Dry preservation involves removing water from samples so that degradative biochemical processes are slowed and extended storage is possible. Recently this approach has been explored as a method for preserving living mammalian cells. The current work explores the use of microwave processing to enhance evaporation rates and to improve drying uniformity, thereby overcoming some of the challenges in this field. Mouse macrophage cells (J774) were pre-incubated in full complement media containing 50 mM trehalose, for 18-h, to allow for endocytosis of trehalose. Droplets of experimental and control (no intracellular trehalose) cell suspensions were placed on coverslips in a microwave cavity. Water was evaporated using intermittent microwave heating (600 W, 30 s intervals). Samples were dried to various moisture levels, rehydrated, and then survival was assessed after a 45-min recovery period using Calcein-AM/PI fluorescence and Trypan Blue exclusion assays. The metabolic activity of dried cells (4.3 gH(2)O/gdw) was assessed after rehydration using a resazurin reduction assay. Apoptosis levels were also measured. Post- rehydration survival correlated with the final moisture content achieved, consistent with other drying methods. Intracellular trehalose provided protection against injury associated with moisture loss. Metabolic assays revealed normal growth in surviving cells, and these survival levels were consistent with results from apoptosis assays (P > 0.05). Brightfield and fluorescence images of microwave-dried samples revealed a uniform distribution of cells within the dried matrix and profilometry analysis demonstrated that solids were uniformly distributed throughout the sample. Microwave-processing successfully facilitated rapid and uniform dehydration of cell-based samples.

A low-cost pulsed laser is used in conjunction with a homebuilt laser confocal-scanning epifluorescence microscope having submicron lateral and axial spatial resolution to determine cytoplasmic viscosity at specific intracytoplasmic locations in J774 mouse macrophage cells. Time-dependent fluorescence anisotropy measurements are made at each location and global deconvolution techniques are used to determine rotational correlation times. These rotational correlation times are related to the hydrated volume of 8-hydroxyperene-1,3,6-trisulfonic acid (HPTS) to calculate viscosity at specific points inside the cell. In the cytoplasmic areas measured, rotational correlation times of HPTS ranged from 0.186 ns to 0.411 ns, corresponding to viscosities ranging from 1.00 +/- 0.03 cP to 2.21+/- 0.05 cP.

Grinding, lapping, and polishing are finishing processes used to achieve critical surface parameters in a variety of precision optical and electronic components. As these processes remove material from the surface through mechanical and chemical interactions, they may induce a damaged layer of cracks, voids, and stressed material below the surface. This subsurface damage (SSD) can degrade the performance of a final product by creating optical aberrations due to diffraction, premature failure in oscillating components, and a reduction in the laser induced damage threshold of high energy optics. As these defects lie beneath the surface, they are difficult to detect, and while many methods are available to detect SSD, they can have notable limitations regarding sample size and type, preparation time, or can be destructive in nature. The authors tested a nondestructive method for assessing SSD that consisted of tagging the abrasive slurries used in lapping and polishing with quantum dots (nano-sized fluorescent particles). Subsequent detection of fluorescence on the processed surface is hypothesized to indicate SSD. Quantum dots that were introduced to glass surfaces during the lapping process were retained through subsequent polishing and cleaning processes. The quantum dots were successfully imaged by both wide field and confocal fluorescence microscopy techniques. The detected fluorescence highlighted features that were not observable with optical or interferometric microscopy. Atomic force microscopy and additional confocal microscope analysis indicate that the dots are firmly embedded in the surface but do not appear to travel deep into fractures beneath the surface. Etching of the samples exhibiting fluorescence confirmed that SSD existed. SSD-free samples exposed to quantum dots did not retain the dots in their surfaces, even when polished in the presence of quantum dots.

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Single molecules of unconjugated Bodipy-Texas Red (BTR), BTR-dimer, and BTR conjugated to cysteine, in aqueous solutions are imaged using total-internal-reflection excitation and through-sample collection of fluorescence onto an intensified CCD camera, or a back-illuminated frame transfer CCD. The sample excitation is provided by the beam from a continuous-wave krypton ion laser, or a synchronously-pumped dye laser, operating at 568 nm. In order to essentially freeze molecular motion due to diffusion and thereby enhance image contrast, the laser beam is first passed through a mechanical shutter, which yields a 3-millisecond laser exposure for each camera frame. The laser beam strikes the fused-silica/sample interface at an angle exceeding the critical angle by about 1 degree. The resultant evanescent wave penetrates into the sample a depth of approximately 0.3 microns. Fluorescence from the thin plane of illumination is then imaged onto the camera by a water immersion apochromat (NA 1.2, WD 0.2 mm). A Raman notch filter blocks Rayleigh and specular laser scatter and a band-pass-filter blocks most Raman light scatter that originates from the solvent. Single molecules that have diffused into the evanescent zone at the time of laser exposure yield near-diffraction-limited Airy disk images with diameters of ~5 pixels. While most molecules diffuse out of the evanescent zone before the next laser exposure, stationary or slowly moving molecules persisting over several frames, and blinking of such molecules, are occasionally observed. Keywords:Single-molecule detection, single-molecule imaging, single-molecule spectroscopy SINGLE MOLECULE DETECTION IN SOLUTIONAlthough this symposium is on multi-photon microscopy and its applications, the experiments reported in this paper use only single-photon induced fluorescence. (The paper was to be presented in a symposium on fluorescence correlation spectroscopy and single molecule methods, to be chaired by R. Rigler and E. Elson, but unfortunately that symposium was canceled and the paper was rescheduled into the present session.) Two-photon excitation has been used for single-molecule detection in solution, as first reported by Webb, et al, 1 but high laser irradiances are generally required because of the low two-photon cross-sections. For such experiments, typically a modelocked laser is focused to a diameter of only 1 micron. However, the goal of our work here is to develop technology to simultaneously image many individual molecules freely moving in solution near a surface over a field of view as wide as 50 microns in diameter. If two-photon excitation were to be used, it would require up to a 2500-fold increase in power to yield the same level of irradiance as in prior experiments. However, if second harmonic generation were to occur at the surface, this would provide a means for one-photon excitation of molecules, and may enable single-molecule detection at surfaces with infrared excitation wavelengths at reduced irradiances. 2The key requirement for fluorescence d...