Exam Mol 3008 May 2009 Subject 1 (15p) ALP (alkaline phosphatase) calibrators were analyzed manually in microtiter plates to find the linearity range by following this protocol: Reaction solutions: 50
More information7/24/2012. DNA Probes. Hybridization and Probes. CLS 420 Immunology & Molecular Diagnostics. Target Sequences. Target Sequences. Nucleic Acid Probes
Hybridization and Probes CLS 420 Immunology & Molecular Diagnostics Molecular Diagnostics Techniques: Hybridization and Probes Nucleic acid probes: A short, known sequence of DNA or RNA Used to detect
More informationSelected Techniques Part I
1 Selected Techniques Part I Gel Electrophoresis Can be both qualitative and quantitative Qualitative About what size is the fragment? How many fragments are present? Is there in insert or not? Quantitative
More informationIntroduction to Fluorescent In Situ Hybridization (FISH)
Robert Driscoll, M.F.S. Heather Cunningham, M.S. Introduction to Fluorescent In Situ Hybridization (FISH) Fluorescent In Situ Hybridization (FISH) FISH is a cytogenetic technique used to detect the presence
More informationChapter 10: Classification of Microorganisms
Chapter 10: Classification of Microorganisms 1. The Taxonomic Hierarchy 2. Methods of Identification 1. The Taxonomic Hierarchy Phylogenetic Tree of the 3 Domains Taxonomic Hierarchy 8 successive taxa
More informationDesign for Manufacturability (DFM) in the Life Sciences
T E C H N I C A L N O T E Design for Manufacturability (DFM) in the Life Sciences Fluorescence Spectroscopy Product Platform Realized with TracePro TM Suite of Opto-Mechanical Design Software Tools Authors:
More informationNEWTON 7.0 BIOLUMINESCENCE & FLUORESCENCE IMAGING IN VIVO - IN VITRO IMAGING
NEWTON 7.0 BIOLUMINESCENCE & FLUORESCENCE IMAGING IN VIVO - IN VITRO IMAGING SMART IMAGING SYSTEM The NEWTON 7.0 system combines high sensitivity with advanced animal-handling features and userfriendly
More informationBacterial Abundance. Objective Measure bacterial numbers and mass per unit volume. Note, we are not concerned with identification here.
Bacterial Abundance Objective Measure bacterial numbers and mass per unit volume. Note, we are not concerned with identification here. Why do we want to know abundance? Allows determination of biomass
More informationDefinitions. What functions does a flow cytometer be able to do? The build of the Flow cytometry and sorting. Flow cytometry
The build of the Flow cytometry and sorting Flow cytometry Seminar Mónika Tóth 11-12-13.04.2011 Definitions Flow cytometry Process or measurement method can measure discrete properties physical, chemical,
More informationseminar
seminar 02.26.-28.2013. Flow cytometry Process or measurement method can measure discrete properties physical, chemical, biochemical, biological parameters of separate particles, e.g. biological cells
More informationThe analysis of fluorescence microscopy images for FRET detection
The analysis of fluorescence microscopy images for FRET detection Ela Claridge, Dale J. Powner and Michael J.O. Wakelam School of Computer Science, The University of Birmingham B5 2TT Institute for Cancer
More informationBIO 315 Lab Exam I. Section #: Name:
Section #: Name: Also provide this information on the computer grid sheet given to you. (Section # in special code box) BIO 315 Lab Exam I 1. In labeling the parts of a standard compound light microscope
More informationLecture 24. Autoimmunity. Origins of autoimmunity. Diseases. Immune Diseases - Chapter 16 - Allergies - Autoimmunity - Immunodeficiency
Lecture 24 Immune Diseases - Chapter 16 - Allergies - Autoimmunity - Immunodeficiency Disease Diagnostics - Chapter 17 - Sample Collections - Phenotypic Method - Genotypic Method - Immunological Method
More informationAzure cseries. A new way to see the light. c600 c500 c400 c300
Azure cseries A new way to see the light. c600 c500 c400 c300 Infrared Laser Excitation for Quantitative Western Blot Imaging in the NIR Improve Your Data Quality Imaging with infrared dyes offers signal
More informationZytoDot SPEC HER2 Probe
ZytoDot SPEC HER2 Probe C-3001-400 C-3001-100 40 (0.4 ml) 10 (0.1 ml) For the detection of the human HER2 gene by chromogenic in situ hybridization (CISH).... In vitro diagnostic medical device according
More informationSpecial Techniques 1. Mark Scott FILM Facility
Special Techniques 1 Mark Scott FILM Facility SPECIAL TECHNIQUES Multi-photon microscopy Second Harmonic Generation FRAP FRET FLIM In-vivo imaging TWO-PHOTON MICROSCOPY Alternative to confocal and deconvolution
More informationMicroscopy, Staining, and Classification
CSLO CHECK CSLO1. Describe distinctive characteristics and diverse growth requirements of prokaryotic organisms compared to eukaryotic organisms. PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell,
More informationQuantum Dot applications in Fluorescence Imaging for Calibration and Molecular Imaging
Quantum Dot applications in Fluorescence Imaging for Calibration and Molecular Imaging Introduction In this application note, we will discuss the application of quantum dots in fluorescence imaging, both
More informationChapter 17: Immunization & Immune Testing. 1. Immunization 2. Diagnostic Immunology
Chapter 17: Immunization & Immune Testing 1. Immunization 2. Diagnostic Immunology 1. Immunization Chapter Reading pp. 505-511 What is Immunization? A method of inducing artificial immunity by exposing
More information1. Immunization. What is Immunization? 12/9/2016. Chapter 17: Immunization & Immune Testing. 1. Immunization 2. Diagnostic Immunology
Chapter 17: Immunization & Immune Testing 1. Immunization 2. Diagnostic Immunology 1. Immunization Chapter Reading pp. 505-511 What is Immunization? A method of inducing artificial immunity by exposing
More informationOverview of Immunohistochemistry
Overview of Immunohistochemistry Immunohistochemistry (IHC) combines anatomical, immunological and biochemical techniques to identify discrete tissue components by the interaction of target antigens with
More informationFluorescence Microscopy
Fluorescence Microscopy Dr. Arne Seitz Swiss Institute of Technology (EPFL) Faculty of Life Sciences Head of BIOIMAGING AND OPTICS BIOP arne.seitz@epfl.ch Fluorescence Microscopy Why do we need fluorescence
More informationINTRODUCTION TO FLOW CYTOMETRY
DEPARTEMENT BIOZENTRUM INTRODUCTION TO FLOW CYTOMETRY F ACS C ore F acility Janine Zankl FACS Core Facility 3. Dezember 2015, 4pm Cellular Parameters Granulocytes Monocytes Basophils Lymphocytes Neutrophils
More informationBiomedical Applications of Molecular Spectroscopy
Biomedical Applications of Molecular Spectroscopy Mike Kayat B&W Tek, Inc 19 Shea Way Newark, DE 19713 United States of America +1 302 368 7824 mikek@bwtek.com 1 Overview Molecular spectroscopy is a large
More informationAbsorption of an electromagnetic wave
In vivo optical imaging?? Absorption of an electromagnetic wave Tissue absorption spectrum Extinction = Absorption + Scattering Absorption of an electromagnetic wave Scattering of an electromagnetic wave
More informationImportance of Molecular Genetics
Molecular Genetic Importance of Molecular Genetics Genetics is playing an important role in the practice of clinical medicine. - Medical genetics involves any application of genetics to medical practice,
More informationHOMOSIL, HERASIL 1, 2 and 3
HOMOSIL, HERASIL 1, 2 and 3 1. GENERAL PRODUCT DESCRIPTION Heraeus HOMOSIL, HERASIL 1, 2, and 3 are optical quartz glass grades manufactured by flame fusion of natural quartz crystals. They combine excellent
More informationab CytoPainter ER Staining Kit Red Fluorescence
ab139482 CytoPainter ER Staining Kit Red Fluorescence Instructions for Use Designed to detect Human endoplasmic reticulum by microscopy. This product is for research use only and is not intended for diagnostic
More informationEPIGENTEK. Methylamp Global DNA Methylation Quantification Ultra Kit. Base Catalog # P-1014B PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
Methylamp Global DNA Methylation Quantification Ultra Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses: The Methylamp Global DNA Methylation Quantification Ultra Kit is suitable for
More informationFluorescence Microscopy
Fluorescence Microscopy Dr. Arne Seitz Swiss Institute of Technology (EPFL) Faculty of Life Sciences Head of BIOIMAGING AND OPTICS BIOP arne.seitz@epfl.ch Fluorescence Microscopy Why do we need fluorescence
More informationZytoDot. 2C SPEC HER2/CEN 17 Probe
ZytoDot 2C SPEC HER2/CEN 17 Probe C-3032-400 C-3032-100 40 (0.4 ml) 10 (0.1 ml) For the detection of the human HER2 gene and alphasatellites of chromosome 17 by chromogenic in situ hybridization (CISH)....
More informationCell Imaging. Cell Imaging 48
Cell Imaging 48 bio-rad.com/zoe Cell Imaging Bio-Rad s suite of tools for fluorescence microscopy and cell imaging includes the ZOE fluorescent cell imager and nuclear dyes. See Also PureBlu Hoechst 33342
More informationFluorescence & UV- Visible Workshop
Fluorescence & UV- Visible Workshop Simple Applications to Sophisticated Analyses Why UV-Vis and Fluorescence? Quantitative measurements in solutions and solids Quality assurance and quality control (QA/QC)
More informationZytoDot. 2C SPEC MDM2/CEN 12 Probe
ZytoDot 2C SPEC MDM2/CEN 12 Probe C-3049-400 C-3049-100 40 (0.4 ml) 10 (0.1 ml) For the detection of the chromosomal region of the human MDM2 gene and alpha-satellites of chromosome 12 by chromogenic in
More informationاالستاذ المساعد الدكتور خالد ياسين الزاملي \مناعة \المرحلة الثانية \ التحليالت المرضية \ المعهد التقني كوت
Types of Antigen Antibody Reactions Serological tests are widely used for detection of either serum antibodies or antigens for diagnosis of a wide variety of infectious diseases. These serological tests
More informationFluorescent In Situ Hybridization (FISH) Assay
Fluorescent In Situ Hybridization (FISH) Assay 1 What is FISH 2 Probes 3 FISH Procedure 4 Application Definition, Principle and Sample Types The core of FISH technology A quick and simple FISH protocol
More informationQImaging Camera Application Notes Multicolor Immunofluorescence Imaging
QImaging Camera Application Notes Multicolor Immunofluorescence Imaging In order to image localization of intracellular proteins with high specificity, it is frequently necessary to multiplex antibody
More informationIntroduction to histology and its methods of study
Introduction to histology and its methods of study Li shulei lishulei@tom.com Department of Histology & Embryology 1 What is histology Definition Cell: smallest units functions in the human body Tissue
More information