In the biomedical sciences, samples are mounted in a wide variety of media for examination by microscope. There are a wide variety of mounting media available with a correspondingly wide range of properties. Using the incorrect mounting medium may cause signal loss and optical aberrations; the correct mounting medium avoids such aberrations and preserves fluorescence signal with “anti-fading” properties. This article introduces mounting media for fluorescence microscopy, providing descriptions of their constituents and their properties, as well as accounts of users' experience

More detailed reviews of antifade reagents have been published by Ono et al. and Longin et al.. Papers describing the effect of refractive index (RI) mismatch have been published by Diaspro et al. and Hell et al..

When making a sculpture, it is the eyes that guide the hands and tools and perceive the outcome. In simple words, “in order to make, you must be able to see.” So too, when making a nanoelectronic device, it is the microscope (eyes) that guides the process equipment (hands and tools) and perceives the outcome. As we emerge into the century of nanotechnology, it is imperative that the eyes on the nanoworld provide an adequate ability to “see.” We have microscopies that resolve 0.02 nm on a surface (scanning tunneling microscope (STM)) or single atoms in a specimen (atom probe tomographs (APT) and transmission electron microscopes (TEM)).

If beauty is in the eye of the beholder, then microscopic photographer Andrew Paul Leonard might have a lot to explain. After all, touting the aesthetic merits of an eosinophil (picture that let alone pronounce it) hardly brings to mind the rugged splendor of Ansel Adam's “El Capitan” or the quirky languidness of Richard Avedon's “Dovima with Elephants.” Yet, one look at these starkly detailed images reveals a textured, beautiful world reminiscent of the fancy of Yellow Submarine and the graphic logic of M.C.Escher.

The methylotropic yeast Pichia pastoris is a model organism for the study of autophagy and peroxisome biogenesis. Being able to look at the organism via transmission electron microscopy (TEM) can yield valuable data on the morphology of the secretory pathway and many other organelles of interest. However, preparing the yeast for TEM work can be very arduous and costly. One of the reasons P. pastoris is so hard to prepare for visualization is because its cell wall is very thick and tough compared to the membrane of a mammalian cell. Thus, P. pastoris is notoriously difficult to infiltrate with fixatives, a step necessary to maintain its ultrastructure. This article outlines an efficient and cost effective way to prepare P. pastoris for TEM without the need for certain specialized equipment. With this protocol, excellent pictures can be obtained by using the buffers, KMnO4, sorbitol, and PIPES, along with glutaraldehyde. These components preserve the ultrastructure of the yeast without any apparent artifactual change in morphology.

The amazing growth of digital imaging in the past several years has blurred the line between "real" images and those that are digitally enhanced. In most of our movies and print advertisements, we see amazing effects that create images that look real but are not.

For the last several decades film has been the predominant method that scientists employ to record the images viewed through their microscopes. Film has been readily accepted as a valid, archival recording medium because it was difficult to alter once the exposure was made and the film or print developed. The improvements in computer power and image resolution, coupled with environmental considerations, have spurred the scientific community to replace photographic processes with digital images. Processes that took hours and days are now performed in minutes. Imaging programs like Adobe Photoshop can perform all of the same photographic steps that required a darkroom, and then can do much more. The image can be “burned” onto a CD or DVD disk which is difficult to alter. Printer technology has advanced so rapidly that inkjet prints rival photographic prints. Images are often distributed digitally and viewed on displays that continue to improve. As the hardware for digital imaging improves, the quality of digital images is approaching photographic quality for a fraction of the cost, and publication quality images are produced in a fraction of the time required for film-based photography.

Just two decades ago, life scientists studied biological structure, developmental anatomy and intracellular processes by describing individual snapshots of kinetic events. Today, with so much bioscience research focusing on dynamic processes that occur on the molecular, cellular and whole organ level, it is important to record events as they happen, over seconds, minutes or hours, in living cells. Photographs and camera lucida drawings of fixed, stained cells have given way to live cell imaging using fluorescent probes, warming trays to promote cell viability and cinemicrography as a method of recording events.

In 1967, Bradbury published a detailed study of the optical properties of early, compound microscopes using modern photo micrographic techniques. This was followed by Bracegirdle's studies on the performance of 17th and 18th C. microscopes and later by Jones's comparative study of a Victorian microscope with an 18th C. Cuff-type microscope, signed ‘ Dollond ’. On the suggestion of the late Dr. S. Bradbury (personal communication), the present study on the assessment of a Ribright microscope includes photomicrographs of specimens in slides similar to the ones he used, i.e. a blow-fly proboscis and diatoms.

One-micron plastic "thick" sections, usually stained with toluidine blue, are indispensable light microscopic adjuvants for the selection and evaluation of tissue blocks prior to electron microscopy. Plastic thick sections may also be the only practical alternative to routine histologic sections when tissue specimens are limited or too small for processing in paraffin. In all cases, however, plastic sections provide superior light microscopic resolution compared to paraffin sections, but lack the compatibility with the wide variety of special stains that make paraffin sections so versatile.

Imagine what a researcher 50 years ago would think of the technology available in today's modern research labs, where the ability to photograph and videotape live cell images in high resolution has spurred numerous advances in the sciences. Additionally, technology's shift from analog to digital image acquisition has enhanced scientific collaboration and analysis abilities, perhaps in ways our predecessors could never have imagined. However, the massive quantities of data that result from these image acquisition techniques, and subsequent storage requirements, have created unforeseen challenges. While we describe life science applications, the methods described in this article are equally applicable in physical sciences laboratories.

Despite the advent of the new PC based digital camera era, there is still a vast archive of film. The obvious thing to do is to scan film optically using a high resolution film scanner and to convert the image into a digital file for digital distribution, PC archiving, and image processing. The likes of NASA can easily afford the best $20k plus PMT drum scanners and pro flatbed scanners for their spaceflight photo archives. See some professional scanners at http://www.imacon.dk, http://www.aztek.com, http://graphics.kodak.com (Creo) and http://www.flatbed-scanner-review.org. You can download selections from the NASA digitised archive at http://grin.hq.nasa.gov.

With new image analysis software for infrared spectroscopy, semi-quantitative studies can be performed without the use of standards providing rapid measurements of component concentrations when an approximate value is suitable or when quantitative standards are not available. Infrared spectroscopy has been used for many years to identify the composition of organic and other materials. Single or multi-component samples can be analyzed and their chemical structures determined by comparing their absorbance peaks to known spectral libraries or fundamental molecular transitions. This technique is used throughout the scientific community and provides rich information.

The problems being addressed by today's microscopists and microanalysts are becoming increasingly complex and data intensive. Thirty five years ago, many of us were challenged by the mere process of recording a spectral profile and then using the limited resources we had at hand to analyze the data to obtain quantitative results. Today, we have within our laptop computers the processing power of yesterday's supercomputers, however, computing power alone will not be sufficient to solve the next generation of problems.

The distribution of components, or microstructure, within a material is critical to its mechanical properties. For example, if a material has too many particles of one particular phase it may become too brittle, yet if there are too few particles it could lose strength. Every material exhibits different and unique characteristics. The goal of a material scientist is to analyze a material's microstructure and to optimize the manufacturing process. Electron microscopists observe this distribution on a very small, but critical, scale. The electron microscope can demonstrate specifically where the particles are distributed and then X-ray microanalysis can be used to identify what the particles are composed of.

This article will discuss the microstructure of an aluminum alloy with copper and iron additions. At the point of development, the mechanical properties of this new alloy were believed to be superior to that of previous materials; however, a number of subsequently conducted heat treatments did not support this theory, producing extremely unsatisfactory properties. In order to understand these results and rectify any problems, X-ray microanalysis was used to analyze the alloy's microstructure and assess the distribution of copper and iron particles within the metal.

There are numerous biological surfaces covered with secretory fluids, such as the adhesive pads of insects, sticky trichomes of plants, gustatory drops on plant leaves, lubricating coatings of vertebrate joints, etc. These fluids can be well visualised using light microscopy methods, however it is difficult to obtain detailed high magnification ultrastructural information on the morphology of these small-volume droplets. Conventional SEM preparation methods fail, because of the presence of vacuum and/or the instability of the subject fluid under the electron beam. Part of the problem can be resolved by the use of an Environmental SEM (ESEM) or an Atomic Force Microscopy (AFM), but the first method is restricted in its resolution and the second method has difficulty with resolving the complex geometry of the substrate and is rather slow for the analysis of these small, fast-evaporating droplets. Moreover, it is desirable in many applications to visualise the fluid behaviour at the contact interface between two surfaces, and this task cannot be resolved via the conventional application of either SEM or AFM methods. Herewith, we describe a Cryo-SEM approach successfully applied for visualisation of droplets of various fluids deposited on a substrate, or located at the interface between two surfaces.

Microscopists only dreamed about seeing atoms until 1951. The concept of atoms is believed to have originated in the writings of Democritus in 430 B.C., actually seeing images of atoms in a microscope became a fact only in 1951 with the publication by Erwin W. Müller, announcing his field ion microscope, FIM. Typical of the great scientist that he was, Müller immediately thought of ways to improve his new microscope and his goal then became achieving atomic resolution microscopy as he thought of it - the ability to not only see individual atoms, but to resolve the adjacent atoms comprising the atomic lattice of a crystal surface.

High-resolution Pt/C shadowing and replication provided important insights into the size and shape of polymers beginning over 40 years ago. The first images of DNA molecules were made this way. However, in my opinion, this methodology has largely been supplanted by the use of Atomic Force Microscopy (AFM), both for direct height measurements (available in Pt/C replicas by measuring shadow lengths when the coating is deposited from one direction only) and for imaging molecular contours.

As an everyday example, consider that making a magnetic read and write head for a hard disk drive requires controlling the relative heights of several different regions to a tolerance of about 1 nm. AFM supports production by providing a rapid means of offline analysis, far faster and more precise than any replica method could be.

There are many techniques for 3D imaging of biological specimens such as confocal, two-photon and, wide-field fluorescence microscopy, CAT scan, MRI, and optical coherence tomography. There are also many derivatives of these techniques, each having its strengths and weaknesses. Due to the differences in resolution, depth-of-field, and field-of-view, it is often difficult to compare images from the relatively high-resolution microscopy methods to the latter lower-resolution high-volume imaging methods. Effectively making this comparison could be very powerful in relating organ or organism level information to cellular level processes.

In science, there are occasions when it is of value to know the number and distribution of different types of particles present in a solution. Calculation of these values is simplified if you can uniformly distribute the number of particles in a known volume over an established surface area. A number of procedures have been proposed. The best which we have used is direct centrifugation of particles to formvar/carbon-formvar coated electron microscope grids in an EM-90 rotor for the Beckman Airfuge®. This instrument makes use of the ability to pellet all material in a suspension using established centrifugation theory.

It is common for microscopists to examine fluorescent samples that have been exposed to a mixed set of fluorophores with specific staining properties. Images are often captured from these samples as a set, using different filter sets to isolate specific elements of the sample. Sometimes the images are captured in gray scale, and in other cases, they are captured with color cameras. In either case, one would often like to combine the images to create a single image that presents the information from all fluorophores at once. This kind of display is particularly useful for determining the relative locations of different components.

This is the second in the series of short articles about Laboratory Information Management Systems (LIMS). This installment will focus on two issues - looking at ontologies and at business process design. The goal is to demonstrate how LIMS is really a combination of several capabilities, and that although each capability should be looked at separately, they ultimately must all work together as seamlessly as possible.

Before diving in, I would like to thank Dr. Q. C. Yu, the director of our Biomedical Imaging Facility here in Pathology and Laboratory Medicine at Penn http://www.med.upenn.edu/bmcrc/morph/?morph for his assistance, encouragement, and sponsorship of my participation in the Honolulu conference.