Transcript Study of Tissue Morphology – Confocal Microscopy

Immunofluorescence
and
Confocal Microscopy
Dr. KW Chan
Part I: Immunofluorescence
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
Principle of Fluorescence
Microcopy
Principle of Fluorescence
Microcopy
Exciter filter
Principle of Fluorescence
Microcopy
Exciter filter
Barrier filter
Fluorescence microscope
 Light source is UV mercury vapor lamp
 UV light is filtered to select excitation light
to pass through
 Excitation light is reflected by a dichroic
mirror to strike on the specimen
 Emission light passes through the dichroic
mirror
Fluorescence microscope
 Barrier filter blocks the excitation light
amid the light path to visualization
 Fluorescent labels are visulized against a
dark background
Auramine for Mycobacterium
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
Epi-fluorescence microscope
Transmitted light flurorescence
microscope
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.
Exciter filter
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
Green fluorescence of FITC
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
Direct Immunofluorescence
 The aim is to identify the presence and
location of an antigen by the use of a
fluorescent labelled specific antibody
One step
Direct Immunofluorescence
Two step
Direct Immunofluorescence
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
Application in renal diseases
IgG
 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
 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
Direct Fluorescent Antibody Test for the
Presence of Immunoglobulin Deposits in Skin
IgG
 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
 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
Double labelling
Lymphoid tissue:
the two Ig light chains
are separately labelled.
Indirect Immunofluorescence
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.
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.
Diagnosis of Bacterial Diseases
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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)
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)
Diagnosis of Protozoal Diseases
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Babesia species (indirect)
Ehrlichia species (indirect)
Toxoplasma gondii (indirect)
Trypanosoma cruzi (indirect)
Cryptosporidia/Giardia (direct)
Encephalitozoon cuniculi (indirect)
Neosporum caninum (direct, indirect)
Some examples
Indirect Immunofluorescence
Indirect Fluorescent Antibody Test for
Antibodies to Toxoplasma gondii
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.
Indirect Fluorescent Antibody Test for
Antibodies to Toxoplasma gondii
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
Indirect Fluorescent Antibody Test
for Antinuclear Antibodies
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.
Indirect Fluorescent Antibody Test
for Antinuclear Antibodies
Advantage over Immunoperoxidase
 Technically easier (fewer steps)
 More sensitive results
Drawbacks
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Microscope is more costly
Frozen sections preferred
Preparations need refrigeration
Preparations cannot be kept for too long
Quick fading of fluorescence under
illumination (bleaching effect)
Part II: Confocal microscopy
Learning Objectives
 To understand the working principles of
confocal scanning microscope
 To know the current use of confocal
scanning microscopy in medical science
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
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
The confocal
concept
Modes of scanning
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Mechanical scanning stage
Beam scanning (by means of mirror)
Combined stage and beam scanning
Slit may be used instead of a pinhole
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Shortens time for scanning an area
Direct vision of real color image made possible
At the expense of a lower resolution
Use of confocal microscope
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Performs optical sectioning of thick samples
Three dimensional image reconstruction
Detects very weak fluorescent signals
Selective photobleaching
Cell ablation
Image modalities
 Autofluorescence
 Single, double or treble fluorescent
labeling:
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immunofluorescence, in-situ hybridization
 Image formed by reflectance intensified
with metallic coating e.g. AgNOR,
immunogold labeling
Application in biomedical science
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Growth of small organisms, cells, embryos
Movement of intracellular structures
Change in membrane permeability
3 dimensional reconstruction
Image analysis
BIO-RAD MRC-1000
 beam scanning LSM
 Zeiss "Axioskop" upright microscope for ordinary
bright field transmitted light and epi-fluorescence
 argon/krypton laser, 15 mW, wavelengths at 488
nm (e.g. FITC), 568 and 647 nm
 reflectance mode
 fluorescence mode:
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simultaneous recording of 2 channels, ie. For double
labels
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 scan
 maximum image size 1024 x 1024 pixels
 scanning speed at maximum of 16 frames/s
at 768 x 30 pixels, and 1 frame/s at 512
lines
BIO-RAD MRC-1000
 beam park function for photobleaching,
ablation
 computer programs for morphometric
analysis, 3-D visualization
IgG
IgG
Confocal microscope
gives a clearer image and
cleaner background over
conventional fluorescent
microscope
Confocal
Microphotography
for weak signals
is much easier
with a confocal
microscope
Microphotography
for weak signals
is much easier
withup
a confocal
picking
weak
microscope
Excellent for
signals
Immunofluorescence
and
Confocal Microscopy
Dr. KW Chan
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
Learning Objectives
 To understand the working principles of
confocal scanning microscope
 To know the current use of confocal
scanning microscopy in medical science