Tips on Preparing a Sample for Confocal Microscopy

Introduction

Processing for confocal microscopy is broken down into 4 basic steps where each step may have several substeps and we are presented with a large variety of opportunities to ruin the entire process! In general, these are the steps one must follow:

1) Fixation

2) Permeabilization

3) Staining

4) Mounting

Be sure to note all steps taken, times, concentrations, supplier, lot numbers and any variations from normal procedure.

 

Tissue Fixation

Processing of most samples begins with fixation to preserve morphology. A fixation method must be chosen that balances two characteristics:

• "Good" preservation of cellular 3-D structure
• "Adequate" access to antigenic sites

The goal is to preserve sufficient cellular organization to allow identifying the features of interest, but yet not destroy the antigenicity of the target. Fixation is also frequently combined with permeabilization to allow the staining solutions used in later steps access to the cytoplasm.

Commonly used histological method of fixation and permeabilization often consist of treating the cells or tissues with solvents, such as alcohol or acetone. While these methods are quick acting precipitating fixatives, and also are good permeabilizing agents, they have one significant negative consequence: cellular shrinkage. The degree of shrinkage may be almost insignificant for monolayers of cells, but will distort tissue samples dramatically.  To provide the ability to take full advantage of the three-dimensional reconstruction ability of the confocal microscope, the use of a fixative that does not destroy in-vivo structure and organization must be found.

Glutaraldehyde is a commonly used fixative used for electron microscopy, and does a terrific job of preserving microscopic structure - and ultrastructure. Unfortunately, glutaraldehyde has the unfortunate side effect of cross-linking (and thereby destroying) many antigenic sites. Therefore, while the tissue may have spectacular three-dimensional morphology, the high degree of glutaraldehyde induced cross linking will render the tissue unlabelable. An additional undesirable glutaraldehyde side effect is enhanced autofluorescence from residual unbound free aldehydes.

A good compromise is the use of paraformaldehyde (PF) as a fixative. PF or the commercially available formalin solution (PF plus added methanol) will preserve most structure resolvable by at the confocal microscope level. Also, usually it will not obscure the epitopes of interest. If using paraformaldehyde fixation, permeabilization with Triton X-100 (or other detergent) is required.

It is important to remember that every different specimen may require a different preparation method. Testing and optimizing each protocol for each new sample type will insure that the best balance between preservation and labeling is obtained.

Permeabilization

If a component of the cytoplasm or nucleus is to be effectively labeled, the plasma membrane must be made permeable to staining solutions. There are several ways to do this, and in part they depend on the fixation method chosen. Cells fixed with solvents do not usually need any additional permeabilizing - the solvent has already extracted enough of the membrane. So, solvent fixation is doubly efficient for this reason. However, cells fixed with crosslinking aldehydes do need to have the plasma membrane integrity breached by the use of chemical agents. Commonly used reagents include DMSO and detergents like Triton X-100, saponin or deoxycholate.

Permeabilization is another area, like fixation where fine tuning is necessary. Different detergents solubilize different molecules within the membrane, so it's necessary to know that what you are interested in isn't being washed away in the permeabilization step! Concentration of detergent is another detail that should be adjusted carefully. The idea is to selectively remove plasma membrane constituents to allow access to the cytoplasm without altering either the antigenicity or morphology of the sample.

Protocols for Labeling

Two basic techniques are used: direct labeling and indirect labeling. Both labeling methods are suitable for confocal microscopy.

Direct labeling consists of using a fluorescently labeled primary antibody or chemical ligand to cause the structure of interest to become fluorescent. Advantages of this method include speed and ease of application. A potential disadvantage is lack of sensitivity (low signal intensity).

The indirect method involves binding a primary antibody to the epitope of interest, followed by a fluorescently labeled secondary antibody. The primary advantage of using this technique is the great amplification of signal possible through an antibody cascade. Disadvantages include increased complexity, more time consuming, and often problems with non-specific antibody reactions.

Choosing a Fluorescent Label

Label choice depends on available equipment (laser lines, emission filters,  transmitted light detectors) and the availability of certain fluors conjugated to required antibodies for use in multiple labeling schemes. In general, the laser lines available dictate which fluorophores may be used. Recent advances in biochemistry have created new families of fluorophores with very favorable signal-to-noise and quantum efficiency (QE) properties. In particular, the  Cy dyes and the Alexa dyes are particularly useful. Both families have high QEs, are very resistant to photobleaching, and are available in a variety of excitation/emission wavelengths.

Fluorescence detection is not the only way to use a confocal, however. The same light that passes through a specimen while imaging a fluorophore may be used to image the specimen by the Nomarski technique. Combining the emitted fluorescence and transmitted Nomarski signal is a particularly powerful technique for illustrating details of a cell layer that may not be fully fluorescent (example). An additional non-fluorescence based technique is reflection-mode confocal microscopy. Light reflected from the point of focus is collected and used as the source of signal for generating the image. Common samples used for reflection mode confocal microscopy are silver-enhanced gold-conjugated antibodies, or materials science samples.

Mounting Samples

Fluorescently labeled cells and tissues exhibit a characteristic photobleaching curve in response to excitation by the laser. Much of the photobleaching can be attributed to generation of free radicals during production of the electronically excited fluor species. Use of free radical scavengers has been shown to greatly decrease the rate of photobleaching. Common scavengers include n-propyl gallate, p-phenylenediamine and DABCO (1,4-diazobicyclo-(2,2,2)-octane). Live systems have reported reduced photobleaching in the presence of vitamin C or Trolox.