Immunofluorescenceand

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 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

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

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

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

Microscope is more costlyFrozen sections preferredPreparations need refrigerationPreparations cannot be kept for too longQuick 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 confocalconcept

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

Use of confocal microscope

Performs optical sectioning of thick samples

Three dimensional image reconstructionDetects very weak fluorescent signalsSelective photobleachingCell ablation

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

Application in biomedical science

Growth of small organisms, cells, embryosMovement of intracellular structuresChange in membrane permeability3 dimensional reconstructionImage analysis

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

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

BIO-RAD MRC-1000

beam park function for photobleaching, ablation

computer programs for morphometric analysis, 3-D visualization

IgG

IgG

Confocal

Confocal microscope gives a clearer image andcleaner background overconventional fluorescentmicroscope

Microphotographyfor weak signalsis much easierwith a confocalmicroscope

Microphotographyfor weak signalsis much easierwith a confocalmicroscope

Excellent for picking up weak signals

Immunofluorescenceand

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