Spectral imaging is a relatively new technique that provides images of a scene at multiple wavelengths and can generate precise optical spectra at every pixel. Mathematical approaches may then be used to extract the maximum information from the spectral image. Spectral imaging is routinely used in “remote sensing”, that is, the analysis of distant landscapes and structures from airplanes or satellites. Minor differences in spectra can be used to detect different crops, or mineral deposits, for example. Closer in, spectral imaging has uses in industrial process control, detection of otherwise invisible bruising in fruit, vascular integrity in transplanted organs, and so on. Finally, spectral imaging has newly been adapted for easy use in microscopy, with applications in surgical pathology, multicolor fluorescence and immunohistochemistry, and in DNA expression arrays, among others. A variety of technologies can be used to perform imaging spectroscopy, including Fourier transform interferometry, tunable filters, line-scanning prism or gratings-based devices, and others based on polarization effects. Recently, CRI has developed a novel approach for brightfield microscopy which uses a spectrally agile light source. The latter has a number of advantages over previous techniques.

The development of the electron microprobe by Raymond Castaing provided a great stimulus to materials science at a critical time in its history. For the first time, accurate elemental analysis could be performed with a spatial resolution of 1 µm, well within the dimensions of many microstructural features. The impact of the microprobe occurred across the entire spectrum of materials science and engineering. Contributions to the basic infrastructure of materials science included more accurate and efficient determination of phase diagrams and diffusion coefficients. The study of the microstructure of alloys was greatly enhanced by electron microprobe characterization of major, minor, and trace phases, including contamination. Finally, the electron microprobe has proven to be a critical tool for materials engineering, particularly to study failures, which often begin on a micro-scale and then propagate to the macro-scale with catastrophic results.

Examples of a few objects are presented, where the electron microprobe has proved that it can provide the conservator and the curator with information that will help in evaluating the authenticity of a particular art object. They will also obtain a better understanding of how an object was fabricated and what materials it was made from. The results of the analysis of paint samples, samples from metal artifacts, as well as specimens from marble sculptures, are described.

For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.

We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.

The muscle nicotinic acetylcholine receptor channel (AChR) is a cylindrical allosteric membrane protein (∼120 x 60 Å Fig. 1) that adopts alternative quaternary conformations (“open” and “closed”) with different functional properties (ion-conducting and ion-impermeable, respectively). We have characterized, residue-by-residue, the dynamics of the conformational change associated with gating using the framework of linear free energy relationships (LFER). The sequence of molecular events that underlies the closed-to-open gating transition was inferred from kinetic measurements of the receptor at the single molecule level.

Specific regions of the AChR were perturbed using site-directed mutagenesis, changes in the membrane potential, or different agonists. Single-channel currents were recorded from cell-attached patches (Fig. 2). For the gain-of-function mutations, choline was used as the agonist because of its low efficacy. The opening rate constant was determined at a saturating concentration of agonist (for choline, 20 mM) in order to isolate gating from binding steps. to avoid bias introduced by fast channel blockade, the closing rate constant was measured at a low concentration (for choline, 200 μM). The diliganded channel opening (β) and closing (α) rate constants were estimated using the QuB suite of kinetic analysis programs. in general, a plot of the log rate constant vs. log equilibrium constant was linear.

The impact of electron probe microanalysis on the study of geological and planetary materials has been tremendous. Electron microprobes evolved into routine analytical instruments in geological research laboratories as instrument capabilities improved and applications to geologic/planetary materials expanded. The contributions of electron probe microanalysis to the characterization of minerals, both terrestrial and extraterrestrial, and to other significant geological research, such as light element analysis, trace element analysis, and element mapping, is described.

A brief description is given of the events surrounding the development of biological X-ray microanalysis during the last 30 years, with particular emphasis on the contribution made by research workers in Cambridge, UK. There then follows a broad review of some applications of biological X-ray microanalysis. A more detailed consideration is given to the main thrust of current procedures and applications that are, for convenience, considered as four different kinds of samples. Thin frozen dried sections which are analyzed at ambient temperatures in a transmission electron microscope (TEM); semithin frozen dried sections which are analyzed at low temperature in a scanning transmission electron microscope (STEM); thick frozen hydrated sections which are analyzed at low temperature in a scanning electron microscope (SEM), and bulk samples which are analyzed at low temperature in the same type of instrument. A brief outline is given of the advantages and disadvantages of performing low-voltage, low-temperature X-ray microanalysis on frozen hydrated bulk biological material. The article concludes with a consideration of alternative approaches to in situ analysis using either high-energy beams or visible and near-visible photons.

RNA molecules must fold into specific three-dimensional shapes to perform their structural and catalytic functions. Unlike proteins, RNAs secondary structural features are usually stable enough to form by themselves in solution. The reason is that in RNA, the stabilization energy gained from the formation of secondary structure is substantially larger than the energies involved in tertiary interactions. As a result, the formation of tertiary interactions is expected to alter only slightly the pre-existing secondary structural contacts. Moreover, secondary structure prediction is robust and can be made without taking into consideration tertiary folding. However, bulk studies of the energetics and kinetics of their secondary and tertiary folding are often frustrated by the presence of multiple species and multiple folding pathways in solution. These problems are circumvented in single-molecule studies in which the folding/unfolding trajectories of the individual molecules can be followed. The T. thermophila group I intron ribozyme is organized into several domains whose mechanical unfolding can be investigated independently, and whose tertiary contacts are stabilized by numerous Mg++ ions.

We have begun characterization of the ribozyme by analysis of the P5abc domain because:

Direct observation of single molecules and single molecular events inside living cells could dramatically improve our understanding of basic cellular processes (e.g., signal transduction and gene transcription) as well as improving our knowledge on the intracellular transport and fate of therapeutic agents (e.g., antisense RNA and gene therapy vectors). However, a key remaining question is whether single-molecule methodologies could be developed to study complex molecular processes in living cells. in contrast to clean and well-controlled conditions in-vitro, the intracellular environment contains a broad collection of biological macromolecules and fluorescent materials such as porphyrins and flavins. This complex environment is known to produce intense background fluorescence, commonly known as autofluorescence. Thus, a major concern is that this intracellular background could overwhelm the relatively weak signals arising from single molecules.

We demonstrate that fluorescence detection of single molecules can be achieved by tightly focusing a laser beam into a living cell (see Figure 1). The observed background fluorescence is indeed higher than that in-vitro (e.g., pure biological buffer), but this background is continuous and stable, and does not significantly interfere with the measurement of single-molecule photon bursts. Specifically, we report single-molecule results on three types of extrinsic fluorescent molecules in cultured human HeLa cells (a cervical cancer cell line).

Biological membranes are not just a passive component of the cells, they actively support their functioning as the imbedded protein machineries carry out a wide range of crucial biochemical processes. Although studies in vivo are becoming more and more accessible to single-molecule optical microscopy, in vitro studies are still very much informative for the understanding of individual biological machineries. One of the major goals is to define the minimum number of components of a machinery that is necessary for a particular step, thereby allowing detailed studies of the mechanism of action. Reconstitution of a transmembrane protein system in artificial membranes (liposomes) is the main method for such a strategy, which thus may provide the option to investigate the functioning of transport proteins, ion channels, fusion machineries, and signal transducers in relation to their environment.

We present a novel procedure in order to reconstitute transmembrane proteins in chemically well-defined and close-to-native lipid bilayers, providing an in vitro system for single-molecule optical microscopy. Furthermore, an application of this technique is shown in the case of a single-molecule study of protein-protein and protein-lipid interactions for the light-induced proton pump bacteriorhodopsin.

In this study, Giant Unilamellar Vesicles (GUV), 10-100 μm sized, are used as lipid bilayer models for several reasons.