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Intravital multiphoton microscopy is a powerful tool to study kidney physiology in living animals. However, certain technical issues have curbed its usage to date, including limited depth of tissue penetration and high background emission of endogenous signals. Most previous studies have used the excitation range 700–1000 nm. Since newer longer wavelength excitation lasers may provide solutions to these problems we constructed a microscope coupled to a laser tunable up to 1300 nm and optimized for kidney imaging. This set-up offers substantial advantages for intravital studies, especially when coupled with newly available far-red probes. First, the background at longer wavelengths is markedly reduced, thus increasing the signal to background ratio. Second, the depth of tissue penetration is significantly increased, enabling detailed imaging of previously inaccessible structures, such as deeper glomeruli. Third, using a combination of two- and three-photon excitation, multiple different fluorescent probes can be imaged simultaneously in the same animal, with clear spectral separation. Application of these techniques helped visualize pathological aspects of tubular cell function in a well-established model of acute kidney injury (maleate toxicity). Thus, utilizing long wavelength excitation offers substantial advantages for intravital kidney imaging, which together enhance the capabilities of this powerful and increasingly used research technique.

Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photo