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Cryoprotectants allow cells to be frozen in liquid nitrogen and cryopreserved for years by minimizing the damage that occurs in cooling and warming processes. Unfortunately, how the specific cryoprotectants keep the cells viable through the cryopreservation process is not entirely evident. This contributes to the arduous process of optimizing cryoprotectant formulations for each new cell line or species that is conserved. Coherent anti-Stokes Raman scattering microscopy facilitates the visualization of deuterated cryoprotectants within living cells. Using this technique, we directly imaged the location of fully deuterated dimethyl sulfoxide (d6-DMSO), the deuterated form of a commonly used cryoprotectant, DMSO, within rice suspension cells. This work showed that d6-DMSO does not uniformly distribute throughout the cells, rather it enters the cell and sequesters within organelles, changing our understanding of how DMSO concentration varies within the cellular compartments. Variations in cryoprotectant concentration within different cells and tissues will likely lead to differing protection from liquid nitrogen exposure. Expanding this work to include different cryoprotectants and mixtures of cryoprotectants is vital to create a robust understanding of how the distributions of these molecules change when different cryoprotectants are used.

Three dimensional single molecule tracking is an essential technique for investigating biomolecular trafficking and signaling. Here we report the latest improvements to our confocal based tracking system. First, we added a Nipkow spinning disk imaging system. This system takes images of the plane of the tracked molecule, thus yielding contextual information about the molecule's environment. Laser illumination is used for the spinning disk system which together with an acousto-optic modulator allows for exposure times of 10 to 50 ms, thus greatly minimizing motion blur effects due to the feedback motion during tracking. Second, we introduced real-time photon time-gating to our tracking setup which allows tracking using only those photons detected within a defined time-window relative to the excitation laser pulse. Gating out detected photons within the first few nanoseconds after the excitation laser pulse greatly reduces Raman scattering and short lived fluorescence, and significantly increases the signal-to-noise ratio for tracked molecules with a long fluorescence lifetime. We also present a model for choosing the optimal time-gate based upon the fluorescence count rates and life times of the background and the tracked molecule.