We developed and tested a confocal scanning optical microscope that fits into a thermally controlled, commercial research cryostat designed for operation from ambient temperature down to below 4 K. The home-built microscope is a fiber-coupled, self-contained instrument based on readily available mechanical and optical components. Its sample module is sealed in a protective stainless steel tube that minimizes vibrations caused by the flow of cryogenic gas. A high numerical aperture microscope objective specifically designed for cryogenic and high-vacuum applications focuses the excitation light onto the sample, while the core of an optical fiber attached to an avalanche photodiode acts as the confocal detection pinhole. The sample is displaced using a piezotube scanner mounted on top of a three-axis, low-temperature nanopositioner assembly for coarse sample positioning. A broadband polarizing cube beam splitter in the emission path allows for polarization-resolved imaging and spectroscopy. Fluorescence excitation scans are acquired with custom-written software that correlates fluorescence photon counts with the output from a high precision wavelength meter, which is part of a narrow-band, tunable dye laser setup. The imaging and spectral data acquisition capabilities of the microscope were confirmed using a variety of samples and excitation wavelengths at temperatures ranging from 5 K to room temperature.

T , respectively. No changes in highresolution static crystallographic T(deoxy)-and R(oxy)-quaternary and tertiary structures of Hb and their heme environment, as well as the axial coordination structures of the deoxy-heme (n Fe-His = 215 /cm ) and the oxy-heme (n Fe-O2 = 567 /cm ) in solution, are observed, despite K T and K R values are changed as much as 100-and 2,000-folds, respectively. Thus, the assumption that the low-affinity state is caused by the inter-dimeric salt-bridge-linked constraints, the out-of-plane shift of the heme Fe, and the allosteric core constraint in the T(deoxy)-Hb is no longer valid. Although these constraints are completely absent in R(oxy)-Hb, its O 2 -affinity is modulated as much as 2,000-folds by its interaction with heterotropic effectors. The effector-linked modulation of thermal fluctuation of the protein may be responsible.

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