Vibrational tags in infrared (IR)-based micro-spectroscopy constitute powerful tools for studies on cellular metabolism. Whereas Raman tags have seen substantial developments, IR tags have not similarly benefitted from systematic design optimization. To improve the utility of IR-based vibrational tags, we modified a series of alkyne-based probes for use in the cell silent region of the IR spectrum. Using density functional theory (DFT) simulations for initial design motifs, the tags were analyzed using linear spectroscopy, and subsequently screened for their utility in cell and tissue imaging. The resulting chemical motifs form a palette of strong vibrational tags for IR-based biological imaging.
KEYWORDS: Brain, Analog electronics, Neuroscience, In vivo imaging, Neuroimaging, Bioluminescence, Biological imaging, Neurophotonics, Visualization, Molecules
Bioluminescence is a popular modality for imaging in living organisms. The platform relies on enzymatically (luciferase) generated light via the oxidation of small molecule luciferins. Since no external light is needed for photon production, there are no concerns with background autofluorescence or photobleaching over time—features that have historically limited other optical readouts. Bioluminescence is thus routinely used for longitudinal tracking across whole animals. Applications in the brain, though, have been more challenging due to a lack of sufficiently bioavailable, bright, and easily multiplexed probes. Recent years have seen the development of designer luciferase and luciferin pairs that address these issues, providing more sensitive and real-time readouts of biochemical features relevant to neurobiology. This review highlights many of the advances in bioluminescent probe design, with a focus on the small molecule light emitter, the luciferin. Specific efforts to improve luciferin pharmacokinetics and tissue-penetrant emission are covered, in addition to applications that such probes have enabled. The continued development of improved bioluminescent probes will aid in illuminating critical neurochemical processes in the brain.
Recent developments have shown the utility of vibrational tags in both Raman-based microscopy and infrared-based microspectroscopy. In this context, the availability of probes that feature a strong response is highly desirable. Here we develop vibrational tags that have strong and narrow vibrational lines in the cell silent region of the spectrum. We screened numerous chemical motifs with density functional theory (DFT). Design criteria included a distinct resonance frequency, a long vibrational lifetime, and overall strength of the vibrational response. We discuss the utility of these optimized probes for cellular imaging studies with stimulated Raman scattering and Fourier transform infrared microspectroscopy.
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