Laser scanning confocal microscopy

8.7 Laser scanning confocal microscopy

8.7.1 Principles of laser scanning confocal microscopy

In this technique a focused laser beam is scanned across a specimen and light emitted from the specimen is collected point by point through a pinhole in front of a photomultiplier. The pinhole ensures that as the laser scans, information is only collected from one particular focal plane, i.e. it removes the out-of-focus information observed in conventional microscopy. An ‘optical slice’ through a specimen is produced, resolving detail within that particular slice and eliminating focus glare. Multiple wavelengths can be collected simultaneously, using filtering systems to direct light to separate photomultipliers. Although in flow cytometry a single 488-nm argon ion laser is used for most purposes, in confocal microscopy it is more common to have two or more lasers, most frequently a ‘blue’ and a ‘green’ laser (see Fig. 8.11). There is a difference in the dyes used. For flow cytometry, dyes are based on the 488-nm excitation laser used. But since the cell spends less than a millisecond in the laser beam, the dyes do not need to be as resistant to quenching as those used in confocal microscopy. Consequently, although fluorescein is suitable for both applications, because phycoerythrin quenches rapidly, other orange-red dyes such as Texas Red and rhodamine are more commonly used in confocal microscopy, i.e. the traditional dyes of the microscopist. Direct labelling of primary antibodies is a less sensitive process than flow cytometry, i.e. a system in which a single measurement is obtained for emission from the entire cell.

For most purposes, more sensitive indirect staining techniques are used. However, use of two monoclonal antibodies simultaneously for direct labelling creates a dilemma, since the indirect anti-mouse Ig labels could react with either of the primary antibodies. Some of the ways around this problem include the use of: • one directly conjugated antibody for the highest-expressing of the two antigens being

detected, combined with one indirect assay. The indirect technique is used first to detect one primary monoclonal antibody and then the directly conjugated primary antibody is used last;

a normal-mouse-serum ‘blocking’ step is used between the two;

8.7LASER SCANNING CONFOCAL MICROSCOPY

Confocal principle

Laser beam (488 nm)

Dichroic mirror

Detector PMT

Emission filter

Pinhole

Scanning mirrors

Sample characteristic of dichroic mirror

Wavelength (nm)

Fig. 8.11 Principles of confocal microscopy.

• different monoclonal immunoglobulin classes or isotypes for the primary antibodies, and then isotype-specific second antibodies, e.g. a combination of IgG and IgM, or an IgG 1 and IgG 2a ; • two different species of primary antibody, e.g. one mouse monoclonal and one rabbit poly- clonal, and the appropriate species-specific antibody. Typically, staining is performed on either cryostat tissue sections or fixed cells on conven-

tional slides. Tissues and cells may be fixed with solvents, such as: (i) cold acetone (generally good for preservation of antigenic sites); (ii) methanol; (iii) conventional agents of insolubilization such as paraformaldehyde; or (iv) cross-linking agents such as glutaraldehyde (poor for antigen preservation). Techniques are available that allow the recovery of antigens inactivated by paraffin embedding. Antibodies have been developed that will react with tissue that has been treated in this way. Antiquenching agents are normally used, to reduce quenching under the laser light, e.g. Vectashield (Vector Labs). A typical double-staining assay is illustrated below. This is used on tis- sue sections for detecting two molecules, using one primary monoclonal antibody and one pri- mary rabbit (polyclonal) antibody. Incubation with free biotin and avidin is performed to neutralize natural avidin-binding activity in some tissues, but is not essential for all purposes, even when using avidin–biotin staining assays. Incubation volumes depend on the size of the area containing the tissue, but 30 µl is frequently enough.

C H A P T E R 8: Lymphocyte structure

8.7.2 Example protocol using laser scanning confocal microscopy as detection

MATERIALS Cells to be investigated Microscope slides and coverslips Acetone Phosphate-buffered saline (PBS) 0.5% w/v bovine serum albumin (BSA) in PBS Avidin D Biotin Primary monoclonal antibody Biotinylated anti-mouse antibody (secondary antibody) Steptavidin–fluorescein isothiocyanate (FITC) Primary rabbit antibody Anti-rabbit antibody tetramethyl rhodamine isothiocyanate (TRITC) Varnish Antiquenching agent

METHOD

1 Air-dry sections for 20 min in a fume hood, then fix with cold acetone for 10 min.

2 Perform serial washes with:

1 × PBS for 1 min;

3 × 0.5% BSA in PBS for 5 min.

3 Add one drop of avidin D ( Vectashield) to the section and incubate for 15 min.

4 Wash in 3 × 0.5% BSA in PBS for 5 min.

5 Add 1 drop of biotin to the section and incubate for 15 min.

6 Wash in 3 × 0.5% BSA/PBS for 5 min.

7 Incubate with 30 µl monoclonal antibody.

8 Wash in 0.5% BSA in PBS for 5 min.

9 Incubate with biotinylated anti-mouse antibody for 30 min.

10 Wash in 0.5% BSA in PBS for 5 min.

11 Incubate with streptavidin–FITC for 30 min.

12 Wash in 0.5% BSA in PBS for 5 min.

13 Incubate with rabbit primary antibody for 30 min.

14 Wash in 0.5% BSA in PBS for 5 min.

15 Incubate with pig anti-rabbit TRITC for 30 min.

16 Wash in 2 × 0.5% BSA/PBS three times for 5 min.

17 Rinse in PBS (this may improve life of antiquencher).

18 Mount in antiquenching agent, and varnish.

It is recommended that the labelling protocol used in laser confocal microscopy is optimized for the individual experiments and cells being investigated.

8.7LASER SCANNING CONFOCAL MICROSCOPY