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.

Chitosan hydrogel encapsulated with LL-37 peptide promotes deep tissue injury healing in a mouse model

Background:LL-37 peptide is a member of the human cathelicidin family,and has been shown to promote the healing of pressure ulcers.However,the low stability of this peptide within the wound environment limits its clinical use.Chitosan(CS)hydrogel is commonly used as a base material for wound dressing material.Methods:CS hydrogel(2.5%w/v)was encapsulated with LL-37.Cytotoxicity of the product was examined in cultured NIH3 T3 fibroblasts.Effects on immune response was examined by measuring tumor necrosis factor-α(TNF-α)release from RAW 264.7 macrophages upon exposure to lipopolysaccharides.Antibacterial activity was assessed using Staphylococcus aureus.Potential effect on pressure ulcers was examined using a mouse model.Briefly,adult male C57 BL/6 mice were subjected to skin pressure using magnets under a 12/12 h schedule for 21 days.Mice were randomized to receive naked LL-37(20μg),chitosan gel containing 20μg LL-37(LL-37/CS hydrogel)or hydrogel alone under the ulcer bed(n=6).A group of mice receiving no intervention was also included as a control.Results:LL-37/CS hydrogel did not affect NIH3 T3 cell viability.At a concentration of 1–5μg/ml,LL-37/CS inhibited TNF-αrelease from macrophage.At 5μg/ml,LL-37/CS inhibited the growth of Staphylococcus aureus.The area of the pressure ulcers was significantly lower in mice receiving LL-37/CS hydrogel in comparison to all other 3 groups on days 11(84.24%±0.25%),13(56.22%±3.91%)and 15(48.12%±0.28%).Histological examination on days 15 and 21 showed increased epithelial thickness and density of newly-formed capillary with naked LL-37 and more so with LL-37/CS.The expression of key macromolecules in the process of angiogenesis(i.e.,hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor-A(VEGF-A))in wound tissue was increased at both the mRNA and protein levels.Conclusion:Chitosan hydrogel encapsulated with LL-37 is biocompatible and could promote the healing of pressure ulcers.

Multidither coherent optical adaptive technique for deep tissue two-photon microscopy

Two-photon microscopy normally suffers from the scattering of the tissue in biological imaging.Multidither coberent optical adaptive technique(COAT)can correct the scattered wavefront in parallel.However,the determination of the corrective phases may not be completely accurate using conventional method,which undermines the performance of this technique.In this paper,we theoretically demonstrate a method that can obtain more accurate corrective phases by determining the phase values from the square root of the fuorescence signal.A numnerical simulation model is established to study the performance of adaptive optics in two-photon micros-copy by combining scalar diffraction theory with vector diffraction theory.The results show that the distortion of the wavefront can be corrected more thoroughly with our method in two-photon imaging.In our simulation,with the scattering from a 450-mn-thick mouse brain tissue,excitation focal spots with higher peak-to background ratio(PBR)and images with higher contrast can be obtained.Hence,further enhancement of the multidither COAT correction performance in two-photon imaging can be expected.

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.

Recent advances in ultrasound-controlled fluorescence technology for deep tissue optical imaging

Fluorescence imaging is a noninvasive and dynamic real-time imaging technique;however,it exhibits poor spatial resolution in centimeter-deep tissues because biological tissues are highly scattering media for optical radiation.The recently developed ultrasound-controlled fluorescence(UCF)imaging is a novel imaging technique that can overcome this bottleneck.Previous studies suggest that the effective contrast agent and sensitive imaging system are the two pivotal factors for generating high-resolution UCF images ex vivo and/or in vivo.Here,this review highlights the recent advances(2015e2020)in the design and synthesis of contrast agents and the improvement of imaging systems to realize high-resolution UCF imaging of deep tissues.The imaging performances of various UCF systems,including the signal-to-noise ratio,imaging resolution,and imaging depth,are specifically discussed.In addition,the challenges and prospects are highlighted.With continuously increasing research interest in this field and emerging multidisciplinary applications,UCF imaging with higher spatial resolution and larger imaging depth may be developed shortly,which is expected to have a far-reaching impact on disease surveillance and/or therapy.

NON-INVASIVE MEASUREMENT OF DEEP TISSUE TEMPERATURE CHANGES CAUSED BY APOPTOSIS DURING BREAST CANCER NEOADJUVANT CHEMOTHERAPY:A CASE STUDY

Treatment-induced apoptosis of cancer cells is one goal of cancer therapy.Interestingly,more heat is generated by mitochondria during apoptosis,especially the uncoupled apoptotic state,^(1,2) compared to the resting state.In this case study,we explore these thermal effects by longitudinally measuring temperature variations in a breast lesion of a pathological complete responder during neoadjuvant chemotherapy(NAC).Diffuse Optical Spectroscopic Imaging(DOSI)was employed to derive absolute deep tissue temperature using subtle spectral features of the water peak at 975 nm.^(3)A significant temperature increase was observed in time windows during the anthracycline and cyclophosphamide(AC)regimen but not in the paclitaxel and bevacizumab regimen.Hemoglobin concentration changes generally did not follow temperature,suggesting the measured temperature increases were likely due to mitochondrial uncoupling rather than a direct vascular effect.A simultaneous increase of tissue oxygen saturation with temperature was observed,suggesting that oxidative stress also contributes to apoptosis.Although preliminary,this study indicates longitudinal DOSI tissue temperature monitoring provides information that can improve our understanding of the mechanisms of tissue response during NAC.

Deep tissue imaging by enhanced photon collection

We have developed a two-photon fluorescence microscope capable of imaging up to 4mm inturbid media with micron resolution.The key feature of this instrument is the innovative de-tector,capable of collecting emission photons from a wider surface area of the sample thandetectors in traditional two-photon microscopes.This detection scheme is extremely efficient inthe collection of emitted photons scattered by turbid media which allows eight fold increase in theimaging depth when compared with conventional two-photon microscopes.Furthermore,thissystem also has in-depth fluorescence lifetime imaging microscopy(FLiM)imaging capabilitywhich increases image contrast.The detection scheme captures emission light in a transmissionconfiguration,making it extremely efficient for the detection of second harmonic generation(SHG)signals,which is generally forward propagating.Here we present imaging experiments oftissue phantoms and in vivo and ea vivo biological tissue performed with this microscope.

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.

Near-infrared-II deep tissue fluorescence microscopy and application

Fluorescence imaging has become an essential tool in biomedical research.However,non-invasive deep-tissue threedimensional optical in vivo imaging with the high spatiotemporal resolution is challenging due to the interaction between photons and tissues.Beam shaping has been used to tailor microscopy techniques to enhance microscope performance.The nearinfrared window(NIR)between 700 and 1,700 nm,generally emphasized as the NIR-II(1,000–1,700 nm)window,has been developed into a promising bio-optical solution chosen as the lower interaction effect in this spectrum,showing potential in basic biological research and clinical application.In this review,we summarize the existing methods to increase penetration depth and extensively describe biological microscopy techniques,NIR-II spectral windows,and fluorophores.Strategies to improve bioimaging performance and NIR-II imaging applications are introduced.Based on the current research achievements,we elucidate the main challenges and provide some recommendations and prospects for deep tissue penetration fluorescence for future biomedical applications.

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.

Early Detection Methods of Deep Tissue Pressure Injuries:A Systematic Review

Deep tissue pressure injuries(DTPIs)have witnessed a growing prevalence in hospitals and other health care units especially among individuals with pathological conditions that give rise to restricted mobility,impaired sensation,and reduced tissue tolerance.The etiology of DTPIs has been a subject of controversy,to which several explanatory models have been proposed,including direct mechanical insult,ischemia-reperfusion,lymphatic occlusion,and inflammatory cytokines.In line with these pathophysiological scenarios,ultrasound,subepidermal moisture detection,and biomarker technologies have been proposed as potential early detection methods of DTPIs.This paper provides a systematic review involving these three methods.The conclusion is that combining and implementing these methods at different time periods during DTPIs development and progression respectively is likely to be the most universal,effective and promising way for DTPIs diagnosis.

Up-converting NaYF4:0.1%Tm^(3+), 20%Yb^(3+) nanoparticles as luminescent labels for deep-tissue optical imaging

Optical imaging plays an important role in biomedical research being extremely useful for early detection, screening and image-guided therapy. Lanthanide-doped up-converting nanoparticles were ideally suited for bioimaging because they could be ex- cited in near infrared （NIR） and emit in NIR or visible （VIS）. Here, we compared lanthanide doped up-converting NaYF4 and organic fluorophores for application in deep-tissue imaging. For that purpose - tissue phantoms mimicking the natural properties of light scat- tering by living tissues were prepared. The studies allowed to quantitatively compare optical resolution of different fluorescent com- pounds, revealing that the NIR photoexcitation was favorable over conventional UV photoexcitation.

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.

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.

High-intensity focused ultrasound with large scale spherical phased array for the ablation of deep tumors

Under some circumstances surgical resection is feasible in a low percentage for the treatment of deep tumors. Nevertheless, high-intensity focused ultrasound （HIFU） is beginning to offer a potential noninvasive alternative to conventional therapies for the treatment of deep tumors. In our previous study, a large scale spherical HIFU-phased array was developed to ablate deep tumors. In the current study, taking into account the required focal depth and maximum acoustic power output, 90 identical circular PZT-8 elements （diameter=1.4 cm and frequency=l MHz） were mounted on a spherical shell with a radius of curvature of 18 cm and a diameter of 21 cm. With the developed array, computer simulations and ex vivo experiments were carried out. The simulation results theoretically demonstrate the ability of the array to focus and steer in the specified volume （a 2 cm×2 cm×3 cm volume） at the focal depth of 15 to 18 cm. Ex vivo experiment results also verify the capability of the developed array to ablate deep target tissue by either moving single focal point or generating multiple foci simultaneously.

dECM restores macrophage immune homeostasis and alleviates iron overload to promote DTPI healing

Due to its highly insidious and rapid progression,deep tissue pressure injury(DTPI)is a clinical challenge.Our previous study found that DTPI may be a skeletal muscle injury dominated by macrophage immune dysfunction due to excessive iron accu-mulation.Decellularized extracellular matrix(dECM)hydrogel promotes skeletal muscle injury repair.However,its role in po-larizing macrophages and regulating iron metabolism in DTPI remains unclear.Here,porcine dECM hydrogel was prepared,and its therapeutic function and mechanism in repairing DTPI were investigated.The stimulus of dECM hydrogel toward RAW264.7 cells resulted in a significantly higher percentage of CD2o6+macrophages and notably decreased intracellular divalent iron levels.In mice DTPI model,dECM hydrogel treatment promoted M1 to M2 macrophage conversion,improved iron metabolism and reduced oxidative stress in the early stage of DTPI.In the remodeling phase,the dECM hydrogel remarkably enhanced revascularization and accelerated skeletal muscle repair.Furthermore,the immunomodulation of dEcM hydrogels in vivo was mainly involved in the P13k/Akt signaling pathway,as revealed by GO and KEGG pathway analysis,which may ameliorate the iron deposition and promote the healing of DTPI.Our findings indicate that dECM hydrogel is promising in skeletal muscle repair,inflammation resolution and tissue injury healing by effectively restoring macrophage immune homeostasis and normalizing iron metabolism.

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.

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.

Extracts of Portulaca oleracea promote wound healing by enhancing angiology regeneration and inhibiting iron accumulation in mice

Objective:To investigate the role of Portulaca oleracea(POL)in promoting revascularization and reepithelization as well as inhibiting iron aggregation and inflammation of deep tissue pressure injury(DTPI).Methods:The hydroalcoholic extract of POL(P)and aqueous phase fraction of POL(PD)were prepared based on maceration and liquid–liquid extraction.The number of new blood vessels and VEGF-A expression level were assessed using H&E stain and Western blot on injured muscle to examine the role of POL different extracts in vascularization.The iron distribution and total elemental iron of injured muscle were detected using laser ablation inductively coupled plasma mass spectrometry(ICP-MS)and Perls’staining to determine whether POL extracts can inhibit the iron accumulation.Besides,the ability of POL extracts to promote wound healing by combining re-epithelization time,inflammation degree and collagen deposition area were comprehensively evaluated.Results:In vitro,we observed a significant increase in HUVEC cell viability,migration rate and the number of the tube after P and PD treatment(P<0.05).In vivo,administration of P and PD impacted vascularization and iron accumulation on injured tissue,evident from more new blood vessels,higher expression of VEGF-A and decreased muscle iron concentration of treatment groups compared with no-treatment groups(P<0.05).Besides,shorter re-epithelization time,reduced inflammatory infiltration and distinct collagen deposition were associated with administration of P and PD(P<0.05).Conclusion:POL extract administration groups have high-quality wound healing,which is associated with increased new blood vessels,collagen deposition and re-epithelization,along with decreased iron accumulation and inflammatory infiltration.Our results suggest that that POL extract is beneficial to repair injured muscle after ischemia–reperfusion,highlighting the potential of POL in the DTPI treatment.

Ce doped polyaniline nanoparticles for absorption and photoacoustic imaging response to GSH in vitro and in vivo

Glutathione(GSH)is an important biological thiol in cells,which is involved in many physiological processes in the organism and regulates pathological processes of cells.Rapid and accurate monitoring of GSH in vitro and in vivo is quite needed in investigating important biochemical events.In this contribution,innovative cerium(Ce)doped polyaniline(Ce–Fe@PANI NPs)were prepared via Fe(III)induced oxidization polymerization method.Upon addition of GSH,the absorption of Ce–Fe@PANI NPs red shifted from the visible to the NIR region,con-firming the excellent absorption response to GSH.Moreover,Ce–Fe@PANI NPs exhibited excellent photoacoustic(PA)imaging enhancement in tube and shifted the PA intensity peak from 680 nm to 820 nm upon addition of GSH.In vitro and in vivo experiment verified that Ce–Fe@PANI NPs can monitor GSH in deep tissues via PA imaging technology.Collectively,this research provides Ce–Fe@PANI NPs would serve as a powerful nano-platform to realize PA imaging detection of GSH in vitro and in vivo.