While today a picture of a serious looking person peering into a microscope may suggest “science” to the general public, we noted in our last column that this was not always so. In the early 19th century, for example, the microscope was seen by many as a hobby, interesting yes, but not as worthy of respect as an amateur's telescope.

Microscopes improved and soon, through the insight of the likes of Ernst Abbe, reached a state of perfection which was not greatly improved upon until recently. Once aberrations were well corrected for and glasses of high refractive index commonly utilized, further improvements in numerical aperture became increasingly difficult to receive. The only way to improve resolution seemed to be to decrease the wavelength of light used to illuminate the sample.

Several well (and also several not so well) publicized incidents of unethical conduct have raised the concerns of scientists, politicians and common folk that the truth and only the truth may not always emerge from scientific investigation. While this is a problem which clearly needs attention, I believe that Science alone of all fields of human endeavor has built in mechanisms which ensure that truth will emerge sooner or later, usually sooner (although some frauds, for example the Piltdown hoax, survived for quite a long time). This self correcting feature arises from the fact that today's interpretations are repeatedly tested against nature, whenever new methods or hypotheses arise. Only correct beliefs survive this repeated scrutiny. The misguided workers who for whatever reasons “cheat” or get so personally involved that they fall in traps made of their own gullibility are eventually discovered and at the very least lose the recognition that they longed for.

In the good old days, when microscopists wanted to show others what they had found, they either drew by hand what they saw or hired someone to do same. Besides manual dexterity, and eye-hand coordination, drawing of course requires artistic interpretation, i.e. at least a smidgen of imagination. As a result one always had to be concerned with the objectively of rendering. Such concerns were more or less put to rest once the proper use of photography became established. The manual dexterity was reduced to twisting the focus knob, squeezing the shutter button without also shaking the camera and doing some handwaving in the dark room (artistic interpretation), surprisingly all non-trivial operations.

Today we have entered a different phase yet in the process of sharing morphological information, a phase characterized by digital recording and along with it, digital image processing. The digits now are not literally fingers anymore, but the numbers they stand for Digital image recording and processing was originally devised to improve the quality of images returned by spacecraft and used to require highly sophisticated computing facilities.

A few months ago I read a most exciting article in a scientific magazine. It seemed to me that the ideas presented had the potential to revolutionize all of microscopy and I got very excited at the vistas opened by this unexpected departure from the accepted dogma. The article was only a summary of the current status of the research in the field and I resolved to probe deeper. Since I read the piece just as I was ready to go to sleep I trusted my memory in the unreasonable belief that, should my memory fail, I could always access a computer database to retrieve the information when I needed it. I even recollect feeling very virtuous when, the very next morning, I entered the data into my files. At least I think I remember that.

As the year ends there is a bumper crop of announcements of advances that I find absolutely amazing. First of course is the continued clever use of light as a veritable tool in manipulating everything from atoms (entrapping them in “atomic molasses”) to having tugs of war with biological motors (using “light tweezers”). But these developments will be for discussion another time. What I want to talk about in this installment are advances in Near Field Scanning Optical Microscopy (NSOM), which has now been used by Chichester and Betzig to visualize single molecules.

In classical (far field) optics, resolution is limited by diffraction to about 1/2 the wavelength of the radiation used for imaging. Near field optics overcome this limitation by use of scanning techniques similar to those employed in Scanning Tunneling or Scanning Force Microscopy.

One of the most pervasive problems in microscopy is that of making sure that what one is observing represents reality. The complex specimen preparation steps which have to be followed to obtainimages of biological samples all produce artifacts, at least in the sense that they modify the sample. The investigator has to somehow show that these changes in the sample reveal true microscopic features, rather than structures which do not exist in the living. This, of course, is why polarized light microscopy, phase and interference microscopy and their derived approaches, all of which reveal detail in live untreated specimen, are so important, in spite of the limited resolution which can be achieved.

When electron microscopy was in its infancy there were, and justifiably so, even more severe fears about the reality of what could be seen. Even visionary people like Gabor had their doubts and the first steps in biological microscopy were taken with the realization that if the sample was burned to a crisp, perhaps examination of its cinders would still give valuable information.

In the Pickwick Papers, Charles Dickens has his character Sam exclaim that he needed “a pair o’ patent double million magnifyin’ gas microscopes of hextra power” Now the general wisdom is that improvements to microscopy are bounded by the laws of optics which tell us that resolution is a function of the wavelength of the radiation used for imaging and inversely proportional to the refractive index of the medium and lens. We have discussed in this space various ways which can and have been used to “circumvent” mother nature, by such tricks as not using lenses and employing scanning techniques instead in order to obtain images.

It turns out that there may yet be other useful “loopholes” in natural laws. Lefs for example consider the fact that the refractive index of a material is a measure of the interaction of photons with the matter they traverse. In that sense refraction is related to absorption, which occurs when materials interact so strongly with photons that the latter never emerge.

Electron beam lithography is a term well known in the world of microelectronics. It provides an effective but costly solution for producing the smallest electronic devices (transistors, memories, etc). Many fields of science and research also require small structures below the one micron dimension. The ability to generate these structures “in house” without going to external sources has been a luxury only the largest corporations and universities could afford. Even when a production lithography system is available, the time allocated for research work can sometimes be extremely limited. Those with the need for these small devices are now realizing there is new way to get the most use from their SEM, STM or STEM.

In the past few years, experimental e-beam systems have been developed by making use of the basic capabilities of the scanning electron microscope. Mow a variety of home built and commercially available systems are being used throughout the world. The mast basic system can take control of the e-beam in the SEM and write a simple pattern on a sample coated with materials sensiiive to electrons.

Slow-scan CCD (charge-coupled device) cameras (SSCs) are based on optical image detection technology introduced in the seventies and first used in astronomy in the eighties. When they are properly configured for the detection of medium energy electrons rather than photons, they are able to capture images that exceed the quality of images recorded on photographic film. Here we discuss aspects of their operation pertinent to routine biological TEM.

Compared to the TV-rate CCD sensors that have become ubiquitous in miniature camcorders, scientific (astronomy-grade) CCDs have several unique characteristics: 1) their pixels are typically 20-27 urn in size rather than the 6 urn typical of TV rate CCDs, 2) their read-out amplifiers are optimized for low noise at read out rates of 1/10 to 1/100 of TV rate, 3) they do not have a separate frame-transfer buffer for CCD read-out, and 4) they are available in large total sizes (up to 2048 x 2048 pixels).

There remains two basic axioms of all microscopists: the first….if you look, you're bound to see something, and the second….not everything you will see is artifact. These axioms apply particularly well to scanning probe microscopy at the molecular and atomic level. Fortunately, coarser resolution images share comforting similarities with images from other established scanning methods. Holes in optical discs look like holes when probed with AFM tips, and these holes look very much like SEM images, a subject with which we have some familiarity. At the molecular and atomic level, however, the scanning probe instruments may or may not be “seeing” the sample, though they are clearly seeing something.

Comparison of surface structure observed with indirect surface structural measurements, for example by LEED (Low Energy Electron Diffraction) or RHEED (Reflection High Energy Electron Diffraction) usually under ultra-high vacuum conditions can lead, by inference, to an understanding of the real bulk or average surface structure.

In his preface to the first edition of The Particle Atlas, Jim Lodge talks of the challenge he posed to Dr. Walter McCrone in the 1950s to come up with a taxonomy for particles to assist in the identification of particles under the microscope. Jim's interest and the support of the Midwest Air Pollution Prevention Association and, laier, the US Public Health Service, gave birth to the first edition of The Particle Ailas. This first edition, published in 1967, was a single volume which combined guidance on particle separation and identification techniques with a collection of optical micrographs of reference particles. The demand for this reference work soon outpaced the supply. In the meantime, however, it had become obvious that reprinting was not the answer as increased application of the microscope, in all its implementations, to environmental pollution and industrial contamination had resulted in a need for a more comprehensive work.