Optical control technologies have been demonstrated with high spatial precision. However, present methods are facing challenges in real-time selection and manipulation. To address these limitations, we present a real-time precision optical control technology (RPOC) which is a close-loop optoelectronic system integrated with a laser scanning confocal fluorescence microscope. This technology allows us to control chemical processes at the target sites in real time with high spatiotemporal precision. Using it, we demonstrated the precise generation of reactive oxygen species (ROS) solely at selected organelles and monitored ROS-induced changes in microtubule polymerization dynamics. We also selectively inhibit tubulin polymerization using RPOC with a photoswitchable inhibitor. Meanwhile, a software-based optical control system is developed for more flexible selections of chemical targets and optical manipulation.
Laser-based optical control technologies have shown promise in achieving high spatiotemporal precision. However, existing methods face challenges in real-time target selection and manipulation. To overcome these limitations, we present a real-time precision opto-control (RPOC) technology, which is a closed-loop optoelectronic system that is built upon a laser scanning confocal fluorescence microscope and integrates chemical-specific optical detection, real-time decision-making, and precise optical manipulation at target sites. Using RPOC, we demonstrated precise inducing reactive oxygen species (ROS) solely at selected targets and monitoring ROS-induced changes in microtubule polymerization dynamics. We also selectively inhibit tubulin polymerization using RPOC paired with a photoswitchable inhibitor.
Phototoxicity is a universal problem in optical imaging. Effective and quantitative evaluation of phototoxicity allows for the optimization of imaging conditions and the minimization of functional perturbation to live biological samples. We found that microtubule polymerization dynamics is a sensitive and reliable readout that can quantify phototoxicity in various imaging conditions. We quantified the photosensitizing effect of common organelle stains under the illumination of different laser wavelengths and compared the phototoxicity of different conditions. By controlling oxygen levels during light treatment, we found that the type I photosensitizing effect is involved in the hypoxia condition. We also evaluated the photoperturbation of IR laser pulses at different average and peak power levels in nonlinear optical imaging. Applying a recently developed real-time precision opto-control system, phototoxicity and reactive oxygen species generated at specific organelles can be studied.
The low-frequency Raman region below 400 wavenumbers has rich chemical information to identify elements and compounds with low vibrational energies. Such low-frequency vibrational modes are usually probed by spontaneous Raman scattering which has low acquisition speed or femtosecond impulsive Raman scattering that requires ultrashort laser pulse and interferometry. High-speed hyperspectral stimulated Raman scattering (hSRS) microscopy has been developed based on spectral focusing but cannot detect low-frequency vibrational transitions. We extended the frequency range of picosecond hSRS microscopy to measure vibrational transitions below 400 cm-1. Using spectral focusing, we can perform hSRS imaging for a ~200 cm-1 spectral window tunable from 200-4000 cm-1.
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