Old embryos are now being looked at in a new way. About a hundred years ago, an embryologist by the name of FranMyn Paine Mall devoted his career to collecting human embryos and fetuses (an embryo becomes a fetus after 2 months of gestation) from miscarriages and abortions. These specimens form the core of what is known today as the Carnegie Collection of Human Embryos, housed in the National Museum of Health and Medicine of the Armed Forces Institute of Pathology in Washington, D.C. Whereas this is a priceless national educational resource, how do we extract the information about our embryonic development from these specimens? Classic techniques involve slicing a specimen as thinly as possible on a microtome, then reconstructing the slices in a model large enough to study. The problem is the specimen is effectively destroyed in the process. What is needed is a technique that allows whole embryos to be examined, but not destroyed.

Having read John Kiernan's contribution in the January 1999 issue of Microscopy Today, I decided to add my two-cents worth (ignoring inflation), I have water-mounted preparations older than 5 years with no indications of threats to their permanence. The procedure is so simple it hardly bears writing about much less printing it for others to read.

I simply make sure any aqueous coversiip preparation on a microscope slide be dry at the edges of an otherwise full (of water) coverslip preparation, I simply add tiny amounts of water to completely fill the space between slide and coversiip, then wait a minute or two for the excess water on the slide outside the coversiip to evaporate. I can speed this step by wicking any excessive excess into a forceps-held small square of filter paper.

The compound microscope forms images in two stages. The objective (first stage) projects a real image of the specimen into the intermediate image plane (IIP) of the microscope at a ratio of magnification (M). The resolution in the IIP is typically between 40 and 100 line pairs permillimeter (Ip/mm). Since the resolution of the human eye is of the order of 5 Ip/mm, additional virtual magnification (V) by the eyepiece (second stage) is necessary to match the resolution of the eye to that of the microscope. The total microscope magnification (VTOTAL) is the product of W x V. The designation VTOTAL is used because the eye views a virtual image in the microscope.

Cover glasses, and by extension mounting media, are the cfe facto front lens of every microscope objective. Those who coverslip are, in effect, completing the assembly of a microscope objective with every cover glass they apply. So while coversliping is a tedious process, its visibly appreciable contribution should not be minimized.

There are optical reasons on which coverslipping do's and don'ts are based. It is optically and physically impossible for alternatives such as liquid substitutes and plastic film to duplicate the specifications and performance of glass cover glasses. Such substitutes are used, however, perhaps because there is so little related useful technical information available to discourage such use.

The use of traditional optics in microscopy has a well-known resolution barrier first presented by Lord Rayleigh. This is the "Rayleigh diffraction limit." In the usual textbook example, the overlapping diffraction rings from two small objects are set so that the central maximum of one pattern falls on the first minimum of the second, The barrier limits the resolution of conventional microscopes to a significant fraction of the wavelength of light.

In the past decade two methods were discovered for surpassing the Rayleigh diffraction barrier. The first was actually an independent rediscovery by D. Pohl er al. of a concept originally proposed by Synge (in a 1928 paper reviewed by Albert Einstein),

If you are one of the many people who are currently considering the purchase of a confocal microscope, you may be asking yourself, "How can 1 be sure that the microscope I choose is the right one?"

Before proceeding, take a step back. Ask yourself: "Why use a confocal?" A confocal microscope is, by definition, a microscope which optically "slices" the sample by showing only those details which are at or near the plane of focus. This results in a clearer image than might be possible with a conventional (also called "wide-field") fluorescence microscope - a microscope in which every pixel in every image is corrupted by light from its neighbors. The optical slicing of the sample done by the confocal microscope lets you construct a 3-D representation of the sample that can be very useful for image visualization or analysis.

Scanning Probe Microscopy (SPU) is a versatile tool for the investigations of surfaces. Additionally to the three-dimensional examination of surface topography, local mechanical or electrical properties can be measured1. In this work we present a short contribution on the performance and reliability of piezoelectric transducers in a SPM, which take care of the fine positioning of the tip relative to the sample. Some of the calibration procedures known in literature [cited in 2] are: laser interferometry, scanning a slightly tilted surface, a grid, a crystallographic or artificial step of a known height or by using a reference piezo. To measure slow and fast piezo response simultaneously, we use a non-contact calibration procedure with a high dynamic range (Angstrom to several hundred micrometers) and high frequency range (D.C. to 200 kHz(-3dB)).

Most of the time an air table is not needed for vibration isolation of electrophysiological equipment and microscopes. In fact, an air table can be a liability in that it is likely to pick up air-borne vibrations. This is especially likefy if you have a solid enclosure, as is often used for the construction of Faraday cages used in electrophysiological investigations of vision research. Sound propagation can be particularly annoying, especially at the lower frequencies. Such important details are often forgotten in discussions of this topic.

A good damping table will help reduce the resonances, but still often can't overcome the difficulty posed by sound. One of the best solutions is proper placement of the entire rig: on the slab in the basement in a quiet place with no heavy vibrations from nearby heating, ventilating, and airconditioning (HVAC) and other equipment.