Stimulated Raman scattering microscopy with phase-controlled light focusing and aberration correction for rapid and label-free, volumetric deep tissue imaging

We report a novel stimulated Raman scattering(SRS)microscopy technique featuring phase-controlled light focusing and aberration corrections for rapid,deep tissue 3D chemical imaging with subcellular resolution.To accomplish phasecontrolled SRS(PC-SRS),we utilize a single spatial light modulator to electronically tune the axial positioning of both the shortened-length Bessel pump and the focused Gaussian Stokes beams,enabling z-scanning-free optical sectioning in the sample.By incorporating Zernike polynomials into the phase patterns,we simultaneously correct the system aberrations at two separate wavelengths(~240 nm difference),achieving a~3-fold enhancement in signal-to-noise ratio over the uncorrected imaging system.PC-SRS provides>2-fold improvement in imaging depth in various samples(e.g.,polystyrene bead phantoms,porcine brain tissue)as well as achieves SRS 3D imaging speed of~13 Hz per volume for real-time monitoring of Brownian motion of polymer beads in water,superior to conventional point-scanning SRS 3D imaging.We further utilize PC-SRS to observe the metabolic activities of the entire tumor liver in living zebrafish in cellsilent region,unraveling the upregulated metabolism in liver tumor compared to normal liver.This work shows that PCSRS provides unprecedented insights into morpho-chemistry,metabolic and dynamic functioning of live cells and tissue in real-time at the subcellular level.

Confocal rescan structured illumination microscopy for real-time deep tissue imaging with superresolution

Structured illumination microscopy(SIM)is an established optical superresolution imaging technique.However,conventional SIM based on wide-field image acquisition is generally limited to visualizing thin cellular samples.We propose combining one-dimensional image rescan and structured illumination in the orthogonal direction to achieve superresolution without the need to rotate the illumination pattern.The image acquisition speed is consequently improved threefold,which is also beneficial for minimizing photobleaching and phototoxicity.Optical sectioning in thick biological tissue is enhanced by including a confocal slit in the system to significantly suppress the out-of-focus background and the associated noise.With all the technical improvements,our method captures threedimensional superresolved image stacks of neuronal structures in mouse brain tissue samples for a depth range of more than 200μm.

Stimulated Raman scattering tomography for rapid three-dimensional chemical imaging of cells and tissue

Three-dimensional(3D)imaging is essential for understanding intricate biological and biomedical systems,yet live cell and tissue imaging applications still face challenges due to constrained imaging speed and strong scattering in turbid media.Here,we present a unique phase-modulated stimulated Raman scattering tomography(PM-SRST)technique to achieve rapid label-free 3D chemical imaging in cells and tissue.To accomplish PM-SRST,we utilize a spatial light modulator to electronically manipulate the focused Stokes beam along the needle Bessel pump beam for SRS tomography without the need for mechanical z scanning.We demonstrate the rapid 3D imaging capability of PM-SRST by real-time monitoring of 3D Brownian motion of polystyrene beads in water with 8.5 Hz volume rate,as well as the instant biochemical responses to acetic acid stimulants in MCF-7 cells.Further,combining the Bessel pump beam with a longer wavelength Stokes beam(NIR-II window)provides a superior scattering resilient ability in PM-SRST,enabling rapid tomography in deeper tissue areas.The PM-SRST technique providestwofold enhancement in imaging depth in highly scattering media(e.g.,polymer beads phantom and biotissue like porcine skin and brain tissue)compared with conventional point-scan SRS.We also demonstrate the rapid 3D imaging ability of PM-SRST by observing the dynamic diffusion and uptake processes of deuterium oxide molecules into plant roots.The rapid PM-SRST developed can be used to facilitate label-free 3D chemical imaging of metabolic activities and functional dynamic processes of drug delivery and therapeutics in live cells and tissue.

Injectable and thermosensitive hydrogels mediating a universal macromolecular contrast agent with radiopacity for noninvasive imaging of deep tissues

It is very challenging to visualize implantable medical devices made of biodegradable polymers in deep tissues.Herein,we designed a novel macromolecular contrast agent with ultrahigh radiopacity(iodinate content>50%)via polymerizing an iodinated trimethylene carbonate monomer into the two ends of poly(ethylene glycol)(PEG).A set of thermosensitive and biodegradable polyester-PEG-polyester triblock copolymers with varied polyester compositions synthesized by us,which were soluble in water at room temperature and could spontaneously form hydrogels at body temperature,were selected as the demonstration materials.The addition of macromolecular contrast agent did not obviously compromise the injectability and thermogelation properties of polymeric hydrogels,but conferred them with excellent X-ray opacity,enabling visualization of the hydrogels at clinically relevant depths through X-ray fluoroscopy or Micro-CT.In a mouse model,the 3D morphology of the radiopaque hydrogels after injection into different target sites was visible using Micro-CT imaging,and their injection volume could be accurately obtained.Furthermore,the subcutaneous degradation process of a radiopaque hydrogel could be non-invasively monitored in a real-time and quantitative manner.In particular,the corrected degradation curve based on Micro-CT imaging well matched with the degradation profile of virgin polymer hydrogel determined by the gravimetric method.These findings indicate that the macromolecular contrast agent has good universality for the construction of various radiopaque polymer hydrogels,and can nondestructively trace and quantify their degradation in vivo.Meanwhile,the present methodology developed by us affords a platform technology for deep tissue imaging of polymeric materials.

Temperature-sensitive polymeric nanogels encapsulating withβ-cyclodextrin and ICG complex for high-resolution deep-tissue ultrasound-switchable fluorescence imaging

One of the thorny problems currently impeding the applications of the fluorescence imaging technique is the poor spatial resolution in deep tissue.Ultrasound-switchable fluorescence(USF)imaging is a novel imaging tool that has recently been explored to possibly surmount the above-mentioned bottleneck.Herein,αβ-cyclodextrin/indocyanine green(ICG)complex-encapsulated poly(N-isopropylacrylamide)(PNIPAM)nanogel was synthesized and studied for ex vivo/in vivo deep tissue/high-resolution near infrared USF(NIR-USF)imaging.To be specific,our results revealed that the average diameter of the as-prepared nanogels was significantly decreased to-32 nm from-335 nm compared to the reported ICG-PNIPAM nanoparticles.Additionally,the excitation/emission characteristics of the ICG itself in present nanogels were almost completely retained,and the resultant nanogel exhibited high physiological stability and positive biocompatibility.In particular,the signal-to-noise ratio of the USF image for the PNIPAM/P-cyclodextrin/ICG nanogel(33.01±2.42 dB)was prominently higher than that of the ICG-PNIPAM nanoparticles(18.73±0.33 dB)in 1.5-cm-thick chicken breast tissues.The NIR-USF imaging in 3.5-cm-thick chicken breast tissues was achieved using this new probe.The e x v iv o NIR-USF imaging of the mouse liver was also successfully obtained.Animal experiments showed that the present nanogels were able to be effectively accumulated into U87 tumor-bearing mice via enhanced permeability and retention effects,and the high-resolution NIR-USF imaging of in v ivo tumor was efficiently acquired.The metabolism and in vivo biodistribution of the nanogels were evaluated.Overall,the results suggest that the current nanogel is a highly promising NIR-USF probe for deep tissue and high-resolution USF imaging.

Reconstruction of 3D light distribution produced by cylindrical diffuser in deep tissues based on photoacoustic imaging

Measurement of light distribution in biological tissue contributes to selecting strategy and optimizing dose for biomedical application. In this letter, a photoacoustic method combined with Monte Carlo simulation was used to estimate the three-dimensional light distribution in biological tissue. The light distribution was produced by a cylindrical diffuser which interposed into tissues. The light profiles obtained by the method were compared to those detected by photo diodes. The experimental results demonstrate the feasibility of this method. The approach can play a significant role for photo-dosimetry in biomedical phototherapy.

In vivo tumor ultrasound-switchable fluorescence imaging via intravenous injections of size-controlled thermosensitive nanoparticles

Near-infrared fluorescence imaging has emerged as a noninvasive,inexpensive,and ionizing-radiation-free monitoring tool for assessing tumor growth and treatment efficacy.In particular,ultrasound switchable fluorescence(USF)imaging has been explored with improved imaging sensitivity and spatial resolution in centimeter-deep tissues.This study achieved the size control of polymer-based and indocyanine green(ICG)encapsulated USF contrast agents,capable of accumulating in the tumor after intravenous injections.These nanoprobes varied in size from 58 to 321 nm.The bioimaging profiles demonstrated that the proposed nanoparticles can efficiently eliminate the background light from normal tissue and show a tumor-specific fluorescence enhancement in the BxPC-3 tumor-bearing mice models possibly via the enhanced permeability and retention effect.In vivo tumor USF imaging further demonstrated that these nanoprobes can effectively be switched“ON”with enhanced fluorescence in response to a focused ultrasound stimulation in the tumor microenvironment,contributing to the high-resolution USF images.Therefore,our findings suggest that ICG-encapsulated nanoparticles are good candidates for USF imaging of tumors in live animals,indicating their great potential in optical tumor imaging in deep tissue.

Optical Brain Imaging： A Powerful Tool for Neuroscience

As the control center of organisms, the brain remains little understood due to its complexity. Taking advantage of imaging methods, scientists have found an accessible approach to unraveling the mystery of neuroscience. Among these methods, optical imaging techniques are widely used due to their high molecular specificity and single-molecule sensitivity. Here, we overview several optical imaging techniques in neuroscience of recent years, including brain clearing, the micro-optical sectioning tomography system, and deep tissue imaging.