Spatiotemporally precise control over complex biological processes in live cells is a long-sought-after goal for researchers. Currently, limited methodologies exist that have the chemical selectivity, spatial precision, or temporal response needed to image and manipulate dynamic biological processes simultaneously. We develop a novel technology, real-time precision opto-control (RPOC), that uses the optical signal generated during laser scanning imaging to activate lasers and control the chemical processes only at the desired pixel locations. We demonstrate the ability of RPOC to precisely manipulate cellular dynamics and as a versatile microsurgery platform for biological applications using a femtosecond laser source.
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.
Periodically patterned photobleaching followed by spatial Fourier transform analysis of the recovery is shown to enable mapping of molecular diffusivity within spatially heterogeneous media.
Image segmentation prior to Fourier transform fluorescence recovery after photobleaching (FT-FRAP) enabled quantitatively evaluating diffusion of macromolecules in spatially and chemically complex media. Notably,multi-harmonic analysis by FT-FRAP was able to definitively discriminate and quantify the roles of internal diffusion and exchange to higher mobility interfacial layers in modeling the recovery kinetics within thin amorphous/amorphous phase separated domains, with interfacial diffusion playing a critical role in recovery.
KEYWORDS: Laser applications, Signal detection, Laser scanners, Molecules, Laser optics, Digital electronics, Chemical reactions, Beam controllers, Signal generators, Process control
The capability to precisely control biomolecules in live cells would give a better understanding of site-specific biofunctions. We developed a real-time precision opto-control (RPOC) platform which uses chemical-specific optical signals to trigger another laser beam only at desired locations to control biomolecules with submicron precision, nanosecond response time, and high chemical specificity. Stimulated Raman scattering and fluorescence signals are used for chemical detection. Using RPOC, we demonstrate real-time and selective control of chemical states of photochromic molecules at different subcellular locations. When used with photoswitchable inhibitors, RPOC can control intercellular microtubule polymerization and lipid droplet dynamics site-specifically based on chemical preference.
Precise control of biochemical reactions in live cells is a long-sought goal for researchers. Currently, there is no method that has the chemical selectivity, spatial accuracy, and temporal response to image and manipulate dynamic cellular processes simultaneously in real-time. We develop a novel technology, real-time precision opto-control (RPOC), that uses the optical signal generated during laser scanning imaging to control the pixel locations at which the opto-control laser is turned “on.” This optical signal is gated using comparator circuitry to command the 1st order output of an acousto-optic modulator for laser activation to minimize off-target manipulation.
Controlling behaviors of biomolecules in living cells is a challenging task. Conventional methods such as treating cells with compounds or using genetic methods do not have spatial precision. Optical tweezers can only manual manipulation of a few targets. Laser ablation methods require pre-scanning and manual target selection. Optogenetics methods also require pre-imaging and only have precision at the single-cell level. We develop a real-time precision opto-control (RPOC) platform that can selectively and precisely control biomolecules using lasers. RPOC can automatically probe and control biomolecules in dynamic living samples with high spatial accuracy, a fast response time, and high chemical selectivity.
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