immunofluorescence and confocal microscopy dr. kw chan
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Immunofluorescenceand
Confocal Microscopy
Dr. KW Chan
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Part I: Immunofluorescence
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Learning Objectives
To understand the working principles of immunofluorescence microscope
To understand the difference between direct and indirect immunofluorescence
To know the current use of immunofluorescence studies in medicine
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Principle of Fluorescence Microcopy
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Principle of Fluorescence Microcopy
Exciter filter
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Principle of Fluorescence Microcopy
Exciter filter Barrier filter
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Fluorescence microscope
Light source is UV mercury vapor lampUV light is filtered to select excitation light
to pass throughExcitation light is reflected by a dichroic
mirror to strike on the specimen Emission light passes through the dichroic
mirror
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Fluorescence microscope
Barrier filter blocks the excitation light amid the light path to visualization
Fluorescent labels are visulized against a dark background
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Auramine for Mycobacterium
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Fluorescence microscope
The combination of exciter filter, dichroic mirror and barrier filter should be selected according to the fluorochrome label
The 3 components are usually built into a single module called the filter block
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Epi-fluorescence microscope
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Transmitted light flurorescence microscope
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The basic principle of immunofluorescence
To use a fluorescent compound (usually fluorescein) to detect the binding of antigen and antibody.
The Ab is labeled with the fluorescent compound and its presence is detected using a fluorescence microscope.
Under a fluorescence microscope, fluorescein appears bright green wherever the binding occurred.
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Exciter filter
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The basic principle of immunofluorescence
To use a fluorescent compound (usually fluorescein) to detect the binding of antigen and antibody
The Ab is labelled with the fluorescent compound
Under a fluorescence microscope, fluorescein appears bright green wherever the binding occurs
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Green fluorescence of FITC
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Using the fluorescence microscope
Select the correct filter block for the fluorescent compound
Fluorescence fades quickly under UV light; try to limit the time of exposure to UV as much as possible
Use high speed films for photography
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Direct Immunofluorescence
The aim is to identify the presence and location of an antigen by the use of a fluorescent labelled specific antibody
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One step Direct Immunofluorescence
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Two step Direct Immunofluorescence
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Medical applications of direct IF
Renal diseases for evidence of immune deposition
Skin diseases for evidence of immune deposition
Detection of specific antigens, especially those of infective organisms
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Application in renal diseases
IgG
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A section of kidney is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away
The presence of fluorescence in the glomeruli indicates that IgG was deposited prior to the biopsy
IgG is deposited in granular clumps along the capillary walls, enabling a diagnosis of membranous glomerulonephritis in this case
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A section of kidney is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away
The presence of fluorescence in the glomeruli indicates that IgG was deposited prior to the biopsy
IgG is deposited in granular clumps along the capillary walls, enabling a diagnosis of membranous glomerulonephritis in this case
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Direct Fluorescent Antibody Test for the Presence of Immunoglobulin Deposits in Skin
IgG
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A section of skin is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away
The presence of fluorescence in the upper layers of the epithelium indicates that IgG was deposited in this skin (prior to the biopsy)
The presence of immunoglobulins deposited around keratinocytes is consistent with a diagnosis of pemphigus
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A section of skin is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away
The presence of fluorescence in the upper layers of the epithelium indicates that IgG was deposited in this skin (prior to the biopsy)
The presence of immunoglobulins deposited around keratinocytes is consistent with a diagnosis of pemphigus
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Double labelling
Lymphoid tissue:the two Ig light chains are separately labelled.
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Indirect Immunofluorescence
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Indirect Immunofluorescence
The aim is to identify the presence of antigen specific antibodies in serum. The method is also be used to compare concentration of the antibodies in sera.
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Indirect Immunofluorescence
A known antigen is placed on a slide; the patient's serum is added, then rinsed away.
A fluorescein-labeled antiglobulin is added, then rinsed away.
The presence of fluorescence over the antigen indicates the presence of antibodies to this antigen in the patient.
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Diagnosis of Bacterial Diseases
Clostridial diseases (direct) Brucella canis (indirect) Afipia catei, cat scratch disease (indirect) Borrelia burgdorferi (indirect) Coxiella burnetii, Q Fever (indirect) Rickettsia rickettsiae, Rocky Mountain
Spotted Fever (indirect)
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Diagnosis of Viral Diseases
rabies virus (direct)bovine immunodeficiency-like virus (indirect)canine coronavirus (indirect)canine distemper (indirect)feline infectious peritonitis (corona-) virus
(direct)porcine respiratory and reproductive syndrome
(indirect)
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Diagnosis of Protozoal Diseases
Babesia species (indirect)Ehrlichia species (indirect)Toxoplasma gondii (indirect)Trypanosoma cruzi (indirect)Cryptosporidia/Giardia (direct)Encephalitozoon cuniculi (indirect)Neosporum caninum (direct, indirect)
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Some examples
Indirect Immunofluorescence
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Indirect Fluorescent Antibody Test for Antibodies to Toxoplasma gondii
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Indirect Fluorescent Antibody Test for Antibodies to Toxoplasma gondii
Toxoplasma organisms are killed and placed on the slide; the patient’s serum is added, then washed away.
A fluorescein-labeled antiglobulin is added, then washed away.
The presence of the green fluorescence outlining the T. gondii organisms indicates the presence of antibodies in the patient's serum.
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Indirect Fluorescent Antibody Test for Antibodies to Toxoplasma gondii
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Immune-Mediated Disorders
antinuclear antibody (ANA) test (for diagnosis of systemic lupus erythematosus)
Direct fluorescent antibody test for deposition of Abs in tissues, e.g. kidney, skin
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Indirect Fluorescent Antibody Test for Antinuclear Antibodies
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Indirect Fluorescent Antibody Test for Antinuclear Antibodies
Cells from a cultured cell line are placed on a slide; the patient's serum is added, then rinsed away.
A fluorescein-labeled antiglobulin is added, then rinsed away.
The presence of fluorescence in the nucleus of these cells indicates the presence of antibodies to nuclear antigens in the patient.
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Indirect Fluorescent Antibody Test for Antinuclear Antibodies
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Advantage over Immunoperoxidase
Technically easier (fewer steps)More sensitive results
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Drawbacks
Microscope is more costlyFrozen sections preferredPreparations need refrigerationPreparations cannot be kept for too longQuick fading of fluorescence under
illumination (bleaching effect)
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Part II: Confocal microscopy
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Learning Objectives
To understand the working principles of confocal scanning microscope
To know the current use of confocal scanning microscopy in medical science
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Principles of confocal microscopy
a focused laser beam serves as a high intensity point source
light reflected or fluorescence emitted by the specimen is allowed to pass through a pinhole that filters light coming from outside (above and below) of the focal plane
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Principles of confocal microscopy
a sensitive detector (photomultipler) behind a pinhole to measure the intensity of light
the laser beam, the pinhole and detector scan through the specimen to build up an image on a monitor
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The confocalconcept
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Modes of scanning
Mechanical scanning stageBeam scanning (by means of mirror)Combined stage and beam scanningSlit may be used instead of a pinhole
Shortens time for scanning an area Direct vision of real color image made possible At the expense of a lower resolution
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Use of confocal microscope
Performs optical sectioning of thick samples
Three dimensional image reconstructionDetects very weak fluorescent signalsSelective photobleachingCell ablation
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Image modalities
AutofluorescenceSingle, double or treble fluorescent
labeling: immunofluorescence, in-situ hybridization
Image formed by reflectance intensified with metallic coating e.g. AgNOR, immunogold labeling
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Application in biomedical science
Growth of small organisms, cells, embryosMovement of intracellular structuresChange in membrane permeability3 dimensional reconstructionImage analysis
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BIO-RAD MRC-1000
beam scanning LSMZeiss "Axioskop" upright microscope for ordinary
bright field transmitted light and epi-fluorescenceargon/krypton laser, 15 mW, wavelengths at 488
nm (e.g. FITC), 568 and 647 nm reflectance mode fluorescence mode:
simultaneous recording of 2 channels, ie. For double labels
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BIO-RAD MRC-1000
upgradable to 3 simultaneous channels in fluorescence mode
computer-controlled stage motor for vertical motion at 0.5µm per step
performs frame scan, line scan, vertical scanmaximum image size 1024 x 1024 pixelsscanning speed at maximum of 16 frames/s
at 768 x 30 pixels, and 1 frame/s at 512 lines
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BIO-RAD MRC-1000
beam park function for photobleaching, ablation
computer programs for morphometric analysis, 3-D visualization
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IgG
IgG
Confocal
Confocal microscope gives a clearer image andcleaner background overconventional fluorescentmicroscope
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Microphotographyfor weak signalsis much easierwith a confocalmicroscope
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Microphotographyfor weak signalsis much easierwith a confocalmicroscope
Excellent for picking up weak signals
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Immunofluorescenceand
Confocal Microscopy
Dr. KW Chan
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Learning Objectives
To understand the working principles of immunofluorescence microscope
To understand the difference between direct and indirect immunofluorescence
To know the current use of immunofluorescence studies in medicine
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Learning Objectives
To understand the working principles of confocal scanning microscope
To know the current use of confocal scanning microscopy in medical science