When I see a drawing by an earty microcopist, I am often Impressed by the amount of detail they illustrated. I am frequently amazed by the resolution they apparently were able to achieve with primitive (by today's standards) unconected optics. Were their instruments and observational skills realty that good, or were they just lucky, correctly guessing what was beneath their lens? In an amazing pictorial published in the April issue of Scientific American, Brian Ford convincingly answers this question.

Ford describes the controversies surrounding descriptions by Antony van Leeuwenhoek in 1674 and Robert Brown in 1827. Both of these pioneer microscopists were often dismissed by their contemporaries and ignored for many years after their deaths. Leeuwenhoek was considered to be a man of fertile imagination whose observations of “animalcules” in pond slime were not appreciated until the time of Louis Pasteur.

Costing laboratory services is a huge job and takes a long time to do properly. I've done this many times, but only once from scratch and that one time took six months to finalize. The whole process depends on what the administration really wants and what you have to work with. There should be guidelines given and help forthcoming from the administration, as the results for the lab won't mean much if everyone does it a different way! I wouldn't even start figuring things until the administration gives guidelines and holds some meetings on how it is to be done.

We have looked at the problem of mothballing EDS detectors for clients going on long leave or over the Christmas period, and in some cases for indefinite periods.

The secret to storing the detectors is the pumping of the detector when you allow it to warm up. This results in pumping off all the contaminants, which are caught up in the molecular sieve in the dewar, before they can settle on the crystal and FET package. For this purpose we made up a T piece pumping port adapter.

There is a red or blue cap on the side or bottom of the detector, depending on the make. Open that and there will be the plug that needs to be removed under vacuum to pump down the detector, The pumping port adapter can be made up by your local engineering workshop.

I use any type of “tin” can, of various sizes, for my isopentane container, and they are free!

Parafilm can be placed between the cork and specimen to prevent it from sticking to the cork. This is helpful when re-embedding for cross-sections. Specimens can also pinned to the cork to maintain correct lengths.

I have frozen large muscle specimens, average size about 5 cm x 2 cm x 1 cm, but they must be left in isopentane for at least one minute or even longer. If the specimen is not completely frozen, it will have “moth-eaten” fiber artifacts in the center.

After freezing large specimens, they can be put in polypropylene centrifuge tubes cooled in liquid nitrogen.

Fifteen years ago, I learned from a neurologist how to embed perfused, glutaraldehyde-fixed rat brain in egg yolk (from store-bought, separated chicken eggs). The original technical note by Adoff (reference below) for whole dog brain tissue was used, and we adapted the procedure for rat brain.

1) Fix the whole brain, minus the meningas, in 10% formalin or glutaraldehydesucrose (1 % glutaraldehyde + 30% sucrose), then place in a specimen container of 30% sucrose, 10% formalin and 0.9% NaCl, The brain is left in this solution until it sinks (less than a week). This step prevents formation of ice crystals when freezing (The meninges must be removed, or the brain will not adhere to the egg yolk.).

If you can recognize a robin or a banana by sight, you should be able to recognize single particles of coal, cement or gypsum when the microscope magnifies them to an equivalent size. This is the thesis of those who identify small particles by microscopy. No other microanalytical method identifies such small samples of such diverse substances so quickly.

Single particles of almost any substance - animal, vegetable, mineral, industrial byproduct, raw material, corrosion product, flyash - can be identified in a few seconds to a few minutes by a microscopist trained in particle identification. The particle must be at least 2 to 3 microns in diameter and 10 picograms or 10-11 gram in weight for identification by light microscopy.

Confocal microscopy has gained great popularity in biology and medical research because of the ability to image three-dimensional objects at greater resolution than conventional optical microscopes. In a typical Laser Scanning Confocal Microscope (LSCM), the specimen stage is stepped up or down to collect a series of two-dimensional images (or slices) at each focal plane. Conventional light microscopes create images with a depth of field, at high power, of 2 to 3 μm. The depth of field of confocal microscopes ranges from 0.5 to 1.5 μm, which allows information to be collected from a well defined optical section rather than from most of the specimen. Therefore, due to this “thin” focal plane, out of focus light is virtually eliminated which results in an increase in contrast, clarity and detection.

We have successfully sectioned decalcified paraffin embedded “inner ears” from mice, rats and humans. We have also prepared guinea pig cochlea for SEM, So, briefly:

Paraffin:

1. 1) Immediately after dissection, place the cochlea in 10% neutral buffered formalin (NSF) and let fix with gentle agitation 24 hours per 2 mm thickness at room temperature.

2. 2) Decalcify by immersing in 5% formic acid at least 4X specimen volume.

3. 3) Gently agitate at room temperature and change at least once per 24 hours. Check for completeness of decalcification by decanting 4 mLs of “spent” formic acid into a clean, clear test tube, Add 1 mL of 5% ammonium oxaiate in distilled water. Let stand undisturbed for 15 to 20 minutes, White precipitate of calcium oxaiate indicates that the sample is not completely decalcified, Change formic acid at least once per 24 hours until the spent formic tests clear.

Microscopes in an electron microscope laboratory are built to be within specific tolerances in relation to resolution and magnification. The final specification from the manufacturer should include detail of magnification accuracy, drift rate, contamination rate and resolution, On delivery, the onus is on the installation engineer to confirm that the microscope meets specification. However, the final responsibility for microscope acceptance to specifications is the responsibility of the lab management.. Energy dispersive instrumentation is also specified as to specific resolution; and should also be confirmed on installation.

In some cases, those with final responsibility for equipment performance might not resolve with the manufacturer's representee, at installation, that the instrumentation meets all specifications.