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Dr.T.V.Rao MD

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Sir George G. Stokes

The phenomenon offluorescence was knownby the middle of the

nineteenth century.British scientist SirGeorge G. Stokes firstmade the observation thatthe mineral fluorsparexhibits fluorescence

when illuminated withultraviolet light, and hecoined the word"fluorescence"

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MD 2

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Discovery of Fluorescence

Microbiology The fluorescence microscope

was devised in the early partof the twentieth century by

August Khler, CarlReichert, and HeinrichLehmann, among others.However, the potential ofthis instrument was notrealized for several decades,and fluorescence

microscopy is now animportant (and perhapsindispensable) tool incellular biology.

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Differences between Conventional

and Fluorescent Microscope

The Conventionalmicroscope usesvisible light (400-700nanometers) toilluminate and

produce amagnified image ofa sample.

A fluorescence microscope,uses a much higherintensity light sourcewhich excites afluorescent species in asample of interest. Thisfluorescent species inturn emits a lower

energy light of a longerwavelength thatproduces the magnifiedimage instead of theoriginal light source.

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What is Fluorescence?

Fluorescence is lightproduced by a

substance when it isstimulated by anotherlight. Fluorescence iscalled "cold light"because it does not

come from a hot sourcelike an incandescentlight bulb.

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.Rao MD 5

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Fluorescence microscopy is a unique way of using a

microscope to discover facts about specimens that often

are not shown by standard bright field microscopy. Inbright field microscopy, specimens are illuminated fromoutside, below or above, and dark objects are seen againsta light background. In fluorescence microscopy,specimens are self-illuminated by internal light, so bright

objects are seen in vivid color against a dark background.Bright objects against dark backgrounds are more easilyseen. This characteristic of fluorescence microscopymakes it very sensitive and specific.

What is FluorescenceMicroscopy?

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Most cellular components are colorless and cannot

be clearly distinguished under a microscope. Thebasic premise of fluorescence microscopy is to stain

the components with dyes. Fluorescent dyes, alsoknown as fluorophores of fluorochromes, aremolecules that absorb excitation light at a givenwavelength (generally UV), and after a short delayemit light at a longer wavelength. The delay between

absorption and emission is negligible, generally onthe order of nanoseconds. The emission light canthen be filtered from the excitation light to reveal thelocation of the fluorophores.

Principle of Fluorescent

Microscopy

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Principle of Fluorescent

Microscopy Fluorescence microscopy

uses a much higher intensitylight to illuminate the

sample. This light excitesfluorescence species in thesample, which then emitlight of a longer wavelength.The image produced isbased on the second lightsource or the emissionwavelength of thefluorescent species -- ratherthan from the lightoriginally used to

illuminate, and excite, thesam le.

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Works on Faster

Transmission of Light Fluorescence, describes

light emission thatcontinues only duringthe absorption of theexcitation light. The timeinterval between absorptionof excitation light and

emission of re-radiated lightin fluorescence is ofextraordinarily shortduration, usually less than amillionth of a second.

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Works on Principles of Light

Pathways Specifically, a dichroic

mirror is used toseparate the excitation

and emission lightpaths. Within theobjective, theexcitation/emissionshare the same optics.

In a fluorescencemicroscope, thedichroic mirrorseparates the lightpaths.

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Fluorescence microscopy is the most popular method for

studying the dynamic behavior exhibited in live cellimaging. This stems from its ability to isolate individual

proteins with a high 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 givefluorescence microscopy a clear advantage over otheroptical imaging techniques, for both in vitro and in vivoimaging.

Advantages of Fluorescent

Microscopy

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Fluorescence Microscope

Fluorescencemicroscopy by

epi-illuminationis the mostcommonly usedmethod today

because it is simpleto do, needsrelatively simpleequipment, and isefficient.

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Epifluorescence Microscopy

Epifluorescence microscopy is amethod of fluorescencemicroscopy that is widely usedin life sciences The excitatory

light is passed from above (or,for inverted microscopes, frombelow), through the objectivelens and then onto thespecimen instead of passing itfirst through the specimen. The

fluorescence in the specimengives rise to emitted light whichis focused to the detector by thesame objective that is used forthe excitation

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The Specimens to be

StainedMost specimens for

fluorescence

microscopy must bestained. Fluorescentstains are called"fluorochromes."

Acridine orange,auramine O, andfluorescent antibody(FA) are thefluorochromes usedmost.

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Fluorescence Microscopy applied inMany Braches of Science and Medicine

uses of fluorescencemicroscopy are many

and varied. They are inmedicine, public health,biological research, andenvironment

monitoring.The most common

application is medicallaboratory

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How to Use a

FluorescenceMicroscope

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How to Use a FluorescenceMicroscope

The object to be studiedis marked with amolecule called a

fluorophore (a dye).When the florescentlight is activated, thelight used forillumination isseparated from theflorescent molecule (thefluorophore), which ismuch weaker. This is done

through an emission filter.Dr.T.V.Rao MD 17

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Step 1 Locate the light switch

on the side of themicroscope that turns

on the light. Turn themicroscope on.

Write down the exacttime you turn on the

light. The florescentlight is mercury-based,and a time log must bekept for exposure anduse of the light.

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Step 2

Locate the toggleswitch on the right

side of themicroscope betweenthe oculars andobjectives. Thisswitch controls the

shutter for themercury light to theobjective lens.

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Step 3

Select the appropriatedye for your object (thiswill depend entirely onwhat you are going tobe studying). The mostcommon dyes includeI3 (for use with CTC,

DTAF and fluorescein),A (for use with DAPIand f420), N21 (for usewith Rhoda mine) and

L3 (for use withfluorescein).Dr.T.V.Rao MD 20

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Step 4

Put the filter

(dye) into the trayoperated by thesilver slidingknob. To remove

the tray, simplypull the silverknob out.

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Step 5

Select the lens youwould like to use. The

63x objective lens willhave the highestnumerical aperture.The 100x objective lenswill have the highest

magnitude that can beused with the mercury-based florescent lightsource.

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Step 6

Turn the light off whenfinished, and mark thetime. Wait 30 minutesbefore turning the lightback on, or the lampcould explode. It is agood idea to keep track ofhow many hours the

lamp is in use and replaceit according to themanufacture's guidelines.

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Step 7

Clean off the

microscope lenswith lenspaper, or if

really dirty, usea cotton swapand glass

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A specific antibody is labeled by chemically attaching a

fluorophore to form what is known as a conjugate, whichis then spread on a microscope slide containing the

suspected presence of a particular antigen known tostimulate production of the antibody. If the antigen ispresent, the labeled antibody conjugate binds to theantigen and remains bound to the specimen after it iswashed. The presence of the chemically attachedfluorescent conjugate and antigen is demonstrated when

the fluorophore is excited at its excitation peak, and thesubsequent emission intensities at various wavelengthscan then be observed visually or captured by a detectorsystem (digital or traditional camera).

Direct Immunofluorescence

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Indirect immunofluorescence here serum possibly containing

unlabeled antibody and its related, but known, antigen areincubated together. A fluorochrome conjugated to an anti-

human antibody (if the specimen being tested is human) is thenplaced on the slide containing the unlabeled antibody-antigen.If indeed, there has been an antigen-antibody reaction, thefluorochrome-labeled anti-human antibody attaches itself to thecomplex formed by the antigen and antibody. Subsequently,the labeled grouping of antigen, antibody, and fluorochromelabeled anti-human antibody is excited at the peak wavelength

intensity for that fluorochrome and any resulting emission isobserved. The indirect immunofluorescence technique reducesthe necessity of keeping in stock large numbers of labeledantibodies, and also usually results in greater fluorescenceintensity. fluorescence,

Indirect Immunofluorescence

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Advantages of fluorescence microscopy are

due to its sensitivity, specificity, rapid

testing, and easy use. It is easy to set up anddo, provides rapid diagnostic tests, and canbe very specific. Modern technology allowsconversion of most compound microscopeseasily and economically into effectivefluorescence microscopes.

Fluorescence Microbiology Modernises theDiagnostic Laboratories

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Fluorescent Staining in

Tuberculosis The Auramine-rhodamine

process uses a yellowfluorescent dye to visualizeMycobacterium tuberculosisunder a fluorescencemicroscope. Potassiumpermanganate or acridineorange can be used as acounterstain. Under the lens,the bacterial cells will appeargreen.

The Auramine-rhodaminestain is more sensitivethan the Zhiel-Neelsonand more cost effective.

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Auramine-phenol solution

Auramine O 25 gEthanol 3000 mlPhenol 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 amagnetic stirrer until solution is complete.

Dissolve 250 g phenol in 5300 ml of distilled water. Mix the phenol solution with alcoholic auraminesolution.

Store in an amber colored bottle at room temp. for upto 3 mths.

Filter before use.

Reagents for staining

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Acid-alcohol for decolorization

Sodium chloride 20 gHydrochloric acid, A.R. 20 mlDistilled water 500 mlEthanol 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.

Reagents for staining

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Counter stain

Potassium permanganate 1 gmDistilled water 1000 ml

Dissolve and store in an amber colored bottle

Stays at room temperature for up to 3 months.

Reagents for staining

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Use of 1000x magnification

Use of oil immersion

Examination of 300 microscopicfields

About 15 minutes to examine onenegative smear

Examination by an experienced

microscopist

Fuchsin-stained smears require

-

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Place the slides on a staining rack, with thesmeared side facing up, the slides not touchingeach other

Flood the slides with freshly filtered auramine-phenol. Let stand for 7-10 min.

Wash well with running water

Decolorize with acid-alcohol for 1-2 min.Wash as before with water and slope the slides

to air dry

Counter stain with 0.1% KMNO4 for 30 seconds

Staining procedure

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Staining procedure

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Avoid under-decolorization. AFBs are difficult to over-

decolorize since the decolorization procedure with acid-alcohol is relatively milder than the 25% H2SO4 used in Z-N.

Thick smears: Interfere with decolorization, and counterstain. Mask the presence of AFB and tendency to flake,resulting in loss of smear material and possible transfer ofmaterial to other slides

Strong counter stain: May mask the presence of AFB

Re Staining: Smears examined by FM may be restained by Z-N to confirm findings. However, Z-N stained smears cannot

be used for FM

Fading: Stained smears may fade on exposure to light. To bestored wrapped in brown or black paper and kept away fromlight

Staining procedure -Precautions

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EXAMINATION PROCEDURE

The mercury vapor lamp : It takes about 10

min. to reach full intensity

Examination: Using the low power objective

(100-150x) first examine a known pos. slide toensure that the microscope is correctly set up

Appearance: Bacilli appear as slender bright

yellow fluorescent rods, standing out clearly

against a dark background

Positive: Minimum of 4 AFB in the entiresmear.

Negative: Less than 4 bacilli ( No.of bacilli to

be recorded)

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Screen the Smear in DefinedPattern

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Identify the Acid Fast Bacilli

with Caution

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EXAMINATION

PROCEDURE The mercury vapor lamp : It takes about

10 min. to reach full intensity

Examination: Using the low power

objective (100-150x) first examine aknown pos. slide to ensure that the

microscope is correctly set up

Appearance: Bacilli appear as slender

bright yellow fluorescent rods, standing

out clearly against a dark background

Positive: Minimum of 4 AFB in theentire smear.

Negative: Less than 4 bacilli ( No.of

bacilli to be recorded)

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Grading is Essential to

Determine Prognosis Morphological Confirmation:

With a high power objective (400-

600x) - To be done with all

doubtful smears as well as scantypositives.

Examination: At least three

horizontal sweeps on the entire

smear.

Gradation: Grade positive smearsinto three degrees of positivity

using the high power objective

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QUANTIFICATION OF FLUOROCHROMESMEAR RESULTS

ZN

1000x

REPORT

FM Magnification

250x 450x 630x

0NO.AFB 0 0 0

1-9/100FIELD EXACT NO

DIVIDE

by 10 by 4 by 2

10-99/FIELD 1+

1-10/FIELD 2+

>10/FIELD 3+

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Examining and Reporting Acid-fast

SmearsNumber 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

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FM Grading

No. of bacilli per HPF GradeLess than 6 per field

6-100 bacilli per field

More than 100 per field or

large clumps

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More than 50 smears examined/day

Continuous availability of power

More sensitive. where small No. of bacilli arepresent

Majority of FM +ve samples are also +ve byculture

Does not yield more false positive than ZN

Doubtful smears to be re examined by ZN

Fluorescence MicroscopyAdvantages in Acid Fast Bacilli

Identification

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The quality of a photomicrograph, either digital or

recorded on film, is dependent upon the quality ofthe microscopy. Film is a stern judge of how good

the microscopy has been prior to capturing theimage. It is essential that the microscope beconfigured using Khler illumination, and that thefield and condenser diaphragms are adjustedcorrectly and the condenser height is optimized.

When properly adjusted, the microscope will yieldimages that have even illumination over the entirefield of view and display the best compromise ofcontrast and resolution.

How to get better Photomicrograph forDocumentation

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QBC Malaria TestUses the Principles of fluorescence

The QBC MalariaTest is afluorescencemicroscopy-based malaria

diagnostic testthat speeds andsimplifies malaria

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The method is centrifugation and therebyconcentration of the red blood cells in a predictablearea of the QBC tube, making detection easy and fast.Red cells containing Plasmodia are less dense than

normal ones and concentrate just below theleukocytes, at the top of the erythrocyte column. Thefloat forces all the surrounding red cells into the 40micron space between its outside circumference andthe inside of the tube. Since the parasites contain DNAwhich takes up the acridine orange stain, they appear

as bright specks of light among the non-fluorescingred cells. Virtually all of the parasites found in the 60microliter of blood can be visualized by rotating thetube under the microscope. A negative test can bereported within one minute and positive result withinminutes.

Quantitative Buffy Coat

(QBC) Test

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MICROSCOPIC EXAMINATION

0.1% Calcofluor White (W/V) Solution

(a) Use commercially available solution cellufluor17352 (Polysciences, Washington, PA), fluorescentbrightener 28.F6259 (Sigma, St. Louis, MO), 1 gm

(b) distilled water, 100 ml

(c) Gently heat if precipitate develops. Filter ifprecipitate persists. Store at 25 C in the dark.

(3) Commercially prepared kits are

EXAMINATION OF SPECIMENS forFungal Diseases

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Need Specific Protocols

Microscope filter system:An epifluorescentmicroscope equipped

with a mercury vaporlamp and either anultraviolet (UV) or blue-violet (BV) excitationfilters to achieve radiation

on the slide below 412 nmshould be used since themaximum absorbance ofCFW is 347 nm.

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Fluorescent stains for demonstratingCryptosporidium spp.

oocysts include Auramine- rhodamine, Auramine CarbolFuschin and Acridine orange . Confirmatory staining ofsuspected oocyst by another method may be required

Casemore 1984

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Fluorescent stains

Cryptosporidium spp. Fluorescent stains for

demonstratingCryptosporidium spp.

oocysts includeAuramine- rhodamine,Auramine CarbolFuschin and Acridineorange . Confirmatorystaining of suspected

oocyst by anothermethod may be required

Casemore 1984

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Immunofluorescent antibody (IFA) procedureemploying cryptosporidium- specific polyclonal or monoclonal antibodies has

been developed

Polyclonal AB, raised against 18 and 20 kDa C.parvum coproantigen, were used to react with C.parvum sporozoites in an immunofluorescence

assay.Monoclonal antibody reagents offer increasedsensitivity and an excellent alternative toconventional staining methods.

These reagents are helpful when screening largenumbers of patients or those with minimalsymptoms.Elimination of the problems of false-positive and

false-negative results with routine staining methods.Dr.T.V.Rao MD 52

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Giardia and Cryptosporidium

The sensitivity andspecificity of theMerifluor DFA test,have beenreportedto be 96 to 100%and 99.8 to 100%,

respectively, for

both Giardia andCryptosporidium

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Several Immuno Diagnostic Methods

are available Using the FluorescentTechniques, which needs specific

testing material and to follow specificprotocols. The Technologists should

be familiar with literature availablewith the Kits

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Enumerable Possibilities withFlorescent Methods

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Created by Dr.T.V.Rao MD for e

learning resources forMicrobiologists in the Developingworld

Email doctortvrao@gmail.com

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