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.
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.
Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam’s position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.
KEYWORDS: Image resolution, In vivo imaging, Fiber optics, Ultrasonography, Mirrors, Imaging systems, Real time imaging, Photoacoustic imaging, Absorption, Image processing, High speed imaging
Intravascular photoacoustic-ultrasound (IVPA-US) imaging is an emerging hybrid modality for the detection of lipidladen plaques by providing simultaneous morphological and lipid-specific chemical information of an artery wall. The clinical utility of IVPA-US technology requires real-time imaging and display at speed of video-rate level. Here, we demonstrate a compact and portable IVPA-US system capable of imaging at up to 25 frames per second in real-time display mode. This unprecedented imaging speed was achieved by concurrent innovations in excitation laser source, rotary joint assembly, 1 mm IVPA-US catheter, differentiated A-line strategy, and real-time image processing and display algorithms. By imaging pulsatile motion at different imaging speeds, 16 frames per second was deemed to be adequate to suppress motion artifacts from cardiac pulsation for in vivo applications. Our lateral resolution results further verified the number of A-lines used for a cross-sectional IVPA image reconstruction. The translational capability of this system for the detection of lipid-laden plaques was validated by ex vivo imaging of an atherosclerotic human coronary artery at 16 frames per second, which showed strong correlation to gold-standard histopathology.
Stimulated Raman scattering (SRS) microscopy is a promising technique for label-free imaging of living systems. We demonstrate microsecond-scale SRS spectral imaging by tuning two spectrally focused pulses temporally through a resonant delay-line. Our platform acquired an SRS spectrum within 42 microseconds and formed a spectral image composed of 40,000 pixels in real-time.
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