Fluorescence Microscopy System Research Articles

A novel variational approach for multiphoton microscopy image restoration: from PSF estimation to 3D deconvolution

In multi-photon microscopy (MPM), a recent in-vivo fluorescence microscopy system, the task of image restoration can be decomposed into two interlinked inverse problems: firstly, the characterization of the point spread function (PSF) and subsequently, the deconvolution (i.e. deblurring) to remove the PSF effect, and reduce noise. The acquired MPM image quality is critically affected by PSF blurring and intense noise. The PSF in MPM is highly spread in 3D and is not well characterized, presenting high variability with respect to the observed objects. This makes the restoration of MPM images challenging. Common PSF estimation methods in fluorescence microscopy, including MPM, involve capturing images of sub-resolution beads, followed by quantifying the resulting ellipsoidal 3D spot. In this work, we revisit this approach, coping with its inherent limitations in terms of accuracy and practicality. We estimate the PSF from the observation of relatively large beads (approximately 1 μm in diameter). This goes through the formulation and resolution of an original non-convex minimization problem, for which we propose a proximal alternating method along with convergence guarantees. Following the PSF estimation step, we then introduce an innovative strategy to deal with the high level multiplicative noise degrading the acquisitions. We rely on a heteroscedastic noise model for which we estimate the parameters. We then solve a constrained optimization problem to restore the image, accounting for the estimated PSF and noise, while allowing a minimal hyper-parameter tuning. Theoretical guarantees are given for the restoration algorithm. These algorithmic contributions lead to an end-to-end pipeline for 3D image restoration in MPM, that we share as a publicly available Python software. We demonstrate its effectiveness through several experiments on both simulated and real data.

Ultrabright quantum dots assisted in vivo NIR-II fluorescence microscopic imaging for brain metastases in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive and often fatal disease, especially since the brain metastasis of TNBC has been a particularly severe manifestation. However, brain metastasis in TNBC at early stages often lacks noticeable symptoms, making it challenging to detect. Near-infrared II (NIR-II) fluorescence microscopic imaging obtains long wavelength, which enables reduced scattering, high spatial resolution and minimal autofluorescence, it is also a favorable imaging method for tumor diagnosis. PbS@CdS quantum dots (QDs) are one of the popular NIR-II fluorescence nanoprobes for well brightness. In this study, NIR-II emissive PbS@CdS QDs were utilized and further encapsulated with thiol-terminated poly(ethylene oxide) (SH-PEG, MW = 5000) to form PbS@CdS@PEG QDs nanoparticles (NPs). The obtained PbS@CdS@PEG QDs NPs were then characterized and further studied in detail. The PbS@CdS@PEG QDs NPs had large absorption spectra, exhibited strong NIR-II fluorescence emission at approximately 1300[Formula: see text]nm, and possessed good NIR-II fluorescence properties. Then, the mice model of early-stage brain metastases of TNBC was established, and the PbS@CdS@PEG QDs NPs were injected into the tumor-bearing mice for NIR-II fluorescence microscopic bioimaging. The brain vessels and tumors of the living mice were detected with high spatial resolution under the NIR-II fluorescence microscopic imaging system with irradiation of 808[Formula: see text]nm laser. The tumor tissues were further restricted and prepared as thin slices. The NIR-II fluorescence signals were collected from the tumor slices with high spatial resolution and signal-to-background ratio (SBR). Thus, the PbS@CdS@PEG QDs NPs-assisted NIR-II fluorescence microscopic system can effectively achieve targeting brain metastases of TNBC imaging, offering a novel and promising approach for TNBC-specific diagnosis.

Polarization-encoded 3D structured light and multifocal spot arrays generation based on metasurface

Fluorescence microscopy possesses the advantages of high resolution, high sensitivity, molecular specificity and noninvasiveness, providing an important tool in life science research. The multifocal array and 3D structured light are two kinds of important light fields that are often used in scanning fluorescence microscopy systems and wide-field fluorescence microscopy systems. However, traditional methods for generating multifocal arrays and 3D structured light illumination rely on various bulk optical components, making it challenging to achieve compact optical systems. Besides, generating these two types of illumination typically requires two separate and independent optical systems, hindering the integration of different types of fluorescence microscopy systems. Here, a dielectric metasurface is proposed that can achieve the switching between multifocal arrays and 3D structured light through polarization state modulation, greatly simplifying the illumination optics of fluorescence microscopy systems and facilitating the integration of different types of fluorescence microscopy systems.

Development and evaluation of an active motion compensation-based optical imaging system for living tissue

Imaging the surgical margin in vivo at microscopic level can help surgeons make a rapid pathological diagnosis intraoperatively. However, the motion of living tissue induced by respiration or heartbeat remains the critical challenge for the intraoperative microscopic imaging system. In this study, we propose an active motion compensation (AMC) based fluorescence microscopic system to reduce the effect of tissue movement, for which the emitted fluorescent signals are not only to construct the microscopic images, but also to measure the motion of living tissue. To validate the proposed method, an experimental prototype was developed. Then, using this prototype, a series of experiments in vitro and in vivo as well as simulation studies have been conducted for evaluation and validation purposes. In this paper, the proposed AMC-based microscopic imaging system might advance the intraoperative pathological diagnosis technologies.

B-110 Validation of Digital and Automated anti-dsDNA IFA Interpretations Using the dIFine Automated Fluorescent Microscope System

Abstract Background Detection of anti-double stranded DNA antibodies (anti-dsDNA) by the indirect fluorescent antibody (IFA) assay is a well-established tool for aiding in the diagnosis of systemic lupus erythematosus (SLE). ZEUS Scientific has manufactured an FDA-cleared anti-dsDNA IFA test system for decades, which relies upon manual interpretation of qualitative results using a standard fluorescent microscope. This same anti-dsDNA IFA test system was recently utilized to train software, in conjunction with a specialized fluorescent microscope (collectively termed dIFine), towards automating qualitative result interpretation. The objective of this study was to validate the reproducibility, sensitivity, and specificity of the ZEUS anti-dsDNA IFA test system, in conjunction with the dIFine automated microscope. Methods Multi-site reproducibility study–2 low-positive, 2 mid-positive, 2 high-positive, and 2 negative serum samples were assembled to create a reproducibility panel. The sample identities of the panel members were scrambled prior to testing. The panel was assayed via the ZEUS anti-dsDNA-IFA test system at a 1:10 screening dilution, in triplicate, twice per day, on five days, at three different laboratories (including ZEUS Scientific). At each laboratory, the processed slides were interpreted manually by two operators, then scanned by dIFine to generate digital images of the wells, as well as an automated qualitative result interpretation. The digital images of each slide well were also interpreted by two operators, resulting in 3 independent interpretations per sample, for each operator and instrument (Manual, Digital, Automated). The number of slide wells processed for interpretation at each laboratory was: 8 samples × 30 replicates each = 240 total. Clinical study–300 serum samples derived from donors previously diagnosed with SLE were assembled. A control cohort consisting of 360 samples from donors previously diagnosed with other rheumatic and non-rheumatic diseases was also assembled. The sample identities were scrambled for both cohorts prior to testing. The 660 samples were assayed at a 1:10 screening dilution using the ZEUS anti-dsDNA IFA test system at ZEUS Scientific. Manual, digital, and automated interpretations were carried out at ZEUS Scientific as described above. Results Reproducibility (Actual/Expected): [Site 1 - Operator A - Manual (240/240), Digital (240/240); Site 1 - Operator B - Manual (240/240), Digital (240/240); Site 1 - Automated - (231/240)]. [Site 2 - Operator A - Manual (240/240), Digital (240/240); [Site 2 - Operator B - Manual (240/240), Digital (240/240); Site 2 - Automated - (236/240)]. [Site 3 - Operator A - Manual (240/240), Digital (238/240); Site 3 - Operator B - Manual (240/240), Digital (238/240); Site 3 - Automated - (231/240)]. Sensitivity for SLE cohort (Positive/Total, %): [Operator A - Manual, (79/300, 26.3%), Digital (79/300, 26.3%)]; [Operator B - Manual, (79/300, 26.3%), Digital (79/300, 26.3%), Automated (81/300, 27.0%)]. Specificity for control cohort (Negative/Total): [Operator A - Manual, (358/360, 99.4%), Digital (358/360, 99.4%)]; [Operator B - Manual, (358/360, 99.4%), Digital (358/360, 99.4%), Automated (358/360, 99.4%)]. Conclusions These validation studies demonstrate that the ZEUS anti-dsDNA-IFA test system, in conjunction with the automated dIFine microscope, yields highly reproducible and accurate results derived from digital and automated interpretations.

Wavefront coding image reconstruction via physical prior and frequency attention.

Wavefront coding (WFC) is an effective technique for extending the depth-of-field of imaging systems, including optical encoding and digital decoding. We applied physical prior information and frequency domain model to the wavefront decoding, proposing a reconstruction method by a generative model. Specifically, we rebuild the baseline inspired by the transformer and propose three modules, including the point spread function (PSF) attention layer, multi-feature fusion block, and frequency domain self-attention block. These models are used for end-to-end learning to extract PSF feature information, fuse it into the image features, and further re-normalize the image feature information, respectively. To verify the validity, in the encoding part, we use the genetic algorithm to design a phase mask in a large field-of-view fluorescence microscope system to generate the encoded images. And the experimental results after wavefront decoding show that our method effectively reduces noise, artifacts, and blur. Therefore, we provide a deep-learning wavefront decoding model, which improves reconstruction image quality while considering the large depth-of-field (DOF) of a large field-of-view system, with good potential in detecting digital polymerase chain reaction (dPCR) and biological images.

Recent developments in photoresponse-based zinc (II) sensors and biomaterials/biomedical applications

The photoresponse-based zinc (II) chemosensors play important roles in several physiological functions in living systems. Due to the unique benefits of photoluminescence imaging, which include high sensitivity and non-invasiveness. It has developed into a potent instrument for the real-time monitoring of Zn2+ trafficking, distribution, and absorption. They have been focused on Zn2+ imaging via photoluminescence in biological systems, which have demonstrated the role of Zn2+ variations in physiological developments. The localization of labile Zn2+ pools as well as the temporal-spatial distribution of labile Zn2+. It has been successful improvements in photoluminescence techniques including fluorescence microscopy and luminescence optical imaging systems.

Development of soft X-ray ptychography and fluorescence microscopy system using total-reflection wolter mirror and application to measurement of drug-treated mammalian cells

We developed a soft X-ray microscopic imaging system, CARROT, that combines ptychography and fluorescence X-ray microscopy using a total-reflection Wolter mirror illumination system. The system offers advantages such as achromaticity and long working distances, enabling seamless application to a wide wavelength range. We demonstrated high-resolution imaging of cellular structures and sub-micrometer-scale changes induced by drug administration in mammalian cells. We further showcased the capability to seamlessly switch from ptychography measurements to X-ray fluorescence measurements at different wavelengths to map elemental distributions within cells. Our findings suggest that this system can be used for investigating the correlation between intracellular structures and elemental distribution, as well as for multimodal measurements of visible light fluorescence, Raman scattering, and soft X-rays to provide valuable insights into understanding intracellular dynamics.

Membrane stretch due to hypoosmolality leads to the development of various pulmonary and cardiovascular diseases

High blood pressures over a long period of time can lead to the development of diseases such as congestive heart failure(CHF). Congestive heart failure also commonly leads to the development of conditions such as hyponatremia, a common electrolyte disorder which increases the body’s water levels, creating hypoosmotic conditions for cells; these hypoosmotic conditions cause cell swelling, which subsequently causes mechanical stress on the cells. In these hypoosmotic environments, specific mechanosensitive ion channels which are activated by these mechanical stresses on cells may play a critical role in the progression of conditions resulting from these hypoosmotic environments, such as pulmonary arterial hypertension (PAH) and CHF. This study aims to look at the connection between the effects of hypoosmotic conditions in cells on the pathogenesis of the development of chronic diseases such as PAH and CHF. Mechanical actions on specific mechanosensitive ion channels such as Piezo1 have already been shown to play a critical role in regulating intracellular Ca2+homeostasis, which play an important part in the regulation of the vascular tone in places such as the pulmonary arteries; mechanical stimulation from hypotonic environments such as those seen in hyponatremia lead to Piezo1-mediated increases of intracellular Ca2+, which are subsequently linked to the increased contraction and proliferation of pulmonary arterial endothelial cells(PAECs). The proliferation of PAECs contribute to the pathogenesis of diseases, such as pulmonary arterial hypertension(PAH) or CHF, which there is currently no cure for. Patch-clamp experiments were conducted to examine the activity of mechanosensitive cation currents; graphed results showed that the activity of mechanosensitive cation currents were significantly increased (p<0.05) in PAECs treated with a hypoosmotic solution when compared with PAECs treated in an isosmotic control solution. Digital fluorescence microscopy using fura-2 AM, a membrane-permeable Ca2+ sensitive fluorescent indicator, and a Nikon digital imaging fluorescent microscopy system was also used to measure intracellular Ca2+ concentration in PAECs; graphed data indicated that the change in intracellular calcium concentration in PAECs was significantly increased(p<0.05) when placed in a hypoosmotic solution, than when placed in an isosmotic solution. The collective data indicates that hypoosmotic conditions increase the activity of mechanosensitive ion channels, as well as intracellular Ca2+ levels, which may prove the role of mechanosensitive ion channels like Piezo1 and TRPC6 in the development of diseases such as PAH and CHF. This work is supported in part by the National Heart, Lung, and Blood Institute of the National Institutes of Health Grants HL135807 and HL146764. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we have developed a cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 110 nm enough for the subsequent cryo-lamella fabrication. We have successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

Computational based time-resolved multispectral fluorescence microscopy

Multispectral imaging and time-resolved imaging are two common acquisition schemes in fluorescence microscopy, and their combination can be beneficial to increase specificity. The multidimensionality of the dataset (space, time, and spectrum) introduces some challenges, such as the acquisition of big datasets and long measurement times. In this work, we present a time-resolved multispectral fluorescence microscopy system with a short measurement time, achieved by exploiting Compressive Sensing (CS) based on the Single-Pixel Camera (SPC) scheme. Data Fusion (DF) with a high-resolution camera allows us to tackle the problem of low spatial resolution, typical of SPC. The combined use of SPC, CS, and DF, in which hardware and algorithms are integrated, represents a computational imaging framework to reduce the number of measurements while preserving the information content. This approach has been exploited to demonstrate a zoom feature without moving the optical system. We describe and characterize the system in terms of spatial, spectral, and temporal properties, along with validation on a cellular sample.

Snapshot temporal compressive light-sheet fluorescence microscopy via deep denoising and total variation priors.

We present a snapshot temporal compressive light-sheet fluorescence microscopy system to capture high-speed microscopic scenes with a low-speed camera. A deep denoising network and total variation denoiser are incorporated into a plug-and-play framework to quickly reconstruct 20 high-speed video frames from a short-time measurement. Specifically, we can observe 1,000-frames-per-second (fps) microscopic scenes when the camera works at 50 fps to capture the measurement. The proposed method can potentially be applied to observe cell and tissue motions in thick living biological specimens.

Multifaceted mirror array illuminator for fluorescence excitation-scanning spectral imaging microscopy.

Hyperspectral imaging (HSI) technologies offer great potential in fluorescence microscopy for multiplexed imaging, autofluorescence removal, and analysis of autofluorescent molecules. However, there are also associated trade-offs when implementing HSI in fluorescence microscopy systems, such as decreased acquisition speed, resolution, or field-of-view due to the need to acquire spectral information in addition to spatial information. The vast majority of HSI fluorescence microscopy systems provide spectral discrimination by filtering or dispersing the fluorescence emission, which may result in loss of emitted fluorescence signal due to optical filters, dispersive optics, or supporting optics, such as slits and collimators. Technologies that scan the fluorescence excitation spectrum may offer an approach to mitigate some of these trade-offs by decreasing the complexity of the emission light path. We describe the development of an optical technique for hyperspectral imaging fluorescence excitation-scanning (HIFEX) on a microscope system. The approach is based on the design of an array of wavelength-dependent light emitting diodes (LEDs) and a unique beam combining system that uses a multifurcated mirror. The system was modeled and optimized using optical ray trace simulations, and a prototype was built and coupled to an inverted microscope platform. The prototype system was calibrated, and initial feasibility testing was performed by imaging multilabel slide preparations. We present results from optical ray trace simulations, prototyping, calibration, and feasibility testing of the system. Results indicate that the system can discriminate between at least six fluorescent labels and autofluorescence and that the approach can provide decreased wavelength switching times, in comparison with mechanically tuned filters. We anticipate that LED-based HIFEX microscopy may provide improved performance for time-dependent and photosensitive assays.

Combining off-axis digital holography with fluorescence for simultaneous fluorescence and quantitative phase imaging.

Off-axis digital holographic microscopy (DHM) is a microscopy technique capable of extracting and translating quantitative phase information (QPI) from the sample into 4D topographical data, which enables the study of phenotypic changes with sub-second temporal resolution. DHM has been widely implemented for long-term live-cell imaging and microelectromechanical systems characterization. However, as a bright field technique, relying on DHM imaging alone can be challenging especially in characterizing sub-cellular structures. Here we report on the design and implementation of a DHM module that can be retrofitted into a commercial dual-deck Olympus IX83 microscope base, transforming the inverted microscope into a hybrid DHM and fluorescence microscopy (DHM-FM) system. In order to achieve simultaneous acquisition of DHM and fluorescence data, the DHM module is designed to integrate illumination, dichroic filter, and interference submodules. The condenser of the microscope was replaced by the DHM illumination module, and the dichroic module was placed in the microscope platform to separate the DHM beam from the fluorescence signal. Finally, the DHM signal was delivered to the interference module outside the microscope body. As the DHM and fluorescence subsystems share the same field of view, this platform can acquire complementary data simultaneously. The capabilities of this coupled platform were explored through a series of pilot studies, including examining phase condensate formation and cellular dynamics. This modular configuration will help others explore the potential of off-axis DHM in their own work and, in particular, the potential of coupled DHM-fluorescence imaging.

Whole-mouse clearing and imaging at the cellular level with vDISCO.

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

An optofluidic platform for interrogating chemosensory behavior and brainwide neural representation in larval zebrafish

Studying chemosensory processing desires precise chemical cue presentation, behavioral response monitoring, and large-scale neuronal activity recording. Here we present Fish-on-Chips, a set of optofluidic tools for highly-controlled chemical delivery while simultaneously imaging behavioral outputs and whole-brain neuronal activities at cellular resolution in larval zebrafish. These include a fluidics-based swimming arena and an integrated microfluidics-light sheet fluorescence microscopy (µfluidics-LSFM) system, both of which utilize laminar fluid flows to achieve spatiotemporally precise chemical cue presentation. To demonstrate the strengths of the platform, we used the navigation arena to reveal binasal input-dependent behavioral strategies that larval zebrafish adopt to evade cadaverine, a death-associated odor. The µfluidics-LSFM system enables sequential presentation of odor stimuli to individual or both nasal cavities separated by only ~100 µm. This allowed us to uncover brainwide neural representations of cadaverine sensing and binasal input summation in the vertebrate model. Fish-on-Chips is readily generalizable and will empower the investigation of neural coding in the chemical senses.

Easy and accessible way to calibrate a fluorescence microscope and to create a microplastic identification key

We present here a technique for setting up detection limits on any fluorescent microscope in conjunction with the fluorophore Nile Red for microplastic identification. Our method also describes a rigorous morphology-specific identification key for microplastics to reduce subjectivity between researchers. The detection limits were established for nine common polymer types and five natural substrates which could result in false-positive signals when using Nile Red for microplastic identification. This method was then applied to real freshwater samples and identified particles were validated with micro-FTIR or Raman spectroscopy. This approach may reduce subjectivity in microplastic identification and counting and enhances transparency, repeatability and harmonization within microplastic research community.•Instructions for calibration of detection limits for microplastics on fluorescence microscope systems described•Microplastic identification key developed and tested to reduce false positive detection•Lower subjectivity for microplastic identification obtained using the detection limits & identification key

AI-boosted CRISPR-Cas13a and total internal reflection fluorescence microscopy system for SARS-CoV-2 detection

Integrating artificial intelligence with SARS-CoV-2 diagnostics can help in the timely execution of pandemic control and monitoring plans. To improve the efficiency of the diagnostic process, this study aims to classify fluorescent images via traditional machine learning and deep learning-based transfer learning. A previous study reported a CRISPR-Cas13a system combined with total internal reflection fluorescence microscopy (TIRFM) to detect the existence and concentrations of SARS-CoV-2 by fluorescent images. However, the lack of professional software and excessive manual labor hinder the practicability of the system. Here, we construct a fluorescent image dataset and develop an AI-boosted CRISPR-Cas13a and total internal reflection fluorescence microscopy system for the rapid diagnosis of SARS-CoV-2. Our study proposes Fluorescent Images Classification Transfer learning based on DenseNet-121 (FICTransDense), an approach that uses TIRF images (before and after sample introduction, respectively) for preprocessing, including outlier exclusion and setting and division preprocessing (i.e., SDP). Classification results indicate that the FICTransDense and Decision Tree algorithms outperform other approaches on the SDP dataset. Most of the algorithms benefit from the proposed SDP technique in terms of Accuracy, Recall, F1 Score, and Precision. The use of AI-boosted CRISPR-Cas13a and TIRFM systems facilitates rapid monitoring and diagnosis of SARS-CoV-2.

Evaluation of MetaSystems Automated Fluorescent Microscopy System for the Machine-Assisted Detection of Acid-Fast Bacilli in Clinical Samples.

Manual reading of fluorescent acid-fast bacilli (AFB) microscopy slides is time-intensive and technically demanding. The aim of this study was to evaluate the accuracy of MetaSystems' automated fluorescent AFB slide scanner and analyzer. Auramine O-stained slides corresponding to 133 culture-positive and 363 culture-negative respiratory (n = 284), tissue (n = 120), body fluid (n = 81), and other (n = 11) sources were evaluated with the MetaSystems Mycobacteria Scanner running the NEON Metafer AFB Module. The sensitivity and specificity of the MetaSystems platform was measured as a standalone diagnostic and as an assistant to technologists to review positive images. Culture results were used as the reference method. The MetaSystems platform failed to scan 57 (11.5%) slides. The MetaSystems platform used as a standalone had a sensitivity of 97.0% (129/133; 95% CI 92.5 to 99.2) and specificity of 12.7% (46/363; 95% CI 9.4 to 16.5). When positive scans were used to assist technologists, the MetaSystems platform had a sensitivity of 70.7% (94/133; 95% CI 62.2 to 78.3) and specificity of 89.0% (323/363; 95% CI 85.3 to 92.0). The manual microscopy method had a sensitivity of 79.7% (106/133; 95% CI 71.9 to 86.2) and specificity of 98.6% (358/363; 95% CI 96.8 to 99.6). The sensitivity of the MetaSystems platform was not impacted by smear grade or mycobacterial species. The majority (70.3%) of false positive smears had ≥2+ smear results with the MetaSystems platform. Further performance improvements are needed before the MetaSystems' automated fluorescent AFB slide reader can be used to assist microscopist in the clinical laboratory.

Large-depth three-photon fluorescence microscopy imaging of cortical microvasculature on nonhuman primates with bright AIE probe In vivo

Multiphoton microscopy has been a powerful tool in brain research, three-photon fluorescence microscopy is increasingly becoming an emerging technique for neurological research of the cortex in depth. Nonhuman primates play important roles in the study of brain science because of their neural and vascular similarity to humans. However, there are few research results of three-photon fluorescence microscopy on the brain of nonhuman primates due to the lack of optimized imaging systems and excellent fluorescent probes. Here we introduced a bright aggregation-induced emission (AIE) probe with excellent three-photon fluorescence efficiency as well as facile synthesis process and we validated its biocompatibility in the macaque monkey. We achieved a large-depth vascular imaging of approximately 1 mm in the cerebral cortex of macaque monkey with our lab-modified three-photon fluorescence microscopy system and the AIE probe. Functional measurement of blood velocity in deep cortex capillaries was also performed. Furthermore, the comparison of cortical deep vascular structure parameters across species was presented on the monkey and mouse cortex. This work is the first in vivo three-photon fluorescence microscopic imaging research on the macaque monkey cortex reaching the imaging depth of ∼1 mm with the bright AIE probe. The results demonstrate the potential of three-photon microscopy as primate-compatible method for imaging fine vascular networks and will advance our understanding of vascular function in normal and disease in humans.