Pressures in the 100 MPa range are known to have an enormous number of effects on the action of proteins, but straightforward means for determining the structural basis of these effects have been lacking. Here, crystallography has been used to probe effects of pressure on sperm whale myoglobin structure. A comparison of pressure effects with those seen at low pH suggests that structural changes under pressure are interpretable as a shift in the populations of conformational substates. Furthermore, a novel high-pressure protein crystal-cooling method has been used to show low-temperature metastability, providing an alternative to room temperature, beryllium pressure cell-based techniques. The change in protein structure due to pressure is not purely compressive and involves conformational changes important to protein activity. Correlation with low-pH structures suggests observed structural changes are associated with global conformational substates. Methods developed here open up a direct avenue for exploration of the effects of pressure on proteins.

Fluorescence lifetime measurements of long excited-state lifetime, oxygen-quenched ruthenium dyes are emerging as methods for intracellular oxygen sensing. Fluorescence lifetime imaging microscopy (FLIM) studies in cells have been reported previously. Many current FLIM systems use high repetition rate (∼10 7 Hz) lasers optimized for nanosecond lifetime measurements, making measurement of long, microsecond lifetime fluorophores difficult. Here, we present an experimental approach for obtaining a large temporal dynamic range in a FLIM system by using a low repetition rate (10 1 Hz), high output, nitrogen pumped dye laser and a wide-field, intensified CCD camera for image detection. We explore the feasibility of the approach by imaging the oxygen-sensitive dye tris(2,2-bipyridyl)dichloro-ruthenium(II) hexahydrate (RTDP) in solution and in living cells. We demonstrate the ability of the system to resolve 60% variations in RTDP fluorescence lifetime upon oxygen cycling in solution. Furthermore, the FLIM system was able to resolve an increase in RTDP fluorescence lifetime in cultured human epithelial cells under diminished oxygen conditions. The technique may be useful in developing methods for quantifying intracellular oxygen concentrations.

We have measured the effect of high pressure on the scattering from the disordered state of a styrene-isoprene diblock copolymer containing 48 wt % styrene (Mw ~16 500). Small-angle X-ray scattering measurements performed at the Cornell High Energy Synchrotron Source (CHESS) show an increase in the aspect ratio of the scattering maximum and a decrease in the angle at which the maximum occurs with increasing pressure. Within the context of the mean-field theory of block copolymer phase behavior originally developed by Leibler,1 this behavior is due to an increase in both the radius of gyration of the molecule (Rg) and the degree of segregation of the system ( ). Examination of the data using the theory of Tang and Freed2 reveals an identical quantitative dependence of Rg and on pressure without indicating a role for the free volume fraction used by these authors to treat compressibility effects. We therefore propose that the observed behavior arises primarily from shifts in the structural characteristics of the blocks, rather than from a reduction in free volume. From comparison of the pressure-induced changes in the structural characteristics of the system with the corresponding temperature-induced ones, we predict the pressure dependence of the order-disorder transition temperature in this material, provided that the phase state of this polymer is determined solely by the structural characteristics examined in this experiment.

Invited paper Lasers and Optics Applied Physics B p. urayama 1 w. zhong 2 j.a. beamish 2 f.k. minn 2 r.d. sloboda 3 k.h. dragnev 4 e. dmitrovsky 4,5 m.-a. mycek 1,u A UV-Visible-NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

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