By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.
Nanometer-sized glass-sealed metal ultramicroelectrodes (UMEs) have been prepared using a laser-based micropipet puller. The tip was exposed to solution either by etching away a small portion of glass insulator or by micropolishing. The characterization of the UMEs was carried out by a combination of steady-state voltammetry, scanning electron microscopy (SEM), and scanning electrochemical microscopy (SECM). The cyclic voltammograms obtained have a regular shape with very small capacitive and resistive background. The effective electrode radii obtained from voltammetry were between 2 and 500 nm. From the SEM micrographs, the shape of polished tips appears to be close to a microdisk, while the geometry of etched electrodes is closer to conical. Accordingly, the SECM current-distance curves (i T -d) obtained with polished electrodes fit well the theory for a disk-shaped tip, while a 20-nm-radius etched electrode was shown to be a fairly sharp cone with a height-to-radius ratio of about 2.5. The experimental data were compared to the theory developed for disk-shaped, conical, and recessed tips to demonstrate suitability of the produced electrodes for quantitative electrochemical experiments. The prospects of steady-state measurements of the rates of fast heterogeneous reactions are discussed. Submicrometer-sized ion selective electrodes (ISEs) were prepared by coating etched Ag tips with silver iodide. The concentration response of such ISEs remained stable and linear after coating of the ISEs with protective Nafion film.
Bacteriorhodopsin is a rhodopsin-like protein found in the cell membrane of Halobacterium halobium. It Halobacterium halobium contains in its cell membrane a rhodopsin-like protein, bacteriorhodopsin, which has a broad absorption band around 570 nm. The absorbed light energy is used by the pigment to translocate protons across the cell membrane (2, 3). Bacteriorhodopsin, like the visual pigments of animals, contains retinal linked as a Schiff base to a lysine residue of the opsin (2, 4, 5). We have studied mammalian rhodopsin with tunable laser resonance Raman spectroscopy (6). The results, together with biochemical and other spectroscopic data on several forms of rhodopsin, indicate that the Schiff base linkage of retinal to opsin is protonated (for recent reviews see refs. 7-9). Mendelsohn (1) used bacteriorhodopsin in similar studies and concluded that the retinal protein linkage in this pigment is unprotonated. This suggested an interesting difference between bacteriorhodopsin and mammalian rhodopsin. We have now extended our study to bacteriorhodopsin using a tunable laser and working at low temperature.Mendelsohn (1) assumed that bacteriorhodopsin is photochemically stable. Our recent data indicate (10, 11) that this is not so. In blue-green light the pigment undergoes cyclic conformational changes through a series of intermediates, including a complex with an absorption maximum at 412 nm (3,10,11 MATERIALS AND METHODSRaman spectra were obtained with a Spex 1401 double monochromator equipped with a home-built stepping motor and a RCA 31034 photomultiplier. A multichannel analyzer was used to record the counts at each wavelength setting of the monochromator. The spectra were averaged and plotted through a PDP-1 1 minicomputer. A Coherent Radiation model 53B argon ion laser equipped with UV mirrors, a model 53B krypton laser, and a Coherent Radiation model 431 tunable dye laser provided the exciting light. Some spectra were obtained with a Liconix model 401 He-Cd laser, which lases at 441.6 nm.The bacteriorhodopsin was used in the form of the purple membrane. This is a preparation of isolated membrane fragments from Halobacterium halobium R1 (12,13) The low-temperature experiments were performed in melting point capillaries surrounded by a glass jacket and cooled with a stream of nitrogen gas. The temperature was moni- 4462$ To whom reprint requests should be addressed.
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