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We present a novel approach of automatically measuring motion in series of microscopic fluorescence images. As a differential method, the three-dimensional structure tensor technique is used to calculate the displacement vector field for every image of the sequence, from which the velocities are subsequently derived. We have used this method for the analysis of the movement of single actin filaments in the in vitro motility assay, where fluorescently labeled actin filaments move over a myosin decorated surface. With its fast implementation and subpixel accuracy, this approach is, in general, very valuable for analyzing dynamic processes by image sequence analysis.

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The influence of ionic strength upon relaxation kinetics from rigor in skinned murine extensor digitorum longus (EDL) skeletal muscle fibres was examined using photolysis of caged-ATP at low Ca2+. The ionic strength was adjusted with either KMeSO3 or ethylene glycolbis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, dipotassium salt (K2EGTA) in the range of tau /2 = 65-215mM, or I.E. 49-194mM, where I.E. denotes ionic equivalent. Following rigor development at a tau /2 of 165-215mM (I.E. 144-194mM), the liberation of approximately 0.5mM ATP resulted in an initial 6-to 10-ms detachment phase with a decline in force of approximately 10-20% followed by a 10-to 30-ms reattachment with up to a 60% increase compared to the corresponding rigor level and a final detachment leading to complete relaxation. Interestingly, when similar ATP concentrations were liberated at lower ionic strengths between a tau /2 of 65mM and 110mM (I.E. 60-100mM), the initial detachment phase was shortened and force decreased by only approximately 5-10%, while the following reattachment phase was lengthened and led to an increased steady-state force of approximately 20-80% without final relaxation. ATP-induced detachment and subsequent reattachment were mainly determined by the currently present ionic strength and were relatively independent of the preceding rigor state which had been developed at higher or lower ionic strengths. The effects of phosphate and apyrase on the force transient suggest that reattachment of ADP- binding crossbridges may contribute to the increase in tension at high and even more at low ionic strengths. The study shows that the kinetics of initial fast relaxation and subsequent redevelopment of force following flash photolysis of similar ATP concentrations are markedly modified by the ionic strength in the narrow range of between 65mM and 215mM.

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In a novel approach, the laser microbeam technique was used to selectively perforate the sarcolemma of skeletal muscle fibers, to prepare fragments of myofibrillar bundles of very small dimensions, and to induce fusion of sarcolemma vesicles. Using a highly focused UV laser microbeam with an effective beam diameter of down to 0.5 micron, very small (< 3 microns) myofibrillar fragments with an intact sarcomere striation pattern were obtained. When small amounts of Ca2+ were released in the vicinity of such a fragment by laser-photolysis of the photolabile compound Ca(2+)-nitr-7 the bundle shortened due to the development of calcium-activated force. We also show that very small selected areas from myopathic single muscle cells can be dissected with a precision unmatched by other current techniques. The microbeam was also used to remove very small patches of the sarcolemma of murine skeletal muscle fibers so giving diffusional access to the myoplasmic interior and thus resulting in a "skinning" of the fiber. To ensure that such laser-skinned fiber segments were physiologically intact we determined the Ca(2+)-activated force and caffeine-induced Ca(2+)-release from the sarcoplasmic reticulum. The fibers showed normal characteristics for force production, Ca(2+)-release and uptake by the sarcoplasmic reticulum. To test the effects of the laser microbeam on the muscle membrane directly, we prepared sarcolemma vesicles of skeletal muscle fibers. The vesicles could be selectively perforated with single laser pulses to allow entry of fluorescein isothiocyanate (FITC)-dextran as a fluorescent marker. Adjacent vesicles were caused to fuse by a few pulses at low intensity of the laser microbeam.(ABSTRACT TRUNCATED AT 250 WORDS)

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In order to obtain information about the activation of macrophages (M phi s) during photodynamic therapy (PDT), the influence of Photofrin II (Pf II) on the viability of thioglycollate-elicited murine M phi s and the subsequent generation of superoxide anion was studied. Irradiations were performed at an energy density of 5 J cm-2, a power density of 150 mW cm-2 and a wavelength of 405 nm. Viability of M phi s was assessed using the acridine orange-ethidium bromide assay. Superoxide anion generation was determined using ferricytochrome c (cyt c) and nitroblue tetrazolium (NBT) reduction. Our results indicate that the M phi s are highly susceptible to PDT as their viability is decreased to approximately 30% by 1 microgram ml-1 Pf II at the energy density indicated above. Within the first 30 min of addition of the photosensitizer, a reducing agent is generated intracellularly by the stimulation of the M phi s. An extracellular release of superoxide anion does not occur, as measured by the cyt c assay. Preincubation of the cells for 1 or 24 h with Pf II and a second challenge with phorbol myristate acetate (PMA) does not enhance the reduction of NBT. Thus, Pf II exerts an immediate effect on the M phi s which could be interpreted as a first step for subsequent reactions.

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A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 microns, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogen-pumped dye laser (366 nm), was collinear with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of approximately 1 microJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.

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