Fluorescence Microscopy For Disease Diagnosis And .
WHO Regional Publications, Eastern Mediterranean Series 28Fluorescence microscopyfor disease diagnosis andenvironmental monitoringWarren R. SanbornSolana Beach, California, USAClaus Chr. HeuckWorld Health OrganizationRaja El AouadInstitut national d’Hygiène, Rabat, MoroccoWulf B. StorchWeinheim, GermanyWorld Health OrganizationRegional Office for the Eastern MediterraneanCairo2005
WHO Library Cataloguing in Publication DataSanborn, Warren R.Fluorescence microscopy for disease diagnosis and environmental monitoring / by Warren R. Sanborn . [etal.] .p. (WHO Regional Publications, Eastern Mediterranean Series; 28)ISBN : 978-92-9021-395-61.Microscopy, Fluorescence 2.Fluorescent Antibody Technique I.Heuck, Claus Chr.II.El Aouad, Raja.III.Storch, Wulf B. IV.Title V. WHO Regional Office for the Eastern Mediterranean VI. Series(NLM Classification: QH 212)Fluorescence microscopy for disease diagnosisand environmental monitoring World Health Organization 2005All rights reserved.The designations employed and the presentation of the material in this publication do not imply the expressionof any opinion whatsoever on the part of the World Health Organization concerning the legal status of anycountry, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.The mention of specific companies or of certain manufacturers’products does not imply that they are endorsedor recommended by the World Health Organization in preference to others of a similar nature that are notmentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initialcapital letters.The World Health Organization does not warrant that the information contained in this publication is completeand correct and shall not be liable for any damages incurred as a result of its use.The named authors alone are responsible for the views expressed in this publication.Publications of the World Health Organization can be obtained from Distribution and Sales, World HealthOrganization, Regional Office for the Eastern Mediterranean, PO Box 7608, Nasr City, Cairo 11371, Egypt(tel: 20 2 670 2535, fax: 20 2 670 2492; email: DSA@emro.who.int). Requests for permission to reproduceWHO EMRO publications, in part or in whole, or to translate them—whether for sale or for noncommercialdistribution—should be addressed to the Regional Adviser, Health and Biomedical Information, at the aboveaddress (fax: 20 2 276 5400; email HBI@emro.who.int).Cover design by John ShimwellPrinted by Toshka, Cairo
.1.11.21.3IntroductionPrinciple of fluorescencePrimary and secondary fluorescenceFluorescence in microscopy specimens131415162.2.12.22.32.4Applications of fluorescence microscopyMedical disease diagnosis and monitoringMonitoring of the environmentFood sanitation and safetyBiological research19212122233.3.13.23.33.4Equipment for fluorescence microscopyBasic fluorescence microscope partsCompound light microscopes for fluorescence microscopyLight sources for fluorescence microscopyLight filters for fluorescence ractical fluorescence microscopyBright-field versus fluorescence microscopyEpi-illumination fluorescence microscopyBasic equipment and supplies for epi-illumination fluorescencemicroscopySet-up and adjusting an epi-illumination fluorescence microscopeUsing an epi-illumination fluorescence microscopeInexpensive transmitted light fluorescence microscopyConcentrating specimens for fluorescence s of fluorochromesFilter sets for commonly used fluorochromes4747496.6.16.2Use of fluorochromesAcridine orange—diagnostic usesFluorescent acid-fast staining for diagnosing acid-fast bacterialinfections5152Principles of immunofluorescence microscopyProcedures for fluorescent antibody stainingFluorochromes for fluorescent antibody conjugatesCounterstains and counterstainingControls of undesirable fluorescence and non-specific reactions85869091937.7.17.27.37.465
4Fluorescence microscopy for disease diagnosis and environmental monitoring7.57.67.77.87.9Control of pHPreventing fluorescence fading (photobleaching)Fixatives and fixation methodsDirect fluorescent antibody tests for infectious disease diagnosisIndirect immunofluorescence tests (IFA)9596991011288.8.18.28.3Fluorescence microscopy for environmental monitoringExamination for microorganisms in the environmentTesting water for microorganismsTesting air for .89.99.109.11Preparation and fluorochrome labelling of antibodiesRepresentative methods for antiserum preparationPurification of gamma globulinsGlobulin concentration with polyethylene glycolConjugation of immunoglobulin with fluorochromesRemoval of unreacted dye from fluorescent antibody conjugatesPreventing non-specific stainingCounterstainImmunohistochemical staining of pre-stained tissue sectionsUnmasking antigens in formaldehyde-fixed paraffin-embedded tissuesStorage of reagents, conjugates, antisera, and blood serum specimensSticking tissue sections to slides17317518819119220120721321721922822910. Characterization of antisera and conjugates10.1 Methods for characterizing conjugates23523511.History of fluorescence sSuppliers of fluorescence microscopesSuppliers of fluorescence conversion kits for microscopesSuppliers of fluorescent antibody conjugates, kits and antiseraSuppliers of chemicals and fluorochromesSuppliers of fluorescence microscopy suppliesSuppliers of auto-immune disease test reagentsMonoclonal fluorescent antibody conjugates to detect infectiousantigensPolyclonal fluorescent antibody conjugates to detect infectious antigensMonoclonal and polyclonal antibodies for indirect fluorescentantibody testsDirect fluorescent antibody test kits for detecting infectious antigensIndirect fluorescent antibody test kits for infectious disease antibodiesAnti-species globulin conjugates for indirect fluorescent antibody testsPreparation of saline solutions, buffers, fluorochrome stains,miscellaneous reagents and 2.1313.13.113.213.3BibliographyFluorochrome dye fluorescence microscopy applicationsFluorescent antibody applicationsSupporting literature256257259261262263264291291307321
ForewordFluorescence microscopy has, for some time now, enhanced the microscopicdiagnosis and monitoring of both communicable and noncommunicable diseases.It has entered many other fields also, especially those in the health sciences,including food safety and research. The latest fluorescent microscopes are muchless expensive and more easily transported than the older versions, making thetechnology affordable now for small laboratory and public health settings indeveloping countries.This comprehensive publication provides laboratory specialists with all theup-to-date information necessary to purchase and use a fluorescence microscope,including suppliers to approach. It addresses, in detail, technical issues such asthe equipment itself and the reagents used, principles of immuno-fluorescencemicroscopy and last, but certainly not least, the use of such microscopy in clinicallaboratory diagnosis.Fluorescence microscopy for diagnosis and environmental monitoringconfirms fluorescence microscopy as a rapid and cost-effective diagnostic tool forcountries with limited resources, and as an invaluable technique that has been testedand validated by experts around the world and, above all, by laboratory practitionersin the field. I strongly recommend its use among central public health laboratoriesin developing countries.FRCSHussein A. Gezairy MDRegional Director for
PrefaceIn countries with limited resources simple, rapid and cost-effective diagnostictechniques are a key element for improving medical and public health laboratoryservices at district and peripheral levels. In these laboratories, bright-fieldmicroscopy still remains the primary diagnostic technique. Rarely has it beendisplaced by other, more modern techniques because these are usually morecomplex and require a continuous line of logistical support. However, there arecertain technologies that do fulfil the technical and logistical requirements of theselaboratories and should enjoy a routine role in improving diagnostic efficiency. Oneof these is fluorescence microscopy.Fluorescence microscopy has proved to be a useful and cost-effectiveprocedure for surveillance of disease outbreaks and for diagnosis of a wide rangeof both communicable and noncommunicable diseases. Fluorescence microscopyis almost as simple and rapid to do as bright-field microscopy, and often it is alsomore specific. However, fluorescence microscopy requires additional investmentin equipment. In the past, the high cost of fluorescence microscopes has preventedthe wider application of this method. More recently, less expensive fluorescencemicroscopes have been developed, and accessories now are available that convert anexisting bright-field microscope into a fluorescence microscope. This developmentplaces fluorescence microscopy in a favourable position as a method that can beused by smaller laboratories with limited resources to enhance their effectivenessat affordable cost.The World Health Organization’s Regional Office for the Eastern Mediterraneanis convinced that laboratories should take more advantage of fluorescencemicroscopy. This manual provides basic information on fluorescence microscopyand an overview of many simple and cost-effective applications for diagnosingdiseases and monitoring environmental contamination by laboratories, even atdistrict or peripheral levels.
AbbreviationsAbAFBAgantibodyacid-fast bacilliantigenBAMbright-field acid-fast microscopyBSAbovine serum albuminCBScarbonate-buffered salineCFUcolony forming unitDANS1-dimethylaminonaphthalene-5-sulfonic acidDAPI4,6-diamidino-2-phenylindol hydrochlorideDFAdirect fluorescent antibodyDNAdeoxyribonucleic acidEMEMEagle’s minimum essential mediumFAfluorescent antibodyFAMfluorescence acid-fast microscopyFDAfluorescein diacetateFITCfluorescein isothiocyanateFMfluorescence microscopyIFimmunofluorescenceIFAindirect fluorescence antibodyim.intramuscular
10Fluorescence microscopy for disease diagnosis and environmental numerical fic stainingPBSphosphate-buffered salineRB 200rhodamine B 200RNAribonucleic acidsc.TRITCsubcutaneoustetramethylrhodamine isothiocyanateURIupper respiratory tract infectionsUVultraviolet
GlossaryAffinity: tendency of certain substances or their compounds to unite with othersubstances and form new compounds.Antigen: a substance, usually a protein or carbohydrate, capable of stimulating animmune response in an animal.Antibody: any of an animal’s immunoglobulins that are produced in response toan antigen and are able to counteract the affects of the stimulating antigen byneutralizing toxins, agglutinating cells or precipitating soluble antigen; i.e., ananimal’s body defence mechanism.Autoantibody: an antibody directed against a patient’s own body tissue.Autofluorescence: synonymous with self, natural or primary fluorescence. Theappearance of fluorescence in tissues or materials that have not been treatedwith labelled conjugates.Fluorochrome: a chemical compound that emits fluorescent light after exposure toshorter wavelength (higher energy) light (usually ultraviolet, purple, blue orgreen).Heterologous: derived from a different substance, i.e. antigen or antibody.Homologous: reaction of an antibody or antigen with the corresponding specificantigen or antibody.Monoclonal antibody: antibody derived from a single cell clone.Monovalent antigen or antibody: antigen or antibody having only one site at whichit can become attached to an antibody or antigen, respectively.Nonspecific staining (NSS): a result of nonimmunological staining by fluorochrome
1. IntroductionFluorescence is light produced by a substance when it is stimulated by anotherlight. It is “cold” light because it does not come from a hot source such as thefilament of an incandescent light bulb. Fluorescence is a short-term event; thefluorescent substance produces light only while it is being stimulated.Fluorescence microscopy is a way to discover facts about specimens that oftenare not visible to bright-field microscopy. In bright-field microscopy, specimens areilluminated from the outside, from below or above. In bright-field microscopy darkobjects are seen against a light background. In fluorescence microscopy, specimensare self-illuminated by internal light, so fluorescence shows bright objects in vividcolour against a dark background. Bright objects against dark backgrounds aremore easily seen. Since the light comes from within the specimen, internal structuresand certain substances can be seen and located. These characteristics makefluorescence microscopy a very sensitive and specific tool.Fluorescence microscopy is used in medicine, environmental studies, foodsanitation, biological research, education and industry. One of the main applicationsis for rapid medical diagnosis. Fluorescent staining is commonly used to improvetuberculosis diagnosis efficiency as well as for malaria diagnosis, for early detectionof bacteria in blood cultures, and to detect and identify nucleic acids by colour.Fluorescent antibodies provide a wide variety of immunologically specific, rapiddiagnostic tests for infectious diseases. In certain cases, detecting and identifyingdisease-causing organisms is only possible by fluorescence microscopy becausesome organisms cannot be cultured easily or the organisms in a specimen are dead.Air and water samples are easily tested for microbial contamination usingfluorescence microscopy. Organisms trapped from these specimens on the surface
14Fluorescence microscopy for disease diagnosis and environmental monitoringof membrane filters can be quickly detected and identified in place, without needfor culture. Environment monitoring by this method is easy and very efficient.Fluorescence microscopy is efficient for direct counting of microorganisms infood and dairy products. It is used to test both raw and pasteurized milk and is about100 times more sensitive than standard culture methods. Fluorescence detection ofmicroorganisms on membrane filters is also used to control the quality of wine andpotable water.The advantages of fluorescence microscopy are due to its sensitivity,specificity, adaptability and easy use. Reliability is high because it is simple and canbe well controlled.This makes fluorescence microscopy a very useful tool for teaching biology.The brilliant colours of fluorescence seen in illuminated specimens are verymemorable for students.The equipment needed for fluorescence microscopy ranges from very complexmicroscopes to relatively simple devices, most bright-field microscopes can be easilyconverted into fluorescence microscopes.Using fluorescence microscopy can reduce laboratory testing costs.Fluorochrome dyes are effective at very dilute concentrations, 1:10 000 or greater.Thus, the cost per test is low. When detecting bacteria in blood cultures,fluorescence microscopy can replace expensive and slow subculturing, saving bothtime and expensive culture media. It considerably reduces time per test, furtherreducing costs. For example, examining a tuberculosis sputum slide by the standardZiehl–Neelsen stain requires 15–20 minutes. Using fluorescent acid-fast stain, thetime for investigation can be reduced to 2–3 minutes. In many other ways, specificand sensitive fluorescence microscopy can replace certain conventionalbacteriological culture uses, reducing costs. Laboratories can easily improve theirservices by using fluorescence microscopy, which saves time in laboratory testingto detect pathogenic microorganisms that other methods may miss.1.1 Principle of fluorescenceLight is electromagnetic energy that moves through space as masslessparticles called photons. Light may also be thought of as moving in waves with high
Introduction15and low points. The distance between a point on one wave, for example the peak,and the same peak on the next wave is the called the wavelength. The energy isinversely proportional to the wavelength—the shorter the wavelength, the moreenergy. Wavelengths of the visible light spectrum range from about 400 nm to 750nm. Different light wavelengths of visible light are seen by humans as differentcolours. The wavelengths of some basic colours are: violet: 420 nm; blue: 480 nm;green: 520 nm; yellow: 590 nm; orange: 630 nm; and red: 660 nm. Longerwavelengths of light above 750 nm are invisible light called infrared Shorterwavelengths of light below 400 nm are a higher energy invisible light calledultraviolet. Near ultraviolet means wavelengths just shorter than 400 nm, and farultraviolet means even shorter wavelengths, about 350 nm and less. Infrared lighthas relatively low energy and low penetrating power, while the shorter-wavelengthultraviolet light has greater energy and penetrating power. The energy spectrumcontinues, and radiation with wavelengths shorter than ultraviolet light, such asgamma rays and X-rays, have still more energy and deeper penetrating power.When light radiation of high enough energy strikes a substance that canfluoresce, the substance absorbs that energy and converts a small part of it intovibrational energy (i.e. heat). The energy that is not absorbed by the substance isemitted again as light (Figure 1.1). The emitted light is called fluorescent light. Thewavelength of the emitted light is always longer than the wavelength of thegenerating light. This phenomenon is called Stokes shift. Fluorescence is shortduration, and fluorescent substances emit light without noticeable delay and onlywhile being stimulated.1.2 Primary and secondary fluorescenceA fluorescent substance can be excited by invisible ultraviolet light, and it willemit longer-wavelength visible light, for example, violet, blue, green or red light.However, many fluorescent substances are also excited by visible violet, blue orgreen light. In fact, visible light is more often used to stimulate fluorescence thaninvisible ultraviolet light. The commonly used dye fluorescein is a good example.Exciting this fluorescent dye with blue light yields yellow-green fluorescence light.Substances that can be activated to fluoresce are called fluorochromes.Fluorochromes may be naturally present in biological materials or may be artificially
16Fluorescence microscopy for disease diagnosis and environmental monitoringFigure 1.1 Scheme showing the origin of fluorescence radiationintroduced into these materials. Fluorescence emission from untreated materials isknown as primary, natural, self- or autofluorescence. When fluorescent substancesare artificially introduced into a specimen, the emitted light is called secondaryfluorescence. Fluorochromes should, when possible, be specific for those structuresor molecules that are of special diagnostic interest.1.3 Fluorescence in microscopy specimensMost biomedical specimens (cells, tissues and microorganisms) do not haveuseful primary fluorescence. Thus, to observe fluorescence in most biologicalspecimens by microscopy, they first must be stained with a fluorochrome. Theultimate goal of staining specimens with fluorochromes is to get specificfluorescence that makes certain areas of a specimen clearly visible.Structures in some plant tissues show primary fluorescence in different coloursmaking these structures stand out in vivid detail. However, at times natural primaryfluorescence (autofluorescence) can interfere with clinical specimen or animal
Introduction17tiss
Practical fluorescence microscopy 37 4.1 Bright-field versus fluorescence microscopy 37 4.2 Epi-illumination fluorescence microscopy 37 4.3 Basic equipment and supplies for epi-illumination fluorescence . microscopy. This manual provides basic information on fluorescence microscopy
Introduction - Optical resolution - Optical sectioning with a laser scanning confocal microscope - Confocal fluorescence imaging Stimulated emission depletion (STED) microscopy Fluorescence resonance energy transfer (FRET) Fluorescence lifetime imaging Two photon excitation microscopy Conclusion @Physics, IIT Guwahati Page 3
FLUORESCENCE MICROSCOPY 177 Overview 177 Applications of Fluorescence Microscopy 178 Physical Basis of Fluorescence 179 . of a microscope under the title “Practical Light Microscopy.” However, the needs of the scientific community for a more comprehensive reference and the furious pace of elec-
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Specialized Fluorescence Techniques 171 Single Molecule Fluorescence 172 Fluorescence Correlation Spectroscopy 173 Forster Resonance Energy Transfer 173 Imaging and Super-Resolution Imaging (Con-ventional and Lifetime) 174 Instrumentation and Laser Based Fluorescence Techniques 175 Nonlinear Emission Processes in Fluorescence Spectroscopy 176
observation to a fluorescence-based experiment. This added dimension can provideinformation on the local environment, fluorescence lifetime and molecular mass. A variety of instruments are utilized in fluorescence polarization studies. These instruments are based on the design of existing fluorescence spectroscopy or microscopy techniques.
Fluorescence is the property of atoms and molecules, so called fluorophores, to absorb light at a particular . Introduction In 1852, the Irish physicist and mathematician Sir George . R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance . Carl Zeiss Microscopy GmbH .
FLUORESCENCE PRACTICAL MANUAL FOR MICROSCOPY TECHNIQUES. 3 chapter. Frequency-domain Fluorescence Lifetime Imaging Microscopy (FD-FLIM) Andrew H.A. Clayton Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology,
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