A mid-infrared laser microscope for the time-resolved study of light-induced protein conformational changes

We have developed a confocal laser microscope operating in the mid-infrared range for the study of light-sensitive proteins, such as rhodopsins. The microscope features a co-aligned infrared and visible illumination path for the selective excitation and probing of proteins located in the IR focus only. An external-cavity tunable quantum cascade laser provides a wavelength tuning range (5.80-6.35 mu m or 1570-1724 cm(-1)) suitable for studying protein conformational changes as a function of time delay after visible light excitation with a pulsed LED. Using cryogen-free detectors, the relative changes in the infrared absorption of rhodopsin thin films around 10(-4) have been observed with a time resolution down to 30 ms. The measured full-width at half maximum of the Airy disk at lambda= 6.08 mu m in transmission mode with a confocal arrangement of apertures is 6.6 mu m or 1.1.. Dark-adapted sample replacement at the beginning of each photocycle is then enabled by exchanging the illuminated thin-film location with the microscope mapping stage synchronized to data acquisition and LED excitation and by averaging hundreds of time traces acquired in different nearby locations within a homogeneous film area. We demonstrate that this instrument provides crucial advantages for time-resolved IR studies of rhodopsin thin films with a slow photocycle. Time-resolved studies of inhomogeneous samples may also be possible with the presented instrument.