A promising approach for quantifying focal stroke modeling and assessing stroke progression:optical resolution photoacoustic microscopy photothrombosis

To investigate the mechanisms underlying the onset and progression of ischemic stroke,some methods have been proposed that can simultaneously monitor and create embolisms in the animal cerebral cortex.However,these methods often require complex systems and the effect of age on cerebral embolism has not been adequately studied,although ischemic stroke is strongly age-related.In this study,we propose an optical-resolution photoacoustic microscopy-based visualized photothrombosis methodology to create and monitor ischemic stroke in mice simultaneously using a 532 nm pulsed laser.We observed the molding process in mice of different ages and presented age-dependent vascular embolism differentiation.Moreover,we integrated optical coherence tomography angiography to investigate age-associated trends in cerebrovascular variability following a stroke.Our imaging data and quantitative analyses underscore the differential cerebrovascular responses to stroke in mice of different ages,thereby highlighting the technique's potential for evaluating cerebrovascular health and unraveling age-related mechanisms involved in ischemic strokes.

Investigation of confocal microscopy for differentiation of renal cell carcinoma versus benign tissue.Can an optical biopsy be performed?

Objective:Novel optical imaging modalities are under development with the goal of obtaining an“optical biopsy”to efficiently provide pathologic details.One such modality is confocal microscopy which allows in situ visualization of cells within a layer of tissue and imaging of cellular-level structures.The goal of this study is to validate the ability of confocal microscopy to quickly and accurately differentiate between normal renal tissue and cancer.Methods:Specimens were obtained from patients who underwent robotic partial nephrectomy for renal mass.Samples of suspected normal and tumor tissue were extracted from the excised portion of the kidney and stained with acridine orange.The stained samples were imaged on a Nikon E600 C1 Confocal Microscope.The samples were then submitted for hematoxylin and eosin processing and read by an expert pathologist to provide a gold-standard diagnosis that can later be compared to the confocal images.Results:This study included 11 patients,17 tissue samples,and 118 confocal images.Of the 17 tissue samples,10 had a gold-standard diagnosis of cancer and seven were benign.Of 118 confocal images,66 had a gold-standard diagnosis of cancer and 52 were benign.Six confocal images were used as a training set to train eight observers.The observers were asked to rate the test images on a six point scale and the results were analyzed using a web based receiver operating characteristic curve calculator.The average accuracy,sensitivity,specificity,and area under the empirical receiver operating characteristic curve for this study were 91%,98%,81%,and 0.94 respectively.Conclusion:This preliminary study suggest that confocal microscopy can be used to distinguish cancer from normal tissue with high sensitivity and specificity.The observers in this study were trained quickly and on only six images.We expect even higher performance as observers become more familiar with the confocal images.

Confocal laser endomicroscopy as a new diagnostic tool for poorly differentiated gastric adenocarcinoma

Gastric cancer(GC)is a multifactorial disease,where both environmental and genetic features can have an impact on its occurrence and development.GC represents one of the leading causes of cancer-related deaths worldwide.GC is most frequent in males and is believed to arise from a series of premalignant lesions.The detection of GC at an early stage is crucial because early GC,which is an invasive stomach cancer confined to the mucosal or submucosal lining,may be curable with a reported 5-year survival rate of more than 90%.Advanced GC usually has a poor prognosis despite current treatment standards.The diagnostic efficacy of conventional endoscopy(with light endoscopy)is currently limited.Confocal laser endomicroscopy is a novel imaging technique that allows real-time in vivo histological examination of mucosal surfaces during endoscopy.Confocal laser endomicroscopy may be of great importance in the surveillance of precancerous gastric lesions and in the diagnosis of GC.In this editorial we commented on the article about this topic published by Lou et al in the recent issue of the World Journal of Clinical Cases.

In vivo evaluation of laser-induced choroidal neovascularization in rats simultaneously using optical coherence tomography and photoacoustic microscopy

Determination of the precise location and the degree of the Choroidal neovascularization(CNV)lesion is essential for diagnosation Neovascular age-related macular degeneration(AMD)and evaluation the efficacy of treatment.Noninvasive imaging techniques with specific contrast for CNV evaluation are demanded.In this paper,two noninvasive imaging techniques,namely Optical coherence tomography(OCT)and Photoacoustic microscopy(PAM),are combined to provide specific detection of CNV for their complimentary contrast mechanisms.In vivo time-serial evaluation of Laser-induced CNV in rats is present at days 1,3,5,7,14,21 after laser photocoagulation is applied to the rat fundus.Both OCT and PAM show that the CNV increases to its maximum at day 7 and decreases at day 14.Quantification of CNV area and CNV thickness is given.The dual-modal information of CNV is consistent with the histologic evaluation by hematoxylin and eosin(H&E)staining.

Organization and Ultra-Structural Components of Endothelial Surface Glycocalyx Revealed by Stochastic Optical Reconstruction Microscopy(STORM)

Introduction The endothelial cells(ECs)lining every blood vessel wall constantly expose to the mechanical forces generated by the blood flow.The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system.In addition to forming a transport barrier between the blood and vessel wall,vascular ECs play important roles in regulating circulation functions.Besides biochemical stimuli,blood flow induced(hemodynamic)mechanical stimuli,such as shear stress,pressure and circumferential stretch,modulate EC morphology and functions by activating mechanosensors,signaling pathways,and gene and protein expressions.The EC responses to the hemodynamic forces(mechano-sensing and transduction)are critical to maintaining normal vascular functions.Failure in the mechano-sensing and transduction leads to serious vascular diseases including hypertension,atherosclerosis,aneurysms and thrombosis,to name a few[1].On the luminal surface of our blood vessels,there is a thin layer called endothelial surface glycocalyx(ESG)which consists of proteoglycans,glycosaminoglycans(GAGs)and glycoproteins.The GAGs in the ESG are heparan sulfate(HS),hyaluronic acid(HA),chondroitin sulfate(CS),and sialic acid(SA)[2].In order to play important roles in vascular functions,such as being a mechanosensor and transducer for the endothelial cells(ECs)to sense the blood flow,a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall,the ESG should have an organized structure at the molecular level.Due to the limitations of optical and electron microscopy,the ultra-structure and organization of ESG has not been revealed until recent development of a super high resolution fluorescence optical microscope,STORM(Stochastic Optical Reconstruction Microscopy).The diffraction of a single fluorescence molecule can be described as the point spread function(PSF).When the light of wavelengthλexcites the fluorophore(emitter),the intensity profile of the spot is defined as the PSF with the width^0.6λ/NA,NA is the numerical aperture of the objective.The diffraction-limited image resolution,for a high numerical aperture objective lens,is^200 nm in the lateral direction and^500 nm in the axial direction,for a conventional fluorescence microscope.The key idea of the single-molecule localization microscopy is to light the molecule,in turn,to achieve the nanometer-level accuracy of their position and reconstruction into a super-resolution image,such as STORM.STORM employs photo-switching mechanisms to stochastically activate individual molecules(photo-switchable or photoactivatable fluorophores)within the diffraction-limited region at different times.Then images with sub-diffraction limit resolution are reconstructed from the measured positions of individual fluorophores[3].To trade the super spatial resolution(accuracy),STORM sacrifices its temporal resolution(efficiency)by switching the state and sequentially exciting the emitters at a high density.Rust et al[3]employed organic dyes and fluorescent proteins as photo-switchable emitters to trade temporal resolution for a super spatial resolution(~20 nm lateral and^50 nm axial at present,can go down to a couple of nanometers if using smaller peptides or antibody fragments instead of currently used whole anti-bodies),which is an order of magnitude higher than conventional confocal microscopy.In the current study,we employed STORM to reveal the major ultra-structural components of the ESG,HS and HA,and their organization at the surface of the cultured EC monolayer[4].Materials and methods We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC(bEnd3,mouse brain microvascular endothelial cells)monolayers.After confluency,the bEnd3 cells were immunolabeled with anti-HS,fol-lowed by an ATT0488 conjugated goat anti-mouse IgG,and with biotinylated HA binding protein,followed by an AF647 conjugated anti-biotin.The ESG was then imaged by the STORM with a 100x/1.49 oil immersed lens.Multiple Reporters of ATT0488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA.The field of 256×256(40×40μm2)of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame.Results HA is a long molecule weaving into a network which covers the endothelial luminal surface.The diameter of the HA segments is 185.3±44.7 nm,155.5±57.2 nm,and 156.9±56.1 nm,respectively,at the top,middle and bottom regions of the cell luminal surface.In contrast,HS is a shorter molecule,perpendicular to the cell surface.HA and HS are partially overlapped with each other at the endothelial luminal surface.We quantified the length,diameter,orientation,and density of HS at the top,middle and bottom regions of the endothelial surface.The diameter of the observed HS is 191.0±46.0 nm,284.3±71.1 nm,and 184.2±59.6 nm,and the length of the HS is 621.0±75.7 nm,651.0±118.0 nm,and 575.2±105.6 nm,respectively,at the top,middle and bottom regions of the cell luminal surface.For the HS orientation,its angle with the cell surface is 92.9±1.9,88.7±8.2,and 96.2±10.9 degree,respectively,at the top,middle and bottom regions.The angle of 90 degree is perfectly perpendicular to the cell surface.For the HS distribution,the average density is0.398 elements/μm2,0.345 elements/μm2 and 0.665 elements/μm2,respectively,and the distance between the adjacent HS is 1 694.4±628.1 nm,1 844.8±758.5 nm,and 1 221.9±450.7 nm,respectively,at the top,middle and bottom regions.Conclusions Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve,due to their organization,ultra-structure and distribution.

High-speed all-optic optical coherence tomography and photoacoustic microscopy dual-modal system for microcirculation evaluation

We propose a high-speed all-optic dual-modal system that integrates spectral domain optical coherence tomography and photoacoustic microscopy(PAM).A 3*3 coupler-based interfer-ometer is used to remotely detect the surface vibration caused by photoacoustic(PA)waves.Three outputs of the interferometer are acquired simultaneously with a multi-channel data ac-quisition card.One channel data with the highest PA signal detection sensitivity is selected for sensitivity compensation.Experiment on the phantom demonstrates that the proposed method can sucessfully compensate for the loss of intensity caused by sensitivity variation.The imaging speed of the PAM is improved compared to our previous system.The total time to image a sample with 256×256 pixels is~20s.Using the proposed system,the microvasculature in the mouse auricle is visualized and the blood flow state is accessed.

Intravital imaging of adriamycin-induced renal pathology using two-photon microscopy and optical coherence tomography

Adriamycin(doxorubicin),a common cancer chemotherapeutic drug,can be used to induce a model of chronic progressive glomerular disease in rodents.In our studies,we evahuated renal changes in a rat model after Adriamydin injection using two photon microscopy(TPM),optical coherence tomography(OCT)and Doppler OCT(DOCT).Taking advantage of deep penetra-tion and fast scanning speed for three dimensional(3D)label-free imaging,OCT/DOCT system was able to reveal glomerular and tubular pathology noninvasively and in real time.By imaging renal pathology following the infusion of fAuorophore-labeled dextrans of different molecular weights,TPM can provide direct views of glomerular and tubular flow dynamics with the onset and progression of renal disease.Specifically,glomerular permeability and filtration,proximal and distal tubular flow dynamics can be revealed.6-8 weeks after injection of Adriamycin,TPM and OCT/DOCT imaging revealed glomerular sclerosis,compromised flow across the glomerular wall,tubular atrophy,tubular dilation,and variable intra-tubular flow dynamics.Our results indicate that TPM and OCT/DOCT provide real-time imaging of renal pathology in vivo that has not been previously available using conventional microscopic procedures.

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.

Deep-learning-based motion-correction algorithm in optical resolution photoacoustic microscopy

In this study,we propose a deep-learning-based method to correct motion artifacts in optical resolution photoacoustic microscopy(OR-PAM).The method is a convolutional neural network that establishes an end-to-end map from input raw data with motion artifacts to output corrected images.First,we performed simulation studies to evaluate the feasibility and effectiveness of the proposed method.Second,we employed this method to process images of rat brain vessels with multiple motion artifacts to evaluate its performance for in vivo applications.The results demonstrate that this method works well for both large blood vessels and capillary networks.In comparison with traditional methods,the proposed method in this study can be easily modified to satisfy different scenarios of motion corrections in OR-PAM by revising the training sets.

Latest advances in soft X-ray spectromicroscopy at SSRF

The BL08U1 A beamline is established as a sophisticated platform at Shanghai Synchrotron Radiation Facility(SSRF), taking advantage of its high spatial resolution(< 30 nm) and high energy resolving power(>10 000),for studying properties of solid, liquid, gas, film and other forms of materials at sub-micron scale. In this paper,we present a review on newly implemented techniques, such as total electron yield(TEY), dual energy contrast imaging, nano-CT, soft X-ray excited optical luminance(SXEOL), and coherent diffraction imaging(CDI)under development. Several research cases in nanomaterials, environmental science and biology are presented to demonstrate capabilities of the beamline.

A SIMULTANEOUS MULTI-PROBE DETECTION LABEL-FREE OPTICAL-RESOLUTION PHOTOACOUSTIC MICROSCOPY TECHNIQUE BASED ON MICROCAVITY TRANSDUCER

We demonstrate the feasibility of simultancous multi-probe detection for an optcal-resolution photoacoustic microscopy(OR-PAM)system.OR-P AM has elicited the attention of biomedical imaging researchers because of its optical absorption contrast and high spatial resolution with great imaging depth.OR-PAM allows label-free and noninvasive imaging by maximizing the optical absorption of endogenous biomolecules.However,given the inadequate absoption of some biomolcules,detection sensitivity at the same incident intensity requires improvement.In this study,a modulated continuous wave with power density less than 3mW/cm^(2)(1/4 of the ANSI safety limit)excited the weak photoacoustic(PA)signals of biological cells.A microcavity traneducer is developed based on the bulk modulus of gas five orders of magnitude lower than that of solid;air pressure variation is inversely proportional to cavity volume at the same temperature increase.Considering that a PA wave expands in various directions,detecting PA signals from different positions and adding them together can increase detection sensitivity and signal-to-noise ratio.Therefore,we employ four detectors to acquire tiny PA signals simul-taneously.Experimental results show that the developed OR-PAM system allows the label-free imaging of cells with weak optical absorption.

Optical polarization response at gold nanosheet edges probed by scanning near-field optical microscopy

Optical properties of metallic edge-like structures known as knife-edges are a topic of interest and possess potential applications in enhanced Raman scattering, optical trapping, etc. In this work, we investigate the near-field optical polar- ization response at the edge of a triangular gold nanosheet, which is synthesized by a wet chemical method. A homemade scanning near-field optical microscope （SNOM） in collection mode is adopted, which is able to accurately locate its probe at the edge during experiments. An uncoated straight fiber probe is used in the SNOM, because it s611 preserves the prop- erty of light polarization though it has the depolarization to some extent. By comparing near-field intensities at the edge and glass substrate, detected in different polarization directions of incident light, the edge-induced depolarization is found, which is supported by the finite differential time domain （FDTD） simulated results. The depolarized phenomenon in the near-field is similar to that in the far-field.

New method for fast morphological characterization of organic polycrystalline films by polarized optical microscopy

A new method to visualize the large-scale crystal grain morphology of organic polycrystalline films is proposed. First,optical anisotropic transmittance images of polycrystalline zinc phthalocyanine（Zn Pc） films vacuum deposited by weak epitaxial growth（WEG） method were acquired with polarized optical microscopy（POM）. Then morphology properties including crystal grain size, distribution, relative orientation, and crystallinity were derived from these images by fitting with a transition dipole model. At last, atomic force microscopy（AFM） imaging was carried out to confirm the fitting and serve as absolute references. This method can be readily generalized to other organic polycrystalline films, thus providing an efficient way to access the large-scale morphologic properties of organic polycrystalline films, which may prove to be useful in industry as a film quality monitoring method.

Optical and atomic force microscopic study on step bunching in BaB_2O_4 crystal growth

The formation of macrostep during high-temperature phase of barium meta-borate （^-BaB204） single crystal growth has been investigated by both optical in-situ observation system and atomic force microscopy （AFM）. The in- si$u observation results demonstrate that the critical linear size of growing facet exceeding the size that the macrostep generates is significantly anisotropic. The critical linear sizes are around 280 ~m and 620 ~m for {1010} and （T010} planes, respectively. AFM study illustrates that macrostep train with a height of 150 nm^200 nm is one typical morphological feature of the as-grown crystal surface. The riser of each macrostep consists of several straight and parallel sub-steps, indicating the occurrence of step bunching. Additionally, triangular sub-steps with heights of several nanometers on the treads of the macrosteps are found to be another typical feature of surface morphology, which implies a microscopically competitive bunching of sub-steps between various crystallographic orientations.

Advanced optical microscopy methods for in vivo imaging of sub-cellular structures in thick biological tissuesl

Optical microscopy has become an indispensable tool for visualizing sub-cellular structures andbiological processes.However,scattering in biological tissues is a major obstacle that preventshigh-resolution images from being obtained from deep regions of tissue.We review commontechniques,such as multiphoton microscopy(MPM)and optical coherence microscopy(OCM),for diffraction limited imaging beyond an imaging depth of 0.5 mm.Novel implementations havebeen emerging in recent years giving higher imaging speed,deeper penetration,and better imagequality.Focal modulation microscopy(FMM)is a novel method that combines confocal spatialfltering with focal modulation to reject out-of-focus background.FMM has demonstrated animaging depth comparable to those of MPM and OCM,near-real-time image acquisition,and thecapability for multiple contrast mechanisms.

Microscopic Study of Defect Luminescence between 0.72 - 0.85 eV by Optical Microscopy

In this contribution, an experimental setup to investigate the defect luminescence between 0.72 - 0.85 eV of single defects in Silicon by optical microscopy is introduced. For this purpose, an optical microscope is equipped with an InGaAs CCD detector and a longpass filter with a cut-off wavelength at 1450 nm in order to filter out the band-to-band luminescence at around 1.1 eV. Grain boundaries showing homogeneous distributed defect luminescence can be localized at a μm-scale.

A microvascular image analysis method for optical-resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy(OR-PAM)has been shown to be an excellent tool for high-resolution imaging of microvasculature,and quantitative analysis of the microvascula-ture can provide valuable information for the early diagnosis and treatment of various vascular-related diseases.In order to address the characteristics of weak signals,discontinuity and small diameters in photoacoustic microvascular images,we propose a method adaptive to the micro-vascular segmentation in photoacoustic images,including Hessian matrix enhancement and the morphological connection operators.The accuracy of our vascular segmentation method is quantitatively evaluated by the multiple criteria.To obtain more precise and continuous mi-crovascular skeletons,an improved skeleton extraction framework based on the multistencil fast marching(MSFM)method is developed.We carried out in vivo OR-PAM microvascular imaging in mouse ears and subcutaneous hepatoma tumor model to verify the correctness and superiority of our proposed method.Compared with the previous methods,our proposed method can extract the microvascular network more completely,continuously and accurately,and provide an ef-fective solution for the quantitative analysis of photoacoustic microvascular images with many small branches.

Internal Defect Measurement of Scattering Media by Optical Coherence Microscopy

Optical coherence microscopy is applied to measure scattering media'sinternal defect, which based on low coherence interferometry and confocal microscopy. Opticalcoherence microscopy is more effective in the rejection of out of focus and multiple scatteredphotons originating further away of the focal plane. With the three-dimension scanning, the internaldefect is detected by measuring the thickness of different points on the sample. The axialresolution is 6 μm and lateral resolution is 1. 2 μm. This method is possessed of the advantagesover the other measurement method of scattering media, such as non-destruction and high-resolution.

SDOCT IMAGE RECONSTRUCTION BY INTERFEROMETRIC SYNTHETIC APERTURE MICROSCOPY

Spectral domain optical coherence tomography(SDOCT)is a noninvasive,cross-sectional imaging technique that measures depth resolved reflectance of tissue by Fourier transforming the spectral interferogram with the scanning of the reference avoided.Interferometric synthetic aperture microscopy(ISAM)is an optical microscopy computed-imaging technique for measuring the optical properties of biological tissues,which can overcome the compromise between depth of focus and transverse resolution.This paper describes the principle of SDOCT and ISAM,which multiplexes raw acquisitions to provide quantitatively meaningful data with reliable spatially invariant resolution at all depths.A mathematical model for a coherent microscope with a planar scanning geometry and spectral detection was described.The two-dimensional fast Fourier transform(FFT)of spectral data in the transverse directions was calculated.Then the nonuniform ISAM resampling and filtering was implemented to yield the scattering potential within the scalar model.Inverse FFT was used to obtain the ISAM reconstruction.One scatterer,multiple scatterers,and noisy simulations were implemented by use of ISAM to catch spatially invariant resolution.ISAM images were compared to those obtained using standard optical coherence tomography(OCT)methods.The high quality of the results validates the rationality of the founded model and that diffraction limited resolution can be achieved outside the focal plane.

Missed Opportunities for Diagnosing Bacilliferous Pulmonary Tuberculosis by Optical Microscopy versus GeneXpert MTB/RIF in Endemic Areas

Objective: To assess the missed opportunities from the diagnosis of bacilliferous pulmonary tuberculosis by optical microscopy compared to GeneXpert MTB/RIF between 2015 and 2019. Methods: This is a retrospective analysis of the diagnostic results of bacilliferous pulmonary tuberculosis in patients suspected of pulmonary tuberculosis at their first episode during the period. GeneXpert MTB/RIF (GeneXpert) and optical microscopy (OM) after Ziehl-Neelsen stained smear were performed on each patient’s sputum or gastric tubing fluid sample. Results: Among 341 patients suspected of pulmonary tuberculosis, 229 patients were declared bacilliferous tuberculosis by the two tests (67%), 220 patients by GeneXpert and 95 patients by OM, i.e. 64.5% versus 28% (p i.e. 58.5% of the positive cases detected by the two tests (134/229 patients) and 39.3% of the patients suspected of tuberculosis (134/341 patients). On the other hand, among 95 patients declared positive by OM, the GeneXpert ignored 9 (9.5%), i.e. 4% of all the positive cases detected by the two diagnostic tests (9/229 patients) and 3% of the patients suspected of tuberculosis (9/341 patients). The differences observed between the results of the two tests were statistically significant at the 5% threshold (p Conclusion: This study reveals missed diagnostic opportunities for bacilliferous pulmonary mycobacteriosis, statistically significant with optical microscopy than GeneXpert. The GeneXpert/optical microscopy couple could be a good contribution to the strategies for the elimination of pulmonary tuberculosis in sub-Saharan Africa.