Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

The near-field scanning optical microscope, or NSOM, provides spectroscopists with resolution beneath the diffraction limit. In the NSOM, an optical aperture smaller than the wavelength λ of the probe radiation is scanned in the near-field of a sample. Pixels are serially gathered and then constituted as a computer-generated image. Spectroscopic NSOM investigations demonstrating sub-λ, resolution include studies of photoluminescence, Raman spectroscopy, and single molecule fluorescence. Results of nano-Raman spectroscopy on semiconducting Rb-doped KTP are shown in figure 1. Figure la is a topographic image of the sample showing a square Rb-doped region in an otherwise undoped sample. Figure lc is a NSOM region of the corner of the doped region, and figure lb is an image of the same region taken within a Raman line. While these data do provide sub-λ spectroscopic resolution and other interesting features, the weak signal provided by current NSOM technologies and the low quantum efficiency of the Raman effect necessitated development of a very low-drift microscope and inconveniently long collection times.

Freeze-fracture techniques have contributed much to our understanding of membrane composition and macromolecular architecture. However, further substantive contributions from freeze fracture have seemed unlikely because: a) replicas typically fragmented, destroying histological orientation within samples; b) histochemical staining techniques were thought to be incompatible with bleach- or acid-cleaned freeze-fracture replicas; c) the limit of resolution was presumed to be 3-5 nm; and d) there were no methods for directing the fracture plane to specific components within a tissue. Recent developments in “grid-mapped freeze fracture” and freeze-fracture immunocytochemistry have addressed each of these limitations.

Using grid-mapped freeze fracture, samples are mapped in three dimensions using confocal microscopy before freeze-fracture (Fig. A), as well as after replication and stabilization in Lexan plastic on a gold “Finder” grid (Fig. B). After replica cleaning, areas of interest are correlated in confocal and TEM images —— for example, cilia of the ependymal layer of rat spinal cord (Fig. C).

Understanding cytoskeletal dynamics in living tissue is prerequisite to understanding mechanisms of injury, mechanotransduction, and mechanical signaling. Real-time visualization is now possible using transfection with plasmids that encode fluorescent cytoskeletal proteins. Using this approach with the muscle-specific intermediate filament protein desmin, we found that a green fluorescent protein–desmin chimeric protein was unevenly distributed throughout the muscle fiber, resulting in some image areas that were saturated as well as others that lacked any signal. Our goal was to analyze the muscle fiber cytoskeletal network quantitatively in an unbiased fashion. To objectively select areas of the muscle fiber that are suitable for analysis, we devised a method that provides objective classification of regions of images of striated cytoskeletal structures into “usable” and “unusable” categories. This method consists of a combination of spatial analysis of the image using Fourier methods along with a boosted neural network that “decides” on the quality of the image based on previous training. We trained the neural network using the expert opinion of three scientists familiar with these types of images. We found that this method was over 300 times faster than manual classification and that it permitted objective and accurate classification of image regions.

Remote ice core, snow, and air filter samples can potentially profile past and present atmospheric characteristics, acting as media to preserve populations of particulate matter. Major constituents of these aerosols derive from combustion processes (fossil and biomass) and airborne minerals. Extraction and characterization of these particles with scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDS) provide morphological and elemental data used in determining the presence and sources of atmospheric aerosols. Due to the minute quantity of carbonaceous particles in the snow and ice samples, ca. 1-10 ng g-1, the microscopist faces several principal challenges to prepare and analyze these samples. First, methodology must be established for particle removal in a clean and quantitative fashion. Secondly, since carbon is contained in many of the particles under analysis, techniques must be established to achieve quantitative carbon X-ray data. The SEM requires optimization in order to analyze the greatest number of particles in the least time.

The resolution of any microscope is usually taken as the benchmark of its quality. When thinking about the scanning electron microscope(SEM) a comparison of its resolution to the resolutions quot-ed for other comparable instruments such as the transmission EM or the scanning tunneling micro-scope has unfortunately led some users to the view that the SEM is only a second best choice. In fact the spatial resolution that can be anticipated for an optimized SEM is highly competitive with its peers making it clearly the microscopy of choice.

The resolution of the SEM depends on two classes of factors; those which are inherent to the use of electrons as an imaging tool, and those which relate to the performance of the instrumentation (and possibly of the operator) used to perform the microscopy. The use of electrons as the scanning probe, and the choice of secondary electrons as the usual imaging mode, potentially affects the achievable resolution in three ways.

The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

In recent years, the magnetic force microscope (MFM) has been used not only for the evaluation of magnetic media but also for the measurement of magnetic characteristics of quantum dots as well as in various fields, because the MFM can visualize magnetic field on the sample surface with the high resolution. The MFM observation in ultra-high vacuum (UHV) requires to reduce the adsorption layer on the sample surface for high resolution observation. The slope detection mode using the variation of vibrating cantilever is basically unstable in UHV due to the increase in the Q factor of the cantilever. Accordingly it has been used for the MFM observation in air. On the other hand, the FM detection technique offers a very high sensitivity because of the high Q factor.

This repot describes the MFM technique to take a magnetic image steadily in UHV. We observed the surface of a magnetic material with MFM technique in UHV, and compared the slope detection technique, the phase detection technique using phase variation of cantilever vibration, and the FM detection technique.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008