Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons (∼700 nm) can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet (∼350 nm). In the fluorescence experiments described here, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g., wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. Three properties of two-photon excitation give this method its advantage over conventional optical sectioning microscopies: (1) the excitation is limited to the focal volume, thus providing inherent three-dimensional resolution and minimizing photobleaching and photodamage; (2) the two-photon technique allows imaging of UV fluorophores with only conventional visible light optics; (3) red light is far less damaging to most living cells and tissues than UV light and permits deeper sectioning, because both absorbance and scattering are reduced. Many cell biological applications of two-photon excitation microscopy have been successfully realized, demonstrating the wide ranging power of this technique.

Results concerning the calibration and use of a new ultrahigh vacuum (UHV) surface preparation and analysis system are reported. This Sample Preparation Evaluation Analysis and Reaction (SPEAR) side chamber system replaces an older surface side chamber that was attached to a Hitachi UHV H-9000 microscope. The system combines the ability to prepare clean surfaces using sample heating, cooling, ion milling, or thin film growth with surface analytical tools such as Auger electron spectroscopy (AES), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM), along with atomic surface structure information available from high-resolution transmission electron microscopy (HREM). The chemical sensitivity of the XPS and AES are demonstrated in preliminary studies of catalytic and semiconductor samples. In addition, the surface preparation capabilities are also demonstrated for the Si(100) and Ge(100) surfaces, including the ability to acquire secondary electron images during milling. During operation, the entire system is capable of maintaining the UHV conditions necessary for surface studies.

We present a novel combined atomic force microscope (AFM) and confocal laser scanning microscope (CLSM). The sample is supported from the side by a boom from a piezo tube scanner, allowing top and bottom access to the sample. The sample is scanned above an inverted microscope objective with a fixed optical path for fluorescent CLSM imaging. An AFM positioned directly above the sample simultaneously measures surface topography. The piezo tube scanner is angled, placing its center of scan curvature directly above the microscope objective. This geometry allows flat scans up to 300 μm on a side. Because the sample is scanned, the AFM and CLSM images are acquired in direct registration.

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about −117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at −155 to −170°C to produce semithin frozen sections (0.5 to 3 μm thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.

A new class of gold immunoprobes has been developed with substantially different characteristics from the usual colloidal gold probes. Instead of chunks of gold metal (colloidal gold) just adsorbed to immunoglobulins by ionic or hydrophobic interactions, well-defined gold compounds are covalently attached to antibodies. Gold, along with several other metals, is known to form organo-cluster compounds with multiple metal atoms. The first gold cluster immunoprobe developed was undecagold (Au11), containing 11 gold atoms in an 0.8-nm sphere (Figure 1), covalently attached to Fab′ antibody fragments (Hainfeld, 1987). A more recent development has been the synthesis of a larger 1.4-nm gold cluster (Hainfeld et al, 1991; Hainfeld and Furuya, 1992).

New instrumental approaches for performing vibrational Raman, near-infrared and mid-infrared spectroscopic imaging microscopy are described. The instruments integrate imaging quality filters such as acousto-optic tunable filters (AOTFs), with visible charge-coupled device (CCD) and infrared focal-plane array detectors. These systems are used in conjunction with infinity-corrected, refractive microscopes for operation in the visible and near-infrared spectral regions and with Cassegrainian reflective optics for operation in the mid-infrared spectral interval. Chemically specific images at moderate spectral resolution (2 nm) and high spatial resolution (1 μm) can be collected rapidly and noninvasively. Image data are presented containing 128 × 128 pixels, although significantly larger format images can be collected in approximately the same time. The instruments can be readily configured for both absorption and reflectance spectroscopies. We present Raman emission images of polystyrene microspheres and a lipid/amino acid mixture and near-infrared images of onion epidermis and a hydrated phospholipid dispersion. Images generated from mid-infrared spectral data are presented for a KBr disk containing nonhomogeneous domains of lipid and for 50-μm slices of monkey cerebellum. These are the first results illustrating the use of infrared focal-plane array detectors as chemically specific spectroscopic imaging devices and demonstrating their application in biomolecular areas. Extensions and future applications of the various vibrational spectroscopic imaging techniques are discussed.

Changes in microstructure of Zr, Ti, and Ti-6%Al-%V resulting from ion-beam sputtering used to prepare samples for transmission electron microscopy have been correlated with hydrogen absorption. Zr was particularly sensitive to this phenomenon, resulting in extensive hydride formation in thin foil samples. Hydrogen enrichment extending to several micrometers in depth could also be produced in bulk samples in a few hours of sputtering. The performance of various sputtering units in different configurations has been examined. It is concluded that hydride formation appears to be caused primarily by the presence of hydrocarbons, for example, from the backstreaming of diffusion pump oil, in the residual vacuum background of the sputtering chamber.

Four leaf preparation techniques (air drying, tetramethylsilane air drying, critical point drying, and freeze substitution) used in scanning electron microscopy (SEM) were evaluated with respect to the degree of cellular distortion they produce in stomatal guard cells of leaves of Dactylis glomerata and Elymus canadensis. Surface morphological distortion and cuticle disruption in the air-dried and tetramethylsilane air-dried leaves, and cuticle disruption within the critical point-dried tissue made it difficult to obtain measurements.The freeze-substituted tissue experienced little cuticle disturbance, and the cellular morphology appeared normal. The length of the guard cells did not significantly differ between the air-dried, tetramethylsilane air-dried, critical point-dried, or freeze-substituted samples. Widths did significantly vary, with the freeze-substituted tissue having lower values than tissues treated with the other treatments. Freeze substitution methodology produced SEM images that appear to be less distorted and allow easy and precise measurement.

An improved, jet electropolishing technique for the production of germanium TEM specimens has been developed. The use of slightly modified, commercially available equipment permits rapid, reliable thinning of foils exhibiting large electron transparent regions. The use of automatic termination of polishing produces cleaner foil surfaces than incremental final polishing did previously.