In a clever application of time-lapse fluorescence microscopy, Li Wang, Scott Seeley, William Wickner, and Alexey Merz looked to see if there was membrane left over after vacuoles fused within a cell. Specifically, It is not known what happens when two vacuoles come together (this is referred to as “docking” ) and join together as one larger vacuole (“fusion“). For reference, the surfaces of the vacuoJes where they have joined is the “boundary” membrane, the membranes on the outside are the “outside” membranes, and the circular junction of the boundary and outside membranes is the “vertex.“

We have all seen beautiful three-dimensional (3D) renderings of molecular models, complete with helices and pleated sheets. And for more than half a century we have also seen electron micrographs of cellular structures, although usually in two dimensions. There has been a gap between these two sets of images. By developing a technique that promises 3D images at resolutions approaching the molecular level, Ohad Medalia, Igor Weber, Achilleas Frangakis, Daniela Nicastro Günther Gerisch, and Wolfgang Baumeister have bridged that gap.

Antoni van Leeuwenhoek showed the practical use of the light microscope in the 1600s after much effort to improve the quality of optical lenses. Pioneering microscopists such as Ernst Abbé, Hermann Ludwig Ferdinand von Helmholtz, Lord John Rayleigh, Carl Zeiss, and August Köhler then brought us to the brink of optimal performance of the light microscope approximately a century ago, Ernst Ruska and Max Knoll showed in the 1930s that high-energy electrons could be used in place of light, giving greatly improved resolution. In the 1970's Albert Crewe and co-workers developed the scanning transmission electron microscope (STEM) and used the Z-contrast method to improve resolution in the electron microscope by about a factor of two. The scanning probe (nonoptical) microscopes aside, there hasn't been a significant advance in spatial resolution since.

Would you believe it might be possible to build organs using an ink-jet printer? In a remarkable pair of articles, Thomas Boland and his collaborators describe a modified printer that promises to be abie to do exactly that.

The first article of this pair describes the software and hardware to arrange cells in two dimensions, just as a printer arranges droplets of ink on a fiat piece of paper. The software that normally controls the position of a drop of ink is modified to position a drop containing one or two cells in suspension onto a substrate.

Temporal resolution has long been a challenge to microscopists. Certainly, spatial resolution has occupied center stage, but we're all concerned about what happens over time in a biologic system, for example, a cell. Tags such as green fluorescent protein (GFP) have been used with confocal microscopy and other light microscopic techniques to achieve outstanding temporal resolution, but good spatial and temporal resolution have proven to be difficult to achieve simultaneously. This has been accomplished in a remarkable study by Guido Gaietta, Thomas Deerinck, Stephen Adams, James Bouwer, Oded Tour, Dale Laird, Gina Sosinsky, Roger Tsien, and Mark Ellisman, who demonstrated a pulse-chase technique that correlates with both fluorescence and electron microscopy.

We are all familiar with the concept of correlating an image acquired by light microscopy (LM) with one obtained by transmission electron microscopy (TEM). This allows us to take advantage of the “wide angle” view of LM and the high resolution of TEM. Correlative microscopy has been taken to a new level by Alvin Lin and Cynthia Goh who have designed a clever device. This device allows repetitive correlative microscopy between TEM and atomic force microscopy (AFM).

Imaging of sub-micron , sub-surface features of thick optically dense materials at high resolution has always been a difficult and/or time consuming task in materials research. For the most part this role has been relegated to technologically complex and expensive instrumentation having highly penetrating radiation, such as the synchrotron- based Scanning Transmission X-ray Microscope (STXM) or involves the careful preparation of thin cross-section slices for study using the Transmission/Scanning Transmission Electron Microscope (TEM/STEM).

With the growing emphasis on nanotechnology, scanning probe microscopy (SPM) is emerging from the surface science laboratories and becoming a mainstream inspection and metrology tool along side optical and SEM microscopes. Scanning probe instrumentation and applications evolved dramatically during the past quarter-century (Table I). By 1998 SPM-related papers were being published at the rateof nearly 5000 per year Here we review the history of scanning probe microscopy, describe its current role as a critical enabler in nanotechnology, discuss why it has become a routine laboratory tool, and present a view of future directions for this advanced technology.

It was Castaing who many years ago lamented “First 1 realized that in massive samples which concerned the metallurgists i would have to give up the splendid resolving power that I had cheerfully envisaged, as 1 became aware of the terrific path that my electrons would perform haphazardly in the sample before agreeing to stop. I had to limit my ambitions to analyzing volumes of a few cubic micrometers. This was a big disappointment. ..”. In the early days most instruments for imaging and microanalysis of bulk samples were restricted to operating only at relatively high beam energies (∼30kV) and for many (and perhaps too many) years this became the accepted operating procedure. However, we now know there are substantial advantages (pros) in using lower, and in some cases much lower, voltages; but also some significant limitations (cons). So what is possible?

In a previous article [1], stereological methods for measuring volume, surface, length, and number were described. The present article will briefly discuss sampling methods, as well as techniques for optimizing the number of animals per group and the number of measurements per animal when planning a morphometric study.

Since early times, the collective understanding of our microscopic universe has been directly tied to the quality of our microscopies. This has been true from the advent of light microscopes through to modern electron microscopes. Indeed, if one is to work on a given scale, one must be able to “see” at that scale. At the beginning of the 21st century, human inquiry is focused on the atomic scale.

Any student of cell biology cannot help but be impressed with the exquisite organization of cellular and subcellular compartments, where Individual proteins and even nucleic acids are targeted to specific domains and held together in macromolecular assemblies to achieve orderly and efficient interactions. Perhaps nowhere is the specialization of cellular compartments more apparent than in the nervous system, where the cell contains multiple, well-defined functional domains, each with its own unique biochemical character.

Many words have been written about filament saturation and filament life, not all with years of experience behind them] Sometimes when visiting laboratories one has the feeling that the life of the filament is far more important than the quality of the results, so let us try to demystify filament life?

The life of a tungsten hairpin filament very much depends upon its use and the applications present in a particular laboratory. Medturn to low magnification applications in the transmission electron microscope (TEM) may only require low emission currents (10 to 15 micro amps) with a long filament life resulting. High resolution studies require higher operating currents (20 to 45 micro amps) but, by demanding more emission current the life of the filament will suffer. Whilst a long filament life is good for the laboratory, it is not worthy of boasting, for those who have a short filament life may well be using the instrument near to the limit of its performance, a far more relevant boast!

During forensic Investigations, determining the time frame of a crime can be an extremely important clue for solving the case. The exact time at which a crime was committed can be especially difficult to determine when considerable time has elapsed. To improve the predictive capabilities of crime scene investigators, we have focused on using spectroscopic methods to Investigate the aging of bloodstains.

The combination of electron tomography (three-dimensional reconstruction of an individual object from a series of EM tilt images) and elemental mapping (based on element-specific differences in interaction between the primary electron beam and the specimen) is at the very beginning of its application. This article reviews the first published results and considers some requirements and potential uses in biology, employing two examples from our own exploratory work.

The first time you stand on the deck of a research icebreaker as it crunches its way through the Antarctic pack Ice, you're surprised at how many colors there are. Ice covers the sea everywhere you look; perhaps here and there an occasional gargantuan iceberg rises majestically above the pack. Everything between you and all three hundred and sixty degrees of the horizon is gently undulating under brilliant sunshine in unison with the sea swell. It's a January mid afternoon, the heart of the southern summer, and you're bundled up against the biting cold. You can't stop smillng.

Many us who use microscopes are interested in the internal structure or components of three-dimensional objects. Often we must section these objects to observe these internal components. For many years, microtomes have been used to make physical sections, but in recent years confocal microscopes, MR imaging, CT scanners, and even standard optical microscopes have been used to obtain “optical” sections. Two-dimensional images of these different types of sections can be used to extract three-dimensional quantitative information about the objects and their internal components, The sectioning process reduces the observed dimensions of the object and components. With apologies to Rene Magritte, the structure portrayed in Figure 1 is not a three-dimensional glomerulus but a two-dimensional profile of a glomerulus. In most cases, interest is on the structure of the three-dimensional object and not the structure in the two-dimensional image. Thus, care must be taken when obtaining and interpreting data from two-dimensional images.

The merger of molecular spectroscopy with microscopy is certainly not a new concept. Microscope attachments for FT-IR spectrometers have been available for nearly two decades and there are literally thousands of FT-IR spectrometers with these devices currently installed. The vast majority of them are applied to contaminant or forensic oriented problems. As such, the microscopes are often used as sophisticated beam condensers, enabling the spectrometer to focus infrared radiation on the samples that are typically larger than 10 microns.

A new class of silicon multi-cathode detector (SMCD) has been developed for microanaiysis spectrometry applications. The detector has excellent energy resolution (< 150 eV FWHM) and high count rate capability (>1 Mcps). An energy resolution of 143 eV FWHM at 5.9 keV was measured with the SMCD at 6 μs peaking time.

This column is, perhaps, a departure from many techniques papers because the accompanying image chosen as “representative“ really is representative, rather than what is seen in many published papers in which the representative image is the one perfect image among hundreds of those that are biologically messy. The image used in this article to illustrate methods is not the best to use when creating regions of interest with Photoshop toots, for it contains elements which make the separation into regions of interest difficult to accomplish without reacquiring the image using better microscope techniques. I'm assuming that an image of this sort CAN bereacquired; if that isn't possible, some head-scratching and, perhaps a shift from a position of absolute accuracy to statistical accuracy might occur, and many would argue, should occur (since the whole matter is statistical anyway). Hopefully the investigator understands that, in some instances, human error can be as confounding as image processing error, except that image processing can create consistency. In other instances, artifacts in the images themselves cart cause too many wrong features to be measured, leaving humans as the best instrument.