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.
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.
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.
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.
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.
The primary goal of this study was to track PS-ASO and GalNAc-PS-ASO uptake in two cell cultures as the first step to understand the observations from the clinical studies. The multimodal imaging setup of CARS and 2PF modalities in conjunction with the image analysis pipeline made it uniquely possible to address these challenges. We report here the time-dependent uptake, internalization, and localization differences between GalNAc-PS-ASOs and PS-ASOs in liver cells. We believe our findings will help us form the basis for further investigations with more complex cellular co-cultures and with tissue and animal models.
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.
Recent advances in tissue engineering and microfabrication have led to development of novel Complex In Vitro models (CIVMs) that more closely mimic pathophysiological functions of human tissues and organs. CIVMs can provide deeper insights into the mechanisms of human disease and pharmacological properties of new drug candidates during early stages of development. In this study, a multimodal optical imaging platform was used for characterizing the structural and functional features of a liver-on-a-chip model (CN Bio Innovations, UK).
Using coherent anti-Stokes Raman scattering (CARS) microscopy, we discovered heterogeneity of mitochondrial chemical compositions in cancer cells after hypothermia. Individual mitochondria associated with higher signals in CARS images are likely the result of fatty acid accumulation, which is caused by a reduced rate of fatty acid β-oxidation. Tracing individual mitochondria after reheating the cells to 37°C reveals degradation of these organelles through the mitophagy process. Further study will continue to unveil how stressed mitochondria would form in various conditions, and the fate of these organelles upon changes of their environment. Our results shed new light on mitochondrial function and cell metabolism.
The current standard for antibiotic susceptibility testing (AST) is based on measuring bacterial growth after 10-24 hours of proliferation. Considering that many life-threatening conditions of infection exist, rapid AST techniques are urgently needed. We developed a rapid AST method based on two-photon fluorescence and coherent anti-Stokes Raman scattering microscopy which can detect antibiotic responses of bacteria within one hour. We used Pseudomonas aeruginosa as a representative pathogen model, and found that antibiotic treatment greatly reduces nicotinamide adenine dinucleotide (phosphate) levels in the bacteria. This enables rapid determination of bacterial susceptibility at the single cell level.
Coherent anti-Stokes Raman scattering (CARS) microscopy was used to disclose dynamic information from lipid droplets (LDs) in living cancer cells. Statistical analyses of LD dynamics show different pools of LDs associated with LD synthesis and degradation processes. We found that each LD population satisfies a log-normal distribution, thus allowing quantitative comparison through fitting of population histograms. Two-dimensional analyses of LD displacements and speeds offer dynamic signatures of cells to monitor responses to stimuli and drugs. We revealed hypothermia-induced cellular metabolic changes and a two-step metabolic response during apoptosis. We also explored dynamic signatures of LDs during starvation and various drug exposures.
It is still unclear how neuronal metabolism is affected by opioids. We developed a multimodal nonlinear optical imaging platform that can resolve single neurons while monitoring endogenous metabolic molecules in a label-free manner. These capabilities enable investigation of morphine-induced metabolic changes in neurons. Additionally, mouse brain slices were used to study the effects of morphine on metabolism in the brain. We found that after morphine exposure, the nucleus accumbens area showed increased metabolic activity compared to the cerebral cortex. Further studies will reveal how opioids alter the metabolism of the brain and individual neurons at the sub-cellular level.
Hyperspectral stimulated Raman scattering (SRS) microscopy allows imaging of complex chemical mixtures and analysis cellular metabolites with high specificity. However, current SRS imaging is not implemented to address the cell heterogeneity issue, which can only be resolved by statistical analysis of a large amount of cells through cytometry. We developed a high-speed hyperspectral SRS image cytometry platform based on multiplex excitation, acquiring a Raman spectrum of 200 wavenumbers in 5 microseconds. This platform enables measurement of <100 cells per second. Multiple chemical signatures, featuring different cellular organelles such as lipids, endoplasmic reticulum, nucleus, and cytoplasm can be segmented. Statistical analysis over a large amount of cells reveals unprecedented details about cell metabolic changes after drug treatment.
Spectroscopic stimulated Raman scattering (SRS) is a label-free chemical imaging modality enabling visualization of molecules in living systems with high specificity. Among various spectroscopic SRS imaging methods, a convenient way is through linearly chirping two femtosecond lasers and tuning their temporal delay, which in turn corresponds to different Raman shifts. Currently, the acquisition speed using a resonant mirror is 3 seconds (80 microseconds per spectrum), which is insufficient for imaging samples with high motility. In this work, we aim to push the imaging speed using a 50-kHz polygon scanner as a delay line tuner, achieving a speed of 20 microseconds per spectrum. At such high speeds, to overcome the signal level decrease due to reduced signal integration time, we apply a U-Net deep learning framework, which first takes pairs of spectroscopic SRS images at different speeds as training samples, with high-speed, low-signal images as input and low speed, high-signal ones as output. After training, the network is capable of rapidly transforming a low-signal spectroscopic image to a high-signal version. Consequently, our design can generate ultrafast spectroscopic SRS image while maintaining the signal level comparable to the output with longer signal integration time.
A multimodal system combining surface sensitive sum frequency generation (SFG) vibrational spectroscopy and total-internal reflection fluorescence (TIRF) microscopy for surface and interface study was developed. Interfacial molecular structural information can be detected using SFG spectroscopy while interfacial fluorescence signal can be visualized using TIRF microscopy from the same sample. As a proof of concept experiment, SFG spectra of fluorescent polystyrene (PS) beads with different surface coverage were correlated with TIRF signal observed. Results showed that SFG signals from the ordered surfactant methyl groups were detected from the substrate surface, while signals from PS phenyl groups on the beads were not seen. Additionally, a lipid monolayer labeled using lipid-associated dye was deposited on a silica substrate and studied in different environments. The contact with water of this lipid monolayer caused SFG signal to disappear, indicating a possible lipid molecular disorder and the formation of lipid bilayers or liposomes in water. TIRF was able to visualize the presence of lipid molecules on the substrate, showing that the lipids were not removed from the substrate surface by water. The integration of the two surface sensitive techniques can simultaneously visualize interfacial molecular dynamics and characterize interfacial molecular structures in situ, which is important and is expected to find extensive applications in biological interface related research.
Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful technique to image the chemical composition
of complex samples in biophysics, biology and materials science. CARS is a four-wave mixing process. The application
of a spectrally narrow pump beam and a spectrally wide Stokes beam excites multiple Raman transitions, which are
probed by a probe beam. This generates a coherent directional CARS signal with several orders of magnitude higher
intensity relative to spontaneous Raman scattering. Recent advances in the development of ultrafast lasers, as well as
photonic crystal fibers (PCF), enable multiplex CARS. In this study, we employed two scanning imaging methods. In
one, the detection is performed by a photo-multiplier tube (PMT) attached to the spectrometer. The acquisition of a
series of images, while tuning the wavelengths between images, allows for subsequent reconstruction of spectra at each
image point. The second method detects CARS spectrum in each point by a cooled coupled charged detector (CCD)
camera. Coupled with point-by-point scanning, it allows for a hyperspectral microscopic imaging. We applied this
CARS imaging system to study biological samples such as oocytes.
KEYWORDS: Digital holography, Holography, 3D metrology, 3D image processing, Holograms, Phase imaging, Microscopy, Phase shift keying, Diffraction, 3D image reconstruction
Digital holography records the superposition of the object and reference waves. The subsequent reconstruction of both
amplitude and phase of the optical field is done by numerically propagating the optical field along the direction
perpendicular to the hologram plane in accordance with the laws of diffraction. Phase changes undergone by a light
wave passing through or reflecting from objects can be converted to the optical thickness or height measurements,
providing the three dimensional structural information about the object. Our dual wavelength phase imaging method
allows three dimensional measurements of a wide variety of biological systems and microstructures.
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