Fluorescent Microscopy

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Fluorescent Microscopy

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Fluorescent Microscopy

  1. 1. Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. Sir George G. StokesThe phenomenon offluorescence was known bythe middle of the nineteenthcentury. British scientist SirGeorge G. Stokes first madethe observation that themineral fluorspar exhibitsfluorescence whenilluminated with ultravioletlight, and he coined theword "fluorescence" Dr.T.V.Rao MD 2
  3. 3. Discovery of Fluorescence Microbiology The fluorescence microscope was devised in the early part of the twentieth century by August Köhler, Carl Reichert, and Heinrich Lehmann, among others. However, the potential of this instrument was not realized for several decades, and fluorescence microscopy is now an important (and perhaps indispensable) tool in cellular biology. Dr.T.V.Rao MD 3
  4. 4. Differences between Conventional and Fluorescent Microscope A fluorescence microscope, The Conventional uses a much higher microscope uses intensity light source visible light (400-700 which excites a fluorescent species in a nanometers) to sample of interest. This illuminate and fluorescent species in turn emits a lower energy light produce a magnified of a longer wavelength image of a sample. that produces the magnified image instead of the original light source. Dr.T.V.Rao MD 4
  5. 5. What is Fluorescence?Fluorescence is lightproduced by a substancewhen it is stimulated byanother light.Fluorescence is called"cold light" because itdoes not come from a hotsource like anincandescent light bulb. Dr.T.V.Rao MD 5
  6. 6. What is Fluorescence Microscopy?Fluorescence microscopy is a unique way of using amicroscope to discover facts about specimens that often are notshown by standard bright field microscopy. In bright fieldmicroscopy, specimens are illuminated from outside, below orabove, and dark objects are seen against a light background. Influorescence microscopy, specimens are self-illuminated byinternal light, so bright objects are seen in vivid color against adark background. Bright objects against dark backgrounds aremore easily seen. This characteristic of fluorescence microscopymakes it very sensitive and specific. Dr.T.V.Rao MD 6
  7. 7. Principle of Fluorescent MicroscopyMost cellular components are colorless and cannot beclearly distinguished under a microscope. The basicpremise of fluorescence microscopy is to stain thecomponents with dyes. Fluorescent dyes, also known asfluorophores of fluorochromes, are molecules that absorbexcitation light at a given wavelength (generally UV), andafter a short delay emit light at a longer wavelength. Thedelay between absorption and emission is negligible,generally on the order of nanoseconds. The emission lightcan then be filtered from the excitation light to reveal thelocation of the fluorophores. Dr.T.V.Rao MD 7
  8. 8. Principle of Fluorescent MicroscopyFluorescence microscopy uses amuch higher intensity light toilluminate the sample. Thislight excites fluorescencespecies in the sample, whichthen emit light of a longerwavelength. The imageproduced is based on thesecond light source or theemission wavelength of thefluorescent species -- ratherthan from the light originallyused to illuminate, and excite,the sample. Dr.T.V.Rao MD 8
  9. 9. Works on Faster Transmission of Light Fluorescence, describes light emission that continues only during the absorption of the excitation light. The time interval between absorption of excitation light and emission of re-radiated light in fluorescence is of extraordinarily short duration, usually less than a millionth of a second. Dr.T.V.Rao MD 9
  10. 10. Works on Principles of Light Pathways Specifically, a dichroic mirror is used to separate the excitation and emission light paths. Within the objective, the excitation/emission share the same optics. In a fluorescence microscope, the dichroic mirror separates the light paths. Dr.T.V.Rao MD 10
  11. 11. Advantages of Fluorescent MicroscopyFluorescence microscopy is the most popular method forstudying the dynamic behavior exhibited in live cell imaging.This stems from its ability to isolate individual proteins with ahigh degree of specificity amidst non-fluorescing material.The sensitivity is high enough to detect as few as 50molecules per cubic micrometer.Different molecules can now be stained with different colors,allowing multiple types of molecule to be trackedsimultaneously. These factors combine to give fluorescencemicroscopy a clear advantage over other optical imagingtechniques, for both in vitro and in vivo imaging. Dr.T.V.Rao MD 11
  12. 12. Fluorescence Microscope Fluorescence microscopy by epi-illumination is the most commonly used method today because it is simple to do, needs relatively simple equipment, and is efficient. Dr.T.V.Rao MD 12
  13. 13. Epifluorescence MicroscopyEpifluorescence microscopy is amethod of fluorescencemicroscopy that is widely used inlife sciences The excitatory light ispassed from above (or, forinverted microscopes, frombelow), through the objective lensand then onto the specimeninstead of passing it first throughthe specimen. The fluorescence inthe specimen gives rise to emittedlight which is focused to thedetector by the same objective thatis used for the excitation Dr.T.V.Rao MD 13
  14. 14. The Specimens to be StainedMost specimens forfluorescence microscopymust be stained.Fluorescent stains arecalled "fluorochromes."Acridine orange,auramine O, andfluorescent antibody (FA)are the fluorochromesused most. Dr.T.V.Rao MD 14
  15. 15. Fluorescence Microscopy applied in Many Braches of Science and Medicine uses of fluorescence microscopy are many and varied. They are in medicine, public health, biological research, and environment monitoring. The most common application is medical laboratory diagnosis. Dr.T.V.Rao MD 15
  16. 16. How to Use aFluorescence Microscope Dr.T.V.Rao MD 16
  17. 17. How to Use a Fluorescence Microscope The object to be studied is marked with a molecule called a fluorophore (a dye). When the florescent light is activated, the light used for illumination is separated from the florescent molecule (the fluorophore), which is much weaker. This is done through an emission filter. Dr.T.V.Rao MD 17
  18. 18. Step 1 Locate the light switch on the side of the microscope that turns on the light. Turn the microscope on. Write down the exact time you turn on the light. The florescent light is mercury-based, and a time log must be kept for exposure and use of the light. Dr.T.V.Rao MD 18
  19. 19. Step 2Locate the toggleswitch on the rightside of the microscopebetween the ocularsand objectives. Thisswitch controls theshutter for themercury light to theobjective lens. Dr.T.V.Rao MD 19
  20. 20. Step 3 Select the appropriate dye for your object (this will depend entirely on what you are going to be studying). The most common dyes include I3 (for use with CTC, DTAF and fluorescein), A (for use with DAPI and f420), N21 (for use with Rhoda mine) and L3 (for use Dr.T.V.Rao MD with fluorescein). 20
  21. 21. Step 4Put the filter (dye)into the trayoperated by thesilver sliding knob.To remove the tray,simply pull thesilver knob out. Dr.T.V.Rao MD 21
  22. 22. Step 5 Select the lens you would like to use. The 63x objective lens will have the highest numerical aperture. The 100x objective lens will have the highest magnitude that can be used with the mercury-based florescent light source. Dr.T.V.Rao MD 22
  23. 23. Step 6Turn the light off whenfinished, and mark the time.Wait 30 minutes beforeturning the light back on, orthe lamp could explode. It isa good idea to keep track ofhow many hours the lamp isin use and replace itaccording to themanufactures guidelines. Dr.T.V.Rao MD 23
  24. 24. Step 7 Clean off the microscope lens with lens paper, or if really dirty, use a cotton swap and glass cleaner. Dr.T.V.Rao MD 24
  25. 25. Direct ImmunofluorescenceA specific antibody is labeled by chemically attaching afluorophore to form what is known as a conjugate, which isthen spread on a microscope slide containing the suspectedpresence of a particular antigen known to stimulate productionof the antibody. If the antigen is present, the labeled antibodyconjugate binds to the antigen and remains bound to thespecimen after it is washed. The presence of the chemicallyattached fluorescent conjugate and antigen is demonstratedwhen the fluorophore is excited at its excitation peak, and thesubsequent emission intensities at various wavelengths canthen be observed visually or captured by a detector system(digital or traditional camera). Dr.T.V.Rao MD 25
  26. 26. Indirect ImmunofluorescenceIndirect immunofluorescence here serum possibly containingunlabeled antibody and its related, but known, antigen are incubatedtogether. A fluorochrome conjugated to an anti-human antibody (ifthe specimen being tested is human) is then placed on the slidecontaining the unlabeled antibody-antigen. If indeed, there has beenan antigen-antibody reaction, the fluorochrome-labeled anti-humanantibody attaches itself to the complex formed by the antigen andantibody. Subsequently, the labeled grouping of antigen, antibody,and fluorochrome labeled anti-human antibody is excited at the peakwavelength intensity for that fluorochrome and any resultingemission is observed. The indirect immunofluorescence techniquereduces the necessity of keeping in stock large numbers of labeledantibodies, and also usually results in greater fluorescence intensity.fluorescence, Dr.T.V.Rao MD 26
  27. 27. Fluorescence Microbiology Modernises the Diagnostic LaboratoriesAdvantages of fluorescence microscopy are due toits sensitivity, specificity, rapid testing, and easyuse. It is easy to set up and do, provides rapiddiagnostic tests, and can be very specific. Moderntechnology allows conversion of most compoundmicroscopes easily and economically intoeffective fluorescence microscopes. Dr.T.V.Rao MD 27
  28. 28. Dr.T.V.Rao MD 28
  29. 29. Fluorescent Staining in TuberculosisThe Auramine-rhodamineprocess uses a yellow fluorescentdye to visualize Mycobacteriumtuberculosis under a fluorescencemicroscope. Potassiumpermanganate or acridine orangecan be used as a counterstain.Under the lens, the bacterial cellswill appear green.The Auramine-rhodaminestain is more sensitive thanthe Zhiel-Neelson and morecost effective. Dr.T.V.Rao MD 29
  30. 30. Reagents for stainingAuramine-phenol solution Auramine O 25 g Ethanol 3000 ml Phenol 250 g Distilled water 5300 ml Suspend 25 g auramine-O in 3000 ml of ethanol in a 5-litre conical flask. Add a magnet and place on a magnetic stirrer until solution is complete. Dissolve 250 g phenol in 5300 ml of distilled water. Mix the phenol solution with alcoholic auramine solution. Store in an amber colored bottle at room temp. for up to 3 mths. Filter before use. Dr.T.V.Rao MD 30
  31. 31. Reagents for stainingAcid-alcohol for decolorization Sodium chloride 20 g Hydrochloric acid, A.R. 20 ml Distilled water 500 ml Ethanol 1500 ml Dissolve sodium chloride in distilled water Add conc. hydrochloric acid, mix thoroughly. Add alcohol Can be stored at room temperature for 3 months. Dr.T.V.Rao MD 31
  32. 32. Reagents for stainingCounter stain Potassium permanganate 1 gm Distilled water 1000 ml Dissolve and store in an amber colored bottle Stays at room temperature for up to 3 months. Dr.T.V.Rao MD 32
  33. 33. Fuchsin-stained smears require -Use of 1000x magnificationUse of oil immersionExamination of 300 microscopic fieldsAbout 15 minutes to examine onenegative smearExamination by an experiencedmicroscopist Dr.T.V.Rao MD 33
  34. 34. Staining procedurePlace the slides on a staining rack, with the smearedside facing up, the slides not touching each otherFlood the slides with freshly filtered auramine-phenol. Let stand for 7-10 min.Wash well with running waterDecolorize with acid-alcohol for 1-2 min.Wash as before with water and slope the slides to airdryCounter stain with 0.1% KMNO4 for 30 seconds Dr.T.V.Rao MD 34
  35. 35. Staining procedure - PrecautionsAvoid under-decolorization. AFBs are difficult to over-decolorizesince the decolorization procedure with acid-alcohol is relativelymilder than the 25% H2SO4 used in Z-N.Thick smears: Interfere with decolorization, and counter stain.Mask the presence of AFB and tendency to flake, resulting in lossof smear material and possible transfer of material to other slidesStrong counter stain: May mask the presence of AFBRe Staining : Smears examined by FM may be restained by Z-N toconfirm findings. However, Z-N stained smears cannot be used forFMFading: Stained smears may fade on exposure to light. To be storedwrapped in brown or black paper and kept away from light Dr.T.V.Rao MD 35
  36. 36. EXAMINATION PROCEDUREThe mercury vapor lamp : It takes about 10 min. toreach full intensityExamination: Using the low power objective (100-150x) first examine a known pos. slide to ensurethat the microscope is correctly set upAppearance: Bacilli appear as slender brightyellow fluorescent rods, standing out clearlyagainst a dark backgroundPositive: Minimum of 4 AFB in the entire smear.Negative: Less than 4 bacilli ( No.of bacilli to berecorded) Dr.T.V.Rao MD 36
  37. 37. Screen the Smear in Defined Pattern Dr.T.V.Rao MD 37
  38. 38. Identify the Acid Fast Bacilli with Caution Dr.T.V.Rao MD 38
  39. 39. EXAMINATION PROCEDUREThe mercury vapor lamp : It takes about 10min. to reach full intensityExamination: Using the low powerobjective (100-150x) first examine a knownpos. slide to ensure that the microscope iscorrectly set upAppearance: Bacilli appear as slenderbright yellow fluorescent rods, standing outclearly against a dark backgroundPositive: Minimum of 4 AFB in the entiresmear.Negative: Less than 4 bacilli ( No.of bacillito be recorded) Dr.T.V.Rao MD 39
  40. 40. Grading is Essential to Determine PrognosisMorphological Confirmation: With ahigh power objective (400-600x) - Tobe done with all doubtful smears aswell as scanty positives.Examination: At least three horizontalsweeps on the entire smear.Gradation: Grade positive smears intothree degrees of positivity using thehigh power objective Dr.T.V.Rao MD 40
  41. 41. QUANTIFICATION OF FLUOROCHROME SMEAR RESULTS FM Magnification ZN REPORT 250x 450x 630x 1000x 0 NO.AFB 0 0 0 DIVIDE1-9/100FIELD EXACT NO by 10 by 4 by 210-99/FIELD 1+1-10/FIELD 2+>10/FIELD 3+ Dr.T.V.Rao MD 41
  42. 42. Examining and Reporting Acid-fast Smears Number of AFB Observed Report 200x,250x 400x,450x No AFB seen 0 0 Doubtful: repeat 1-2/30F* 1-2/70F 1+ 1-9/10F 2-18/50F 2+ 1-9/F 4-36/10F 3+ 10-90/F 4-36/F 4+ >90/F >36/F * number of acid-fast bacilli observed per microscopic field Dr.T.V.Rao MD 42
  43. 43. FM Grading No. of bacilli per HPF Grade Less than 6 per field 1+ 6-100 bacilli per field 2+More than 100 per field or large 3+ clumps Dr.T.V.Rao MD 43
  44. 44. Fluorescence Microscopy Advantages in Acid Fast Bacilli Identification More than 50 smears examined/day Continuous availability of power More sensitive. where small No. of bacilli are present Majority of FM +ve samples are also +ve by culture Does not yield more false positive than ZN Doubtful smears to be re examined by ZN Dr.T.V.Rao MD 44
  45. 45. How to get better Photomicrograph for DocumentationThe quality of a photomicrograph, either digital orrecorded on film, is dependent upon the quality of themicroscopy. Film is a stern judge of how good themicroscopy has been prior to capturing the image. It isessential that the microscope be configured using Köhlerillumination, and that the field and condenserdiaphragms are adjusted correctly and the condenserheight is optimized. When properly adjusted, themicroscope will yield images that have even illuminationover the entire field of view and display the bestcompromise of contrast and resolution. Dr.T.V.Rao MD 45
  46. 46. QBC Malaria TestUses the Principles of fluorescenceThe QBC MalariaTest is afluorescencemicroscopy-basedmalaria diagnostictest that speeds andsimplifies malariadetection Dr.T.V.Rao MD 46
  47. 47. Quantitative Buffy Coat (QBC) Test The method is centrifugation and thereby concentration ofthe red blood cells in a predictable area of the QBC tube,making detection easy and fast. Red cells containingPlasmodia are less dense than normal ones and concentratejust below the leukocytes, at the top of the erythrocytecolumn. The float forces all the surrounding red cells intothe 40 micron space between its outside circumference andthe inside of the tube. Since the parasites contain DNAwhich takes up the acridine orange stain, they appear asbright specks of light among the non-fluorescing red cells.Virtually all of the parasites found in the 60 microliter ofblood can be visualized by rotating the tube under themicroscope. A negative test can be reported within oneminute and positive result within minutes. Dr.T.V.Rao MD 47
  48. 48. EXAMINATION OF SPECIMENS for Fungal DiseasesMICROSCOPIC EXAMINATION0.1% Calcofluor White (W/V) Solution(a) Use commercially available solution cellufluor 17352(Polysciences, Washington, PA), fluorescent brightener28.F6259 (Sigma, St. Louis, MO), 1 gm(b) distilled water, 100 ml(c) Gently heat if precipitate develops. Filter if precipitatepersists. Store at 25 C in the dark.(3) Commercially prepared kits are Dr.T.V.Rao MD 48
  49. 49. Need Specific ProtocolsMicroscope filter system: Anepifluorescent microscopeequipped with a mercuryvapor lamp and either anultraviolet (UV) or blue-violet (BV) excitation filtersto achieve radiation on theslide below 412 nm shouldbe used since the maximumabsorbance of CFW is 347nm. Dr.T.V.Rao MD 49
  50. 50. Fluorescent stains for demonstrating Cryptosporidium spp.oocysts include Auramine- rhodamine, Auramine CarbolFuschin and Acridine orange . Confirmatory staining ofsuspected oocyst by another method may be required Casemore 1984 Dr.T.V.Rao MD 50
  51. 51. Fluorescent stains Cryptosporidium spp. Fluorescent stains for demonstrating Cryptosporidium spp. oocysts include Auramine- rhodamine, Auramine Carbol Fuschin and Acridine orange . Confirmatory staining of suspected oocyst by another method may be required Casemore 1984 Dr.T.V.Rao MD 51
  52. 52. Immunofluorescent antibody (IFA) procedure employingcryptosporidium- specific polyclonal or monoclonal antibodies has been developed•Polyclonal AB, raised against 18 and 20 kDa C. parvumcoproantigen, were used to react with C. parvumsporozoites in an immunofluorescence assay.•Monoclonal antibody reagents offer increasedsensitivity and an excellent alternative to conventionalstaining methods.•These reagents are helpful when screening largenumbers of patients or those with minimal symptoms.•Elimination of the problems of false-positive and false-negative results with routine staining methods. Dr.T.V.Rao MD 52
  53. 53. Giardia and CryptosporidiumThe sensitivity andspecificity of theMerifluor DFA test,have been reported tobe 96 to 100% and99.8 to 100%,respectively, for bothGiardia andCryptosporidium Dr.T.V.Rao MD 53
  54. 54. Enumerable Possibilities with Florescent MethodsSeveral Immuno Diagnostic Methods are available Using the FluorescentTechniques, which needs specific testingmaterial and to follow specific protocols. The Technologists should be familiar with literature available with the Kits Dr.T.V.Rao MD 54
  55. 55. Created by Dr.T.V.Rao MD for ‘ e ‘learning resources for Microbiologists in the Developing world Email doctortvrao@gmail.com Dr.T.V.Rao MD 55

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