It has been dogma for more than a hundred years that the light microscope cannot resolve structures that are much closer together than the length of the wavelength of light. This limit is on the order of a few hundred nanometers. This rule was “broken” by using the electron beam as the illumination source. The shorter wavelength of the electron beam gave correspondingly better resolution, down in the nanometer range. The rule was “broken” again when non-optical scanning probe microscopes (such as the scanning tunneling and atomic force microscopes) were developed. This technology gave us resolution in the sub-nanometer range, imaging individual atoms and molecules. However, scanning probe microscopes scan the surface of the specimen, excluding what's underneath from our view.

There is much discussion on the best way to fix biological specimens, often advocating cryofixation or microwave fixation. These discussions sometimes become acrimonious, and we at the P.R.I, fear the eventual division of microscopists into rival lab gangs, the “Waves” and the “Cryos”.

Wishing to forestall this, and because Leonard Bernstein is no longer with us to write West Lab Story, we have been working on a method of combining the two fixation methods. Less romantic than rival gang members falling in love perhaps, but more practical.

Valuable information concerning the relative amounts of the end proucts of histochemical and immunochemical reactions present in sections may be provided by qualitative evaluations, however, greater reliance is often placed on quantitative evaluations. Many quantitative evaluations are based on the use of image analysis and optical density readings of the visible end products. An important question is whether these quantitative measurements are reliable, accurate and reproducible, and if quantitation of these reactions offers any real advantage over qualitative evaluations.

Artifacts in preparations are sample contaminants and may constitute a nuisance if they are not recognized, or an error if they are mistaken as part of the sample. Most artifacts have two characteristics: they are common substances and they are usually minor or trace components of the sample. Occasionally, artifacts are found as a major part of what is assumed to be the sample; for example, microscopical analysis of a plugged pneumatic valve showed cornstarch as the major component. It was soon discovered that the valve had been sent in a plastic bag dusted with cornstarch. Only one percent of the particles were actual sample.

A major goal of Atomic Force Microscopy (AFM) is to achieve nanometer resolution on surface topography, Vibrating cantilever mode (VCM) is an important configuration of an AFU instrument, It was proposed in the first AFM paper.

VCM in ultra-high vacuum (UHV) results in true AFM atomic resolution, which reveals atomic scale surface defects such as a single missing atom in a lattice. However, the VCM operation in air has many difficulties due to the surface contamination on the sample and the AFM tip. The most popular operation modes of the VCM are the non-contact mode and the Tapping mode. Both of these have limited lateral resolution in air.

Microscopists frequently find that they need to regulate temperature during the course of their experiments. This is especially true for cell biologists working with living cells, Excellent commercial equipment does exist to control the stage temperature of light microscopes, and regulation by such devices is tightly controlled; ± 0.1°C is routinely reported. However, such equipment is often quite expensive and may not provide the investigator with the certainty that the experiment is being performed at exactly the temperature reported by the readout of the thermoelectric control, since the temperature sensor is often at a distance from the actual site of observation. The temperature also varies considerably from the stage heater/cooler to the critical, observed portion of the slide.

Silicone rubber embedding molds may be inexpensive In North America, but are not necessarily so elsewhere in the world. Further, commercial molds are available only in standard sizes and only with standard sizes and shapes of cavities for specimen embedding, If, however, one needs to embed specimens of n on standard size or shape, wants more embedding cavities per mold than usually available, or is working where commercial molds are expensive or not easily available, then all is not lost, Embedding molds for specimen blocks can easily be made in the lab from silicone rubber. Further, making embedding blocks allows the use of different silicone rubbers which would be of value with unusual embedding resins, or when embedding must be done in unusual environments (such as extreme cold or heat).

The use of antigen retrieval techniques has been the foundation of the development of immunocytochemistry (ICC). After the pioneering work done on cryostat sections, the use of enzyme digestion made formalin-fixed archival and current material available for study by ICC. However, the relationship between the duration of fixation and the digestion time necessary for optimum results is a major drawback associated with enzyme digestion. The longer the tissue has been in fixative, the longer the required digestion time will be. Even after a standardised fixation time, some antigens require greatly extended digestion times. Whereas 10-15 minutes in trypsin may be a standard protocol after overnight fixation, immunoglobulin deposits in renal glomeruli may require 60-90 minutes.