Low Magnification Research Articles

CDK19 as a diagnostic marker for high-grade prostatic intraepithelial neoplasia

High-grade prostatic intraepithelial neoplasia (HGPIN) is a facultative precursor lesion of prostate cancer (PCa). Multifocal HGPIN in needle biopsies in the absence of PCa indicates a higher risk of cancer detection in subsequent biopsies. Therefore, a reliable diagnosis of HGPIN is of high clinical relevance guiding the management of patients with cancer-negative biopsies. Detection of HGPIN is merely based on morphological features while biomarkers aiding in the diagnosis of HGPIN and its differentiation from benign glands and other glandular lesions are lacking yet. Here, we investigated the expression of cyclin-dependent kinase 19 (CDK19) by immunohistochemistry on prostate needle biopsies of 140 patients who were all diagnosed with PCa using whole-tissue sectionsand compared CDK19 levels between HGPIN, PCa, and adjacent benign glands. In addition, CDK19 was compared with AMACR expression in a subset of intraductal carcinomas (IDCs) on radical prostatectomy (RP) specimens. HGPIN was present in 65.7% of biopsies and in 88% associated to adjacent PCa. CDK19 overexpression defined as moderate to high CDK19 expression visible at low magnification was found in 82.6% of HGPIN. In contrast, 89.3% of benign glands were CDK19-negative or demonstrated only low CDK19 expression highlighting a high sensitivity and specificity to accurately detect HGPIN based on CDK19 expression levels. CDK19 was overexpressed in 59% of PCa but did not correlate significantly with the expression of intermingled HGPIN. On RP, CDK19 and AMACR showed no significant difference in the detection rate of IDC. In summary, assessment of CDK19 facilitates accurate and simplified diagnosis of HGPIN with high sensitivity and specificity and aides the differentiation to non-neoplastic glandular alterations. Considering the high clinical significance of diagnosis HGPIN that still has a limited reproducibility among pathologists, we suggest CDK19 as diagnostic biomarker for HGPIN.

Prediction of BRCA Gene Mutation in Breast Cancer Based on Deep Learning and Histopathology Images.

BackgroundBreast cancer is one of the most common cancers and the leading cause of death from cancer among women worldwide. The genetic predisposition to breast cancer may be associated with a mutation in particular genes such as gene BRCA1/2. Patients who carry a germline pathogenic mutation in BRCA1/2 genes have a significantly increased risk of developing breast cancer and might benefit from targeted therapy. However, genetic testing is time consuming and costly. This study aims to predict the risk of gBRCA mutation by using the whole-slide pathology features of breast cancer H&E stains and the patients’ gBRCA mutation status.MethodsIn this study, we trained a deep convolutional neural network (CNN) of ResNet on whole-slide images (WSIs) to predict the gBRCA mutation in breast cancer. Since the dimensions are too large for slide-based training, we divided WSI into smaller tiles with the original resolution. The tile-based classification was then combined by adding the positive classification result to generate the combined slide-based accuracy. Models were trained based on the annotated tumor location and gBRCA mutation status labeled by a designated breast cancer pathologist. Four models were trained on tiles cropped at 5×, 10×, 20×, and 40× magnification, assuming that low magnification and high magnification may provide different levels of information for classification.ResultsA trained model was validated through an external dataset that contains 17 mutants and 47 wilds. In the external validation dataset, AUCs (95% CI) of DL models that used 40×, 20×, 10×, and 5× magnification tiles among all cases were 0.766 (0.763–0.769), 0.763 (0.758–0.769), 0.750 (0.738–0.761), and 0.551 (0.526–0.575), respectively, while the corresponding magnification slides among all cases were 0.774 (0.642–0.905), 0.804 (0.676–0.931), 0.828 (0.691–0.966), and 0.635 (0.471–0.798), respectively. The study also identified the influence of histological grade to the accuracy of the prediction.ConclusionIn this paper, the combination of pathology and molecular omics was used to establish the gBRCA mutation risk prediction model, revealing the correlation between the whole-slide histopathological images and gRCA mutation risk. The results indicated that the prediction accuracy is likely to improve as the training data expand. The findings demonstrated that deep CNNs could be used to assist pathologists in the detection of gene mutation in breast cancer.

DeepBatch: A hybrid deep learning model for interpretable diagnosis of breast cancer in whole-slide images

The gold standard for breast cancer diagnosis, treatment, and management is the histological analysis of a suspected section. Histopathology consists in analyzing the characteristics of the lesions using tissue sections stained with hematoxylin and eosin. However, pathologists are currently subjected to high workloads, mainly due to the fundamental role of histological analysis in the patient’s treatment. In this context, methods able to reduce histological analysis time, provide a second opinion, or even point out suspicious locations as a screening tool become increasingly important for pathologists. This article proposes a model based on Convolutional Neural Networks (CNN) to provide a refined and multiclass segmentation of Whole Slide Imaging (WSI) for breast cancer. The methodology is divided into four modules: pre-processing, ROI detection, ROI sampling, and region segmentation. These modules are organized to decode the information learned using CNNs in interpretable predictions for pathologists. The preprocessing module is responsible for removing background and noise from WSI. At ROI detection, we use the U-Net convolutional architecture to identify suspicious regions in low magnification WSI. The sampling module maps the identified suspected areas from low magnifications to 40× magnifications. region segmentation module segments high-magnification areas using a ResNet50/U-Net. To validate the methodology, we use data sets from different sources that can be used together or separately in each module, depending on its purpose. We used 205 breast cancer WSI for training, validation, and testing. For the detection of suspicious regions by ROI detection, we obtained an IoU of 93.43%, accuracy of 91.27%, sensitivity of 90.77%, specificity of 94.03%, F1 score of 84.17%, and an AUC of 0.93. For the refined segmentation of WSI by the region segmentation module, we obtained an IoU of 88.23%, accuracy of 96.10%, sensitivity of 71.83%, specificity of 96.19%, F1 score of 82.94%, and an AUC of 0.88. In short, the model provides refined segmentation of breast cancer WSIs using a cascade of CNNs. This segmentation can assist pathologists in interpreting the diagnosis by accurately presenting the regions considered during the inference of WSI. Our results indicate the possibility of using the model as a second screening system.

Evaluation of tear film instability pre and post corneal collagen cross-linking procedure in keratoconus patients

Purpose: To evaluate tear film instability in keratoconus patients pre and post CXL procedure. Study design: Prospective cross-sectional study. Place and duration of study: Study was conducted at Al Ehsan Welfare eye hospital Lahore from 15th September, 2020 to 15th June, 2021. Materials and methods: Tear break-up time (TBUT) test was used for determining the stability of the tear film out of 30 patients diagnosed with mild, moderate and severe keratoconus and tear film stability was assessed before and after CXL procedure by measuring tear break up time. Data was collected by using non-probability convenient sampling method. The patients were excluded if they had any previous dry eye syndrome, tear film instability, refractive surgery, other ocular surface abnormalities, cataract surgery and contact lens use before the CXL procedure. Pre and post-operative tear break up time was measured by performing TUBT test. Patients were selected between the age 25 to 35 years. In TBUT, sodium fluorescein dye was used and the tear film was observed with slit lamp under low magnification while the patient was instructed to avoid blinking while time was calculated until first dry spot appearance. SPSS version 21 was used for data analysis and Mcneemar’s test (kappa statistics) was applied with significance of p <0.05. Results: Results shows that pre CXL tear breakup time of 22 patients was normal (10-15) sec while 8 patients were observed with borderline tear break up time less than 10 sec with percentage of 73.3% and 26.7 % respectively. Results of Post TBUT after one week of CXL procedure shows variation in values of tear break up time which shows decrease of tear break up time less than 10 sec of 12 patients and 18 patients with significantly reduced tear break up time less than 5 sec was recorded with percentage of 40.% and 60. % respectively. After two weeks of procedure it was recorded that tear break up time of 4 patients was normal (10-15 sec) post tear break up time, while tear break up time of 18 patients was noted borderline (<10 sec) and 8 patient with reduced post tear break up time (<5sec) with percentage of 13.3 % , 60 % and 26.7% respectively. After one month of CXL the tear break up time was significantly improved as compared to one and two weeks of postoperative procedure of CXL as the corneal epithelium was regenerated. Conclusion: This study concludes that tear breakup time was significantly reduced after CXL procedure. TBUT was normal before the CXL but after CXL due to destruction of corneal epithelium the TBUT was reduced. Thus results reveals instability of tear films occurs after CXL procedure.

Retracted Rotator Cuff Repairs Heal With Disorganized Fibrogenesis Without Affecting Biomechanical Properties: A Comparative Animal Model Study

To determine the differences in the scar tissue formation during the healing of the repaired retracted cuff tear from that of the nonretracted tear. Eighteen right rabbit shoulders received a 1-cm transverse cuff incision over the footprint to simulate "nonretracted cuff tears" before the transosseous repairs (group A). A 1-cm tendinous portion was excised from 18 left shoulders to create defects to simulate "retracted cuff tears" before repairing the defects (group B). At week 12 postrepair, 20 and 16 shoulders underwent histologic and biomechanical analyses, respectively. Eight shoulders were used as a control group for biomechanical analyses. All specimens showed good healing and continuity of the repaired tendons. At low magnification, fibrous tissue firmly held the tendon-to-bone junctions in group A; however, all specimens in group B showed medially retracted tendons with fibrous tissue continuity between the tendon stumps and footprints. At medium magnification, more irregular collagen fiber orientation was observed in group B. Polarized light microscopy showed fibrous tissue continuity with medially retracted tendons in group B. When we quantified collagen fiber orientation using ImageJ software, group B had inferior grayscale measurements when compared with group A (P= .001). At week 12, no statistical differences existed in mean loads-to-failure at the repair sites between the groups (P= .783). In the nonretracted cuff tears, fibrous tissue bound the tendon-to-bone junction with healing. After the healing of the retracted cuff tears, continuity of nontendinous tissue was observed adjacent to the medially retracted tendon, which comprised more disorganized immature fibrous tissue than that in the nonretracted cuff tears. Unlike the healing of nonretracted rotator cuff tear, repairing of the "retracted" tendon end of cuff tear still resulted in retraction of the tendon back to its original position but being held down with fibrous tissue to the footprint.

In situ water radiolysis in a scanning electron microscope and observation of micro- and nanobubbles formation

Nanobubble formation by radiolysis of water has been observed in a transmission electron microscope using very high electron dose ratios. Here, surface micro- and nanobubbles were formed in situ in a conventional scanning electron microscope at relatively low magnifications of ∼1000. Bubble formation was observed at a low dose ratio of 1.3 × 1011 electrons/cm2 s, which is 5–7 orders of magnitude smaller than in previous studies using transmission electron microscopes. Our observations suggest that the pressure in the liquid cell is one of the important factors for the formation of bubbles. The contact angles obtained by direct observation of 69 bubbles were 50°–90°, with an average of 69°. These angles are much larger than those of the bubbles previously observed using an atomic force microscope. Despite the large contact angles (and large Laplace pressures), the micro- and nanobubble lifetimes were at least three days. Scanning electron microscopy shows potential as an important analytical tool for micro- and nanobubbles.

Improved resolution in x-ray tomography by super-resolution.

In this paper, super-resolution imaging is described and evaluated for x-ray tomography and is compared with standard tomography and upscaling during reconstruction. Blurring is minimized due to the negligible point spread of photon counting detectors and an electromagnetically movable micro-focus x-ray spot. Scans are acquired in high and low magnification geometry, where the latter is used to minimize penumbral blurring from the x-ray source. Sharpness and level of detail can be significantly increased in reconstructed slices to the point where the source size becomes the limiting factor. The achieved resolution of the different methods is quantified and compared using biological samples via the edge spread function, modulation transfer function, and Fourier ring correlation.

Morphological characterization using scanning electron microscopy of fly artifacts deposited by Calliphora vomitoria (Diptera: Calliphoridae) on household materials

Insects found at a crime scene can produce traces referred to as fly artifacts (FA) due to their movement over the corpse and the manner in which they feed upon it. These can be detrimental for carrying out criminal investigations. Confusing a FA with a genuine bloodspot can lead to misinterpretations, also taking into consideration that FA may contain a human DNA profile. The aim of the present study was to employ scanning electron microscopy (SEM) for the analysis of FA produced by Calliphora vomitoria on hard surfaces and fabrics that are commonly present at crime scenes. FA and control bloodstains were produced under experimental conditions on metal, glass, plaster, cotton, and polyester. After macroscopic analysis, FA were examined at standard low (20–40 ×), medium low (300–600 ×), and high ultrastructural (1200 ×) magnification through a SEM Stereoscan 360, Leica, Cambridge. SEM analysis enabled the identification of distinctive features of FA on hard surfaces, namely, amorphous crystals, micro-crystals with a morphology similar to those of uric or micro-crystals with a comparable morphology to cholesterol, absent in controls. Moreover, red blood cells (RBC) were absent in FA but were always present in controls. On cotton, for both FA and controls, the drop was almost completely absorbed and thus indistinguishable from the underlying fabric texture. On polyester, FA showed amorphous/crystal-like deposits and no RBC, as observed on hard surfaces, except for those showing a completely flat surface. SEM analysis appeared to be suitable for differential diagnosis between FA and genuine bloodstains on hard surfaces, although the results may be inconclusive on tested fabrics.

Optical mesoscopy, machine learning, and computational microscopy enable high information content diagnostic imaging of blood films.

Automated image-based assessment of blood films has tremendous potential to support clinical haematology within overstretched healthcare systems. To achieve this, efficient and reliable digital capture of the rich diagnostic information contained within a blood film is a critical first step. However, this is often challenging, and in many cases entirely unfeasible, with the microscopes typically used in haematology due to the fundamental trade-off between magnification and spatial resolution. To address this, we investigated three state-of-the-art approaches to microscopic imaging of blood films which leverage recent advances in optical and computational imaging and analysis to increase the information capture capacity of the optical microscope: optical mesoscopy, which uses a giant microscope objective (Mesolens) to enable high-resolution imaging at low magnification; Fourier ptychographic microscopy, a computational imaging method which relies on oblique illumination with a series of LEDs to capture high-resolution information; and deep neural networks which can be trained to increase the quality of low magnification, low resolution images. We compare and contrast the performance of these techniques for blood film imaging for the exemplar case of Giemsa-stained peripheral blood smears. Using computational image analysis and shape-based object classification, we demonstrate their use for automated analysis of red blood cell morphology and visualization and detection of small blood-borne parasites such as the malarial parasite Plasmodium falciparum. Our results demonstrate that these new methods greatly increase the information capturing capacity of the light microscope, with transformative potential for haematology and more generally across digital pathology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

New giant planet beyond the snow line for an extended MOA exoplanet microlens sample

ABSTRACT Characterizing a planet detected by microlensing is hard if the planetary signal is weak or the lens-source relative trajectory is far from caustics. However, statistical analyses of planet demography must include those planets to accurately determine occurrence rates. As part of a systematic modelling effort in the context of a >10-yr retrospective analysis of MOA’s survey observations to build an extended MOA statistical sample, we analyse the light curve of the planetary microlensing event MOA-2014-BLG-472. This event provides weak constraints on the physical parameters of the lens, as a result of a planetary anomaly occurring at low magnification in the light curve. We use a Bayesian analysis to estimate the properties of the planet, based on a refined Galactic model and the assumption that all Milky Way’s stars have an equal planet-hosting probability. We find that a lens consisting of a $1.9^{+2.2}_{-1.2}\, \mathrm{M}_\mathrm{J}$ giant planet orbiting a $0.31^{+0.36}_{-0.19}\, \mathrm{M}_\odot$ host at a projected separation of $0.75\pm 0.24\, \mathrm{au}$ is consistent with the observations and is most likely, based on the Galactic priors. The lens most probably lies in the Galactic bulge, at $7.2^{+0.6}_{-1.7}\,\mathrm{kpc}$ from Earth. The accurate measurement of the measured planet-to-host star mass ratio will be included in the next statistical analysis of cold planet demography detected by microlensing.

Analysis of Surface Morphology and Elemental Composition on Zirconia Implants Before and After Photofunctionalization by Scanning Electron Microscopy and Energy Dispersive X ray Spectroscopy - An In vitro Study.

Background:The purpose of this study is to analyze the surface morphology and elemental composition of zirconia implants before and after photofunctionalization.Materials and Methods:Ten zirconia implants (white sky implant system– Bredent Company) five each in the study group and control group was taken. Study group samples were treated with ultraviolet light for 48 h. Microstructured surface of the study and control group blanks at abutment and thread regions were documented by Scanning Electron Microscope The semi-quantitative element composition was analyzed using Energy-dispersive X-ray (EDX) spectrum.Results:SEM images of the study and control group divulged a varied array of topographical configuration of the abutment area and thread region at different magnifications. At low magnification, both study and control group revealed plain compact surface and wavy porous area, whereas higher magnification showed dense grainy regions of various sizes and intensities disrupted by pores. EDX spectrum analysis for elemental composition showed increased oxygen concentration in the study group (42.8%) than the control group (29.09%), whereas carbon concentration was lower in photofunctionalized group (34.34%) than in the control group (45.41%).Conclusion:In zirconia implants, photofunctionalization is a viable method to effectively enhance the surface topography and hydrophilicity of bone-implant interface.

MO319BEDSIDE URINE SEDIMENT EXAMINATION IN IMMUNOGLOBULIN A NEPHROPATHY PATIENTS PERFORMED BY NEPHROLOGISTS

Abstract Background and Aims Urine sediment microscopy is mostly abandoned by nephrologists nowadays, however it is an important diagnostic tool in kidney and urinary tract diseases. The aim of this study is to emphasize the benefits of urine microscopy performed by a nephrologist. Method A prospective cohort study at Pauls Stradins Clinical University Hospital Nephrology center included patients with histologically confirmed diagnosis of IgA nephropathy from 1st January 2020 till December 2020. Appropriately collected urine samples were examined using manual microscopy within an hour after sample collection and by automated urinalysis. Samples were centrifuged at 4000 rpm for 4 minutes, the supernatant urine was carefully decanted, 1 - 1,5 ml of the left urine was mixed by gentle agitation and placed on a standard glass slide with a cover slip. Sample examination was performed using low (magnification x10) and high power (magnification x40) using brightfield microscopy with a minimum of 10 fields. Results A total of 37 patients (24 men, mean age 42.7 ± 10.9 years) were included in the study. 59.5 % of patients (n = 22) had hematuria based on automated urine sediment analysis and 62.2 % (n = 23) of patients had hematuria based on manual urine microscopy. 45.9 % of patients (n=17) had dysmorphic erythrocytes, 13.5 % of patients (n = 5) had isomorphic red blood cells (RBC) and 40.5 % of patients (n = 15) did not have RBC in urine samples by manual urine microscopy. 54.2 % (13/24) of men and 30.8 % (4/13) of women had dysmorphic RBC in urine. Conclusion Manual urine sediment examination was more sensitive than automated analysis. Majority of IgA nephropathy patients have active urine sediment with hematuria and dysmorphic RBC. Manual microscopy remains an effective and reliable method that can be easily and quickly performed by nephrologists.

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.

Forestry control in the Brazilian Amazon II: charcoal anatomy of 21 species

Abstract The production of illegal charcoal, associated with other local human practices, is responsible for the degradation of native forests, especially in the Brazilian Amazon. The need for market control and charcoal production is accompanied by the lack of comparative charcoal materials from the Amazon. Here, we describe charcoal samples of 21 Brazilian species and provide SEM images that can facilitate the charcoal identification. It is possible to distinguish the species on the basis of anatomy. Features such as vessel groupings, vessel–ray pitting, perforation plates, axial parenchyma, ray cellular composition, storied structure and secretory elements were chosen as primary diagnostic features for the identification of species. We highlight vessel groupings, axial parenchyma type and in some cases even perforation plates, which were easily observed in low magnification SEM images and can assist in the supervision by government agents. From our descriptions, evaluations and photomicrographs, it will be possible to compare charcoal from commercial species from the Amazon.

Large Field of View Scanning Electron Microscopy of the Paraventricular Nucleus of the Hypothalamus During Diet‐Induced Obesity

The paraventricular nucleus of the hypothalamus (PVN) is critical in autonomic and endocrine control. Previous work has indicated that PVN dysregulation is associated with a variety of cardiometabolic diseases including those related to obesity. However, investigations have focused primarily on molecular alterations. Importantly, changes in the anatomical structure of brain networks also leads to functional alterations. While electron microscopy (EM) can provide nanometer resolution of normal/abnormal brain structures, an inherent limitation of traditional transmission EM is the random sampling of an object within a large region of interest (i.e. PVN). To overcome the inherent limitations of EM, we introduce an approach that utilizes large field of view high-resolution focused ion beam scanning electron microscopy (SEM) of the PVN. Six week old male C57Bl/6J mice were fed a high fat diet (HFD) or normal chow for 10 weeks (n=4/group), and brains prepared for SEM imaging. Due to the COVID-19 pandemic, which required social distancing, SEM imaging and analysis was conducted remotely (i.e. at home) using a virtual network computing approach. We first acquired interactive, zoomable maps of the PVN at a low magnification. This image was then used to navigate precisely to the PVN and acquire a high-resolution image encompassing ~30-40% of the nucleus (Figure). With this approach, we could rapidly scan for any visually apparent anatomical alterations. This initial evaluation revealed more electron dense regions within PVN neuronal nuclei, potentially indicative of heterochromatin clusters, in HFD mice relative to normal chow counterparts. Thus, we performed subsequent analysis on 80-120 neurons from each diet group by outlining neuronal nuclear envelopes and quantifying pixel distribution. This provided an indirect estimate of heterochromatin (i.e. dark pixels) and euchromatin (lighter pixels), with a shift towards darker pixels suggestive of increased chromatin “clustering”. The area and perimeter of analyzed PVN neuronal nuclei between groups was similar (e.g. area: 4.0±2.9 vs 3.7±1.4 µm2x106, normal chow vs HFD, p<0.05). However, histogram analysis of pixel intensities revealed positive kurtosis, indicating that HFD resulted in a redistribution of nuclear pixels to dark and light pixel intensities (-0.05±0.03 vs 0.12±0.03 a.u., normal chow vs HFD, p<0.05). Measures of skew, an indicator of curve symmetry, further revealed a leftward pixel shift (i.e. more negative skew toward dark pixels) in diet-induced obese mice (-0.48±0.01 vs -0.64±0.01 a.u., normal chow vs HFD, p<0.05). Finally, we created a PVN “map” to examine where the most altered neurons were located. In short, following HFD feeding, “altered” neurons were distributed throughout the entire PVN and not located in discrete PVN sub-nuclei. Collectively, these findings present an approach that allows for the precise placement of PVN cells within the overall region (i.e. locating a house within a city). Moreover, they suggest that HFD feeding may disrupt PVN neuronal chromatin structure, which may be reflective of epigenetic modifications and/or transcriptional alterations.

Rapid Antimicrobial Susceptibility Testing on Clinical Urine Samples by Video-Based Object Scattering Intensity Detection.

To combat the ongoing public health threat of antibiotic-resistant infections, a technology that can quickly identify infecting bacterial pathogens and concurrently perform antimicrobial susceptibility testing (AST) in point-of-care settings is needed. Here, we develop a technology for point-of-care AST with a low-magnification solution scattering imaging system and a real-time video-based object scattering intensity detection method. The low magnification (1-2×) optics provides sufficient volume for direct imaging of bacteria in urine samples, avoiding the time-consuming process of culture-based bacterial isolation and enrichment. Scattering intensity from moving bacteria and particles in the sample is obtained by subtracting both spatial and temporal background from a short video. The time profile of scattering intensity is correlated with the bacterial growth rate and bacterial response to antibiotic exposure. Compared to the image-based bacterial tracking and counting method we previously developed, this simple image processing algorithm accommodates a wider range of bacterial concentrations, simplifies sample preparation, and greatly reduces the computational cost of signal processing. Furthermore, development of this simplified processing algorithm eases implementation of multiplexed detection and allows real-time signal readout, which are essential for point-of-care AST applications. To establish the method, 130 clinical urine samples were tested, and the results demonstrated an accuracy of ∼92% within 60-90 min for UTI diagnosis. Rapid AST of 55 positive clinical samples revealed 98% categorical agreement with both the clinical culture results and the on-site parallel AST validation results. This technology provides opportunities for prompt infection diagnosis and accurate antibiotic prescriptions in point-of-care settings.

Evaluation of the color measurement based on a microscopic hyperspectral imaging system

AbstractIn order to make a color assessment for micron‐grade materials such as a single fiber, a non‐destructive and push‐broom microscopic hyperspectral imaging system (MHIS) was set up consisting of a stereomicroscope, an imaging spectrograph, and a digital detector. The performance of this system for acquiring accurate and repeatable information, such as spectral and colorimetric values, was investigated. The experimental results show that the system has an excellent spatial resolution, repeatability, and accuracy. The spatial resolution with a ×1.5 auxiliary objective lens was down to 90.244 lp mm−1 (5.54 μm). The average ΔE00 of the tested patches in the ColorChecker Mini chart was between 0.15 and 0.40, and the color measurement repeatability was acceptable. Color difference ΔE00 and the standard variation of spectral wavelength between ×2.5 and ×4.5 magnification were larger than those at lower magnifications. It can be concluded that the MHIS can identify colorimetric values of the materials and detect the color changes sensitively with a high spatial resolution at micron‐grade.

Fast, cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas

Fast and wide field-of-view imaging with single-cell resolution, high signal-to-noise ratio, and no optical aberrations have the potential to inspire new avenues of investigations in biology. However, such imaging is challenging because of the inevitable tradeoffs among these parameters. Here, we overcome these tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of ∼16,000 neurons at 7.5Hz from a 9mm2 contiguous image plane, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Network analysis based on single-cell activities revealed that the brain exhibits small-world rather than scale-free behavior. The FASHIO-2PM is expected to enable studies on biological dynamics by simultaneously monitoring macroscopic activities and their compositional elements.

Quantification and Imaging of Nanoscale Contact with Förster Resonance Energy Transfer.

Adhesion is caused by molecular interactions that only take place if the surfaces are in nanoscale contact (NSC); i.e., the distance between the surfaces is in the range of 0.1–0.4 nm. However, there are several difficulties measuring the NSC between surfaces, mainly because regions that appear to be in full contact at low magnification may show no NSC when observed at higher magnifications. Thus, the measurement area of NSC is very small with imaging techniques, and an experimental technique to evaluate NSC for large contact areas has not been available thus far. Here, we are proposing Förster resonance energy transfer (FRET) spectroscopy/microscopy for this purpose. We demonstrate that NSC in a distance range of 1–10 nm can be evaluated. Our experiments reveal that, for thin films pressed under different loads, NSC increases with the applied pressure, resulting in a higher FRET signal and a corresponding increase in adhesion force/energy when separating the films. Furthermore, we show that local variations in molecular contact can be visualized with FRET microscopy. Thus, we are introducing a spectroscopic technique for quantification (FRET spectroscopy) and imaging (FRET microscopy) of NSC between surfaces, demonstrated here for the application of surface adhesion. This could be of interest for all fields where adhesion or nanoscale surface contact are playing a role, for example, soft matter, biological materials, and polymers, but also engineering applications, like tribology, adhesives, and sealants.

Compressed sensing for highly efficient imaging transcriptomics.

Recent methods for spatial imaging of tissue samples can identify up to ~100 individual proteins1-3 or RNAs4-10 at single-cell resolution. However, the number of proteins or genes that can be studied in these approaches is limited by long imaging times. Here we introduce Composite In Situ Imaging (CISI), a method that leverages structure in gene expression across both cells and tissues to limit the number of imaging cycles needed to obtain spatially resolved gene expression maps. CISI defines gene modules that can be detected using composite measurements from imaging probes for subsets of genes. The data are then decompressed to recover expression values for individual genes. CISI further reduces imaging time by not relying on spot-level resolution, enabling lower magnification acquisition, and is overall about 500-fold more efficient than current methods. Applying CISI to 12 mouse brain sections, we accurately recovered the spatial abundance of 37 individual genes from 11 composite measurements covering 180 mm2 and 476,276 cells.