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.

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.

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.

3D micro-devices for enhancing the lateral resolution in optical microscopy

Although optical microscopy is a widely used technique across various multidisciplinary fields for inspecting small-scale objects,surfaces or organisms,it faces a significant limitation:the lateral resolution of optical microscopes is fundamentally constrained by light diffraction.Dielectric micro-spheres,however,offer a promising solution to this issue as they are capable of significantly enhancing lateral resolution through extraordinary phenomena,such as a photonic nanojet.Building upon the potential of dielectric micro-spheres,this paper introduces a novel approach for fabricating 3D micro-devices designed to enhance lateral resolution in optical microscopy.The proposed 3D micro-device comprises a modified coverslip and a micro-sphere,facilitating easy handling and integration into any existing optical microscope.To manufacture the device,two advanced femtosecond laser techniques are employed:femtosecond laser ablation and multi-photon lithography.Femtosecond laser ablation was employed to create a micro-hole in the coverslip,which allows light to be focused through this aperture.Multi-photon lithography was used to fabricate a micro-sphere with a diameter of 20μm,along with a cantilever that positions the above the processed micro-hole and connect it with the coverslip.In this context,advanced processing strategies for multi-photon lithography to produce a micro-sphere with superior surface roughness and almost perfect geometry(λ/8)from a Zr-based hybrid photoresist are demonstrated.The performance of the micro-device was evaluated using Mirau-type coherence scanning interferometry in conjunction with white light illumination at a central wavelength of 600 nm and a calibration grid(Λ=0.28μm,h>50 nm).Here,the 3D micro-device proved to be capable of enhancing lateral resolution beyond the limits achievable with conventional lenses or microscope objectives when used in air.Simultaneously,it maintained the high axial resolution characteristic of Mirau-type coherence scanning interferometry.The results and optical properties of the micro-sphere were analyzed and further discussed through simulations.

When optical microscopy meets all-optical analog computing:A brief review

As a revolutionary observation tool in life science,biomedical,and material science,optical microscopy allows imaging of samples with high spatial resolution and a wide field of view.However,conventional microscopy methods are limited to single imaging and cannot accomplish real-time image processing.The edge detection,image enhancement and phase visualization schemes have attracted great interest with the rapid development of optical analog computing.The two main physical mechanisms that enable optical analog computing originate from two geometric phases:the spin-redirection Rytov-Vlasimirskii-Berry(RVB)phase and the Pancharatnam-Berry(PB)phase.Here,we review the basic principles and recent research progress of the RVB phase and PB phase based optical differentiators.Then we focus on the innovative and emerging applications of optical analog computing in microscopic imaging.Optical analog computing is accelerating the transformation of information processing from classical imaging to quantum techniques.Its intersection with optical microscopy opens opportunities for the development of versatile and compact optical microscopy systems.

Nonlinear optical imaging by detection with optical parametric amplification(invited paper)

Nonlinear optical imaging is a versatile tool that has been proven to be exceptionally useful in various research fields.However,due to the use of photomultiplier tubes(PMTs),the wide application of nonlinear optical imaging is limited by the incapability of imaging under am-bient light.In this paper,we propose and demonstrate a new optical imaging detection method based on optical parametric amplification(OPA).As a nonlinear optical process,OPA in-trinsically rejects ambient light photons by coherence gating.Periodical poled lithium niobate(PPLN)crystals are used in this study as the media for OPA.Compared to bulk nonlinear optical crystals,PPLN crystals support the generation of OPA signal with lower pump power.Therefore,this characteristic of PPLN crystals is particularly beneficial when using high-repetition-rate lasers,which facilitate high-speed optical signal detection,such as in spec-troscopy and imaging.A PPLN-based OPA system was built to amplify the emitted imaging signal from second harmonic generation(SHG)and coherent anti-Stokes Raman scattering(CARS)microscopy imaging,and the amplified optical signal was strong enough to be detected by a biased photodiode under ordinary room light conditions.With OPA detection,ambient-light-on SHG and CARS imaging becomes possible,and achieves a similar result as PMT detection under strictly dark environments.These results demonstrate that OPA can be used as a substitute for PMTs in nonlinear optical imaging to adapt it to various applications with complex.light ing conditions.

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.

Stimulated Raman scattering microscopy on biological cellular machinery

Beneting from the developments of advanced optical microscopy techniques,the mysteries of biological functions at the cellular and subcellular levels have been continuously revealed.Stimulated Raman scattering(SRS)microscopy is a rapidly growing technique that has attracted broad attentions and become a powerful tool for biology and biomedicine,largely thanks to its chemical specicity,high sensitivity and fast image speed.This review paper introduces the principles of SRS,discusses the technical developments and implementations of SRS microscopy,then highlights and summarizes its applications on biological cellular machinery andnally shares our visions of potential breakthroughs in the future.

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.

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.

Multi-prior physics-enhanced neural network enables pixel super-resolution and twin-imagefree phase retrieval from single-shot hologram

Digital in-line holographic microscopy(DIHM)is a widely used interference technique for real-time reconstruction of living cells’morphological information with large space-bandwidth product and compact setup.However,the need for a larger pixel size of detector to improve imaging photosensitivity,field-of-view,and signal-to-noise ratio often leads to the loss of sub-pixel information and limited pixel resolution.Additionally,the twin-image appearing in the reconstruction severely degrades the quality of the reconstructed image.The deep learning(DL)approach has emerged as a powerful tool for phase retrieval in DIHM,effectively addressing these challenges.However,most DL-based strategies are datadriven or end-to-end net approaches,suffering from excessive data dependency and limited generalization ability.Herein,a novel multi-prior physics-enhanced neural network with pixel super-resolution(MPPN-PSR)for phase retrieval of DIHM is proposed.It encapsulates the physical model prior,sparsity prior and deep image prior in an untrained deep neural network.The effectiveness and feasibility of MPPN-PSR are demonstrated by comparing it with other traditional and learning-based phase retrieval methods.With the capabilities of pixel super-resolution,twin-image elimination and high-throughput jointly from a single-shot intensity measurement,the proposed DIHM approach is expected to be widely adopted in biomedical workflow and industrial measurement.

Development of Morus alba Reinforced Poly-Lactic Acid with Elevated Mechanical and Thermal Properties

This research investigates the mechanical and thermal properties of Morus alba combined with polylactic acid in comparison with other natural fibers. The study uses three different fiber and PLA compositions - 20%, 30%, and 40% respectively - to produce composite materials. In addition, another composite with the same fiber volume is treated with a 4% NaOH solution to improve mechanical properties. The composites are processed by twin-screw extrusion, granulation, and injection molding. Tensile strength measurements of raw fibers and NaOH-treated fibers were carried out using a single-fiber tensile test with a gauge length of 40 mm. It was observed that the NaOH surface treatment increases the resistance against tensile loading and exhibited improved properties for raw fiber strands. The diameter of the fibers was measured using optical microscopy. During this research, flexural tests, impact tests, differential scanning calorimetry (DSC), and heat deflection temperature measurements (HDT) were conducted to evaluate the mechanical and thermal properties of the developed composite samples. The results indicate that the mechanical properties of NaOH-treated Morus alba-reinforced polylactic acid outperform both virgin PLA samples and untreated Morus alba samples.

Super-resolution imaging of low-contrast periodic nanoparticle arrays by microsphere-assisted microscopy

We use the label-free microsphere-assisted microscopy to image low-contrast hexagonally close-packed polystyrene nanoparticle arrays with diameters of 300 and 250 nm.When a nanoparticle array is directly placed on a glass slide,it cannot be distinguished.If a 30-nm-thick Ag film is deposited on the surface of a nanoparticle array,the nanoparticle array with nanoparticle diameters of 300 and 250 nm can be distinguished.In addition,the Talbot effect of the 300-nm-diameter nanoparticle array is also observed.If a nanoparticle sample is assembled on a glass slide deposited with a 30-nm-thick Ag film,an array of 300-nm-diameter nanoparticles can be discerned.We propose that in microsphere-assisted microscopy imaging,the resolution can be improved by the excitation of surface plasmon polaritons(SPPs) on the sample surface or at the sample/substrate interface,and a higher near-field intensity due to the excited SPPs would benefit the resolution improvement.Our study of label-free super-resolution imaging of low-contrast objects will promote the applications of microsphere-assisted microscopy in life sciences.

Multimodal label-free microscopy

This paper reviews the different multimodal applications based on a large ext ent of label-freeinaging modalities,ranging from linear to nonlinear optics,while also inchuding spectroscopicmeasurements.We put specific emphasis on multimodal measurements going across the usual boundaries between imaging modalities,whereas most multimodal platforms combine techniquesbased on similar light interactions or similar hardware implementations.In this review,we limitthe scope to focus on applications for biology such as live cells or tissues,since by their nat ure ofbeing alive or fragile,we are often not free to take liberties with the image acquisition times andare forced to gather the maximum amount of information possible at one time.For such samples,imaging by a given label-free method usually presents a challenge in obt aining suficient opticalsignal or is limited in terms of the types of observable targets.Multimodal imaging is thenparticularly attractive for these samples in order to maximize the amount of measured infor-mation.While multimodal imaging is always useful in the sense of acquiring additional infor-mation from additional modes,at times it is possible to attain information that could not bediscovered using any single mode alone,which is the essence of the progress that is possible usinga multimodal approach.

Applications of nanostructures in wide-field, label-free super resolution microscopy

Super resolution imaging capable of resolving details beyond the diffraction limit is highly desired in many scientific and application fields, including bio-medicine, nanomaterial science, and opto-electronic integration. Up to now, many different methods have been proposed, among which wide-field, label-free super resolution microscopy is indispensable due to its good applicability to diverse sample types, large field of view（FOV）, and high imaging speed. In recent years,nanostructures have made a crucial contribution to the wide-field, label-free subdiffraction microscopy, with various working mechanisms and configuration designs. This review summarizes the recent applications of the nanostructures in the wide-field, label-free super resolution microscopy, with the emphasis on the designs of hyperlens with hyperbolic dispersion, microsphere with ＂nano-jets＂, and nanowire ring illumination microscopy based on spatial frequency shift effect. The bottlenecks of the current techniques and possible solutions are also discussed.

Influence of the probe-sample interaction on scanning near-field optical microscopic images in the far field

We have studied the influence of probe-sample interaction in a scanning near-field optical microscopy （SNOM） in the far field by using samples with a step structure. For a sample with a step height of - λ/4, the SNOM image contrast between the two sides of the step changes periodically at different scan heights. For a step height of-λ/2, the image contrast remains approximately the same. The probe-sample interaction determines the SNOM image contrast here. The influence of different refractive indices of the sample has been also analysed by using a simple theoretical model.

ADVANCED OPTICAL TECHNIQUES TO EXPLORE BRAIN STRUCTURE AND FUNCTION

Understanding brain structure and function,and the complex relationships bet ween them,is one of the grand challenges of contemporary sciences.Thanks to their fexiblity,optical techniques could be the key to explore this complex network.In this manuscript,we briefly review recent adv ancements in optical methods applied to three main issues:anatomy,plasticity and func-tionality.We describe novel implement ations of light-sheet microscopy to resolve neuronal anat-omy in whole fixed brains with cellular resolution.Moving to liv ing samples,we show how real-time dynamics of brain rewiring can be visualized through two-photon microscopy with the spatial resolution of single synaptic contacts.The plasticity of the injured brain can also be dissected through cut ting edge optical methods that specifically ablate single neuronal processes.Finally,we report how nonlinear microscopy in combination with novel voltage sensitive dyes allow optical registrations of action potential across a population of neurons opening promising prospective in understanding brain functionality.The knowledge acquired from these complementary optical methods may provide a deeper comprehension of the brain and of its unique features.

Hematite, Biotite and Cinnabar on the Face of the Turin Shroud: Microscopy and SEM-EDX Analysis

The Turin Shroud, recently accessible for hands-on scientific research, is now extensively investigated. Its pinkish red blood stains that seem anomalous ones are studied by modern techniques (notably by resolute optical microscopy and scanning electron microscopy coupled with energy dispersive X-ray). Exploration by these techniques of a blood stain located on the face permits us to discover some red-colour particles (hematite, biotite and cinnabar) of exogenous material in this stain. We finally characterize these red-colour particles and try to explain their presences in the blood stain. Globally, all these red-colour particles cannot explain all of the reddish appearance of the area under study.

Super-resolution microscopy and its applications in neuroscience

Optical microscopy promises researchers to soe most tiny substances directly.However,the resolution of conventional microscopy is resticted by the diffraction limit.This makes it a challenge to observe subcellular processes happened in nanoscale.The development of super-resolution microscopy provides a solution to this challenge.Here,we briefly review several commonly used super-resolution techniques,explicating their basic principles and applications in biological science,especially in neuroscience.In addition,characteristics and limitations of each techrique are compared to provide a guidance for biologists to choose the most suitable tool.