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.
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.
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.
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.
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.
Cancer cells in the hypoxic environment alter their metabolism to address the low oxygen stress. Such metabolic changes contribute to cancer resistance to chemotherapy and radiotherapy treatment. Understanding the hypoxia-induced metabolic changes allows for finding new targets to treat hypoxic cancer cells. We applied stimulated Raman scattering spectroscopic imaging to explore the metabolic changes of cancer cells exposed to hypoxia. We found spatial redistribution and content changes of lipid droplets (LDs) in various cancer types. We also studied the sources contributing to the formation of LDs and discovered the relationship between LD formation and endoplasmic reticulum stress.
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.
We developed a pulse-picking multimodal nonlinear optical microscope that increases the sensitivity of label-free chemical imaging at low average laser power. Using a function-generator-controlled acousto-optic modulator, we collinearly combine two excitation wavelengths and can flexibly control the number of pulses at each pixel. The pulse-picking method gives over 1000x signal improvement for CARS and 20x for two-photon excitation fluorescent and second-harmonic generation at the same low average power. By varying the peak and average power of laser pulses, we evaluated the laser phototoxicity and found the optimal power window with the best sensitivity and minimum phototoxicity for tissue samples and live cells.
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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