Standard metadata for 3D microscopy.

Recent advances in fluorescence microscopy techniques and tissue clearing, labeling, and staining provide unprecedented opportunities to investigate brain structure and function. These experiments' images make it possible to catalog brain cell types and define their location, morphology, and connectivity in a native context, leading to a better understanding of normal development and disease etiology. Consistent annotation of metadata is needed to provide the context necessary to understand, reuse, and integrate these data. This report describes an effort to establish metadata standards for three-dimensional (3D) microscopy datasets for use by the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative and the neuroscience research community. These standards were built on existing efforts and developed with input from the brain microscopy community to promote adoption. The resulting 3D Microscopy Metadata Standards (3D-MMS) includes 91 fields organized into seven categories: Contributors, Funders, Publication, Instrument, Dataset, Specimen, and Image. Adoption of these metadata standards will ensure that investigators receive credit for their work, promote data reuse, facilitate downstream analysis of shared data, and encourage collaboration.

Characterization of an artificial intelligence model for ranking static images of blastocyst stage embryos.

OBJECTIVE: To perform a series of analyses characterizing an artificial intelligence (AI) model for ranking blastocyst-stage embryos. The primary objective was to evaluate the benefit of the model for predicting clinical pregnancy, whereas the secondary objective was to identify limitations that may impact clinical use. DESIGN: Retrospective study. SETTING: Consortium of 11 assisted reproductive technology centers in the United States. PATIENT(S): Static images of 5,923 transferred blastocysts and 2,614 nontransferred aneuploid blastocysts. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Prediction of clinical pregnancy (fetal heartbeat). RESULT(S): The area under the curve of the AI model ranged from 0.6 to 0.7 and outperformed manual morphology grading overall and on a per-site basis. A bootstrapped study predicted improved pregnancy rates between +5% and +12% per site using AI compared with manual grading using an inverted microscope. One site that used a low-magnification stereo zoom microscope did not show predicted improvement with the AI. Visualization techniques and attribution algorithms revealed that the features learned by the AI model largely overlap with the features of manual grading systems. Two sources of bias relating to the type of microscope and presence of embryo holding micropipettes were identified and mitigated. The analysis of AI scores in relation to pregnancy rates showed that score differences of ≥0.1 (10%) correspond with improved pregnancy rates, whereas score differences of <0.1 may not be clinically meaningful. CONCLUSION(S): This study demonstrates the potential of AI for ranking blastocyst stage embryos and highlights potential limitations related to image quality, bias, and granularity of scores.

Performance of Abbott Alinity hq hematology analyzer for automated cell counting of body fluids.

INTRODUCTION: Body fluid cell counting and differentiation provide essential information for diagnosis and monitoring of diverse pathologies. We evaluated the performance of the newly launched Abbott Alinity hq hematology analyzer for automated cell counting in body fluids and compared red blood cell (RBC) and total nucleated cell (TNC) counts with the Cell-Dyn Sapphire automated hematology analyzer. Differential counts were compared with microscopic differentiation on cytocentrifuged preparations. METHODS: Background concentration limits, limit of detection (LOD), linearity, imprecision, functional sensitivity and carryover were evaluated. For method comparison, we collected 172 body fluids (17 continuous ambulatory peritoneal dialysis fluids, 56 cerebrospinal fluids and 99 serous fluids). RESULTS: Background concentration limits were ≤1000 cells/µL for RBC counts and ≤3 cells/µL for TNC counts. The LOD was 1000 RBC/µL and 5 TNC/µL. Results from linear regression analysis revealed excellent linearity. Functional sensitivity was 3000 cells/µL for RBC counts and 50 cells/µL for TNC counts. Carryover was 0.6% and 0.1% for TNC and RBC, respectively. The Alinity hq shows good clinical performance. CONCLUSION: We demonstrated comparable performance for body fluid cell counting between the Alinity hq analyzer and the Cell-Dyn Sapphire. The Alinity hq can be very useful as a screening tool for body fluid cell counting.

Robust, fiducial-free drift correction for super-resolution imaging.

We describe a robust, fiducial-free method of drift correction for use in single molecule localization-based super-resolution methods. The method combines periodic 3D registration of the sample using brightfield images with a fast post-processing algorithm that corrects residual registration errors and drift between registration events. The method is robust to low numbers of collected localizations, requires no specialized hardware, and provides stability and drift correction for an indefinite time period.

OME-NGFF: a next-generation file format for expanding bioimaging data-access strategies.

The rapid pace of innovation in biological imaging and the diversity of its applications have prevented the establishment of a community-agreed standardized data format. We propose that complementing established open formats such as OME-TIFF and HDF5 with a next-generation file format such as Zarr will satisfy the majority of use cases in bioimaging. Critically, a common metadata format used in all these vessels can deliver truly findable, accessible, interoperable and reusable bioimaging data.

Multiplex RT-PCR provides improved diagnosis of skin and nail dermatophyte infections compared to microscopy and culture: a laboratory study and review of the literature.

Dermatophytes are the most common cause of superficial mycosis, estimated to affect 20% to 25% of the general population. We assessed the performance of a novel real-time polymerase chain reaction (RT-PCR) multiplex assay for diagnosis of dermatophytosis. To evaluate sensitivity and specificity, 10 known bacteria and 10 known fungi commonly found on skin, as well as 105 samples with culture confirmed dermatophytosis were tested using Dermatophyte and Fungi assay (AusDiagnostics, Sydney, Australia), a novel multiplex assay for diagnosis of dermatophytosis in skin and nail. This was followed by prospective evaluation of 195 clinical samples for dermatophytosis by both conventional methods and RT-PCR. RT-PCR showed almost two-fold higher sensitivity and high specificity in the diagnosis of skin and nail dermatophytosis compared to traditional microscopy and culture. In addition, RT-PCR demonstrated markedly reduced turnaround time from 4 to 6 weeks to 4 to 6 hours and ability for high throughput.

3D test sample for the calibration and quality control of stimulated emission depletion (STED) and confocal microscopes.

Multiple samples are required to monitor and optimize the quality and reliability of quantitative measurements of stimulated emission depletion (STED) and confocal microscopes. Here, we present a single sample to calibrate these microscopes, align their laser beams and measure their point spread function (PSF) in 3D. The sample is composed of a refractive index matched colloidal crystal of silica beads with fluorescent and gold cores. The microscopes can be calibrated in three dimensions using the periodicity of the crystal; the alignment of the laser beams can be checked using the reflection of the gold cores; and the PSF can be measured at multiple positions and depths using the fluorescent cores. It is demonstrated how this sample can be used to visualize and improve the quality of STED and confocal microscopy images. The sample is adjustable to meet the requirements of different NA objectives and microscopy techniques and additionally can be used to evaluate refractive index mismatches as a function of depth quantitatively.

Fringe analysis: single-shot or two-frames? Quantitative phase imaging answers.

Conditions of the digital recording of the fringe pattern determine the phase reconstruction procedure, which in turn directly shapes the final accuracy and throughput of the full-field (non-scanning) optical measurement technique and defines the system capabilities. In this way, the fringe pattern analysis plays a crucial role in the ubiquitous optical measurements and thus is under constant development focused on high temporal/spatial resolution. It is especially valuable in the quantitative phase imaging technology, which emerged in the high-contrast label-free biomedical microscopy. In this paper, I apply recently blossomed two-frame phase-shifting techniques to the QPI and merge them with advanced adaptive interferogram pre-filtering algorithms. Next, I comprehensively test such frameworks against classical and adaptive single-shot methods applied for phase reconstruction in dynamic QPI enabling highest phase time-space-bandwidth product. The presented study systematically tackles important question: what is the gain, if any, in QPI realized by recording two phase-shifted interferograms? Counterintuitively, the results show that single-shot demodulation exhibited higher phase reconstruction accuracy than two-frame phase-shifting methods in low and medium interferogram signal-to-noise ratio regimes. Thus, the single-shot approach is promoted due to not only high temporal resolution but also larger phase-information throughput. Additionally, in the majority of scenarios, the best option is to shift the paradigm and employ two-frame pre-filtering rather than two-frame phase retrieval. Experimental fringe analysis in QPI of LSEC/RWPE cell lines successfully corroborated all novel numerical findings. Hence, the presented numerical-experimental research advances the important field of fringe analysis solutions for optical full-field measurement methods with widespread bio-engineering applications.

Counting mitoses: SI(ze) matters!

Mitoses are often assessed by pathologists to assist the diagnosis of cancer, and to grade malignancy, informing prognosis. Historically, this has been done by expressing the number of mitoses per n high power fields (HPFs), ignoring the fact that microscope fields may differ substantially, even at the same high power (×400) magnification. Despite a requirement to define HPF size in scientific papers, many authors fail to address this issue adequately. The problem is compounded by the switch to digital pathology systems, where ×400 equivalent fields are rectangular and also vary in the area displayed. The potential for error is considerable, and at times this may affect patient care. This is easily solved by the use of standardized international (SI) units. We, therefore, recommend that features such as mitoses are always counted per mm2, with an indication of the area to be counted and the method used (usually "hotspot" or "average") to obtain the results.

Evaluation of microscopy, serology, circulating anodic antigen (CAA), and eosinophil counts for the follow-up of migrants with chronic schistosomiasis: a prospective cohort study.

BACKGROUND: An accurate test for the diagnosis and post-treatment follow-up of patients with schistosomiasis is needed. We assessed the performance of different laboratory parameters, including the up-converting reporter particle technology lateral flow assay to detect circulating anodic antigen (UCP-LF CAA), for the post-treatment follow-up of schistosomiasis in migrants attending a dedicated outpatient clinic in a non-endemic country. METHODS: Routine anti-Schistosoma serology results and eosinophil counts were obtained of patients with positive urine/stool microscopy and/or PCR (confirmed cases) or only positive serology (possible cases), and at least one follow-up visit at 6 (T6) or 12 (T12) months after praziquantel treatment. All sera samples were tested with the UCP-LF CAA assay. RESULTS: Forty-eight patients were included, 23 confirmed and 25 possible cases. The percentage seropositivity and median antibody titers did not change significantly during follow-up. UCP-LF CAA was positive in 86.9% of confirmed and 20% of possible cases. The percentage positivity and median CAA levels decreased significantly post-treatment, with only two patients having positive CAA levels at T12. CONCLUSIONS: The UCP-LF CAA assay proved useful for the diagnosis of active infection with Schistosoma spp. and highly valuable for post-treatment monitoring in migrants, encouraging the development of a commercial test.

Mitotic count: A need for standardization / Recuento mitótico: una necesidad de estandarización

PURPOSE: The mitotic count (MC), number of mitosis per unit area, is a very important parameter frequently used for classification and grading of some tumors. Traditionally, the MC has been expressed in terms of number of mitoses per high power field. The size of the field of view can vary greatly among different microscopes. In order to avoid under or overestimation of mitotic count, a conversion needs to be made. METHODS: A simple formula based on a simple rule of three has been devised to standardize the mitotic count to the reference area by multiplying the number of mitotic figures by a correction factor which has been calculated for the most frequently used microscopes and various common tumors. RESULTS AND CONCLUSIONS: We propose this simple method, which involves only a single multiplication, to standardize the mitotic count to the reference area

OBJETIVO: El recuento mitótico o número de mitosis por unidad de área, es un parámetro muy importante utilizado frecuentemente para clasificar y estadificar ciertos tumores. Tradicionalmente se ha expresado el recuento mitótico en términos de número de mitosis por campos de alta frecuencia. El tamaño del campo de visión puede variar ampliamente entre los diferentes microscopios. A fin de evitar la infraestimación o sobreestimación del recuento mitótico, debe realizarse una conversión. MÉTODOS: Por medio de una simple regla de tres, se ha obtenido una fórmula simple para estandarizar el recuento mitótico. Multiplicando el número de mitosis por un factor de corrección, se obtiene el recuento mitótico estandarizado al área de referencia. Se han calculado los factores de corrección para los microscopios más habituales y para los diferentes tumores comunes. RESULTADOS Y CONCLUSIONES: Proponemos este método simple (únicamente uno por multiplicación) para estandarizar el recuento mitótico con respecto al área de referencia

A method to prepare large resin sections for counting myelinated axons in rodent CNS and PNS structures.

We present a method that allows preparing histological sections from large blocks of nervous tissue embedded in epoxy resin. Resin-embedding provides excellent resolution especially for the myelin-rich white matter and is often being used for visualizing the myelinated axons in peripheral nerves. However, because of the limited penetration of the reagents, only very small tissue specimens can be processed in this way. Here, we describe a method that enables to embed large specimens and their sectioning on a standard sliding microtome. To process the large specimens, modifications in several steps of the processing technique had to be made. In this paper we demonstrate, that with this technique 1-3 µm thick transversal sections can be prepared from the resin-embedded specimens as large as rat brain hemisphere. Such a large section allows simultaneously: 1.) overviewing and delineating the gross anatomical structures, and 2.) observing the subcellular details at the highest possible optical magnifications. Such a large section with excellent resolution allows application of unbiased stereological methods and reliable quantification of very small objects within the area of interest.

Performance evaluation of Baermann techniques: The quest for developing a microscopy reference standard for the diagnosis of Strongyloides stercoralis.

BACKGROUND: Soil-transmitted helminths (STH) are common in low and middle income countries where there is lack of access to clean water and sanitation. Effective diagnosis and treatment are essential for the control of STH infections. However, among STH parasites, Strongyloides stercoralis is the most neglected species, both in diagnostics and control strategies. Diagnostic methods cover different approaches, each with different sensitivities and specificities, such as serology, molecular techniques and microscopy based techniques. Of the later, the Baermann technique is the most commonly used procedure. In the literature, several ways have been described to perform the Baermann method, which illustrates the overall lack of a '(gold) reference standard' method for the diagnosis of S. stercoralis infection. In this study we have evaluated the performance of three Baermann techniques in order to improve the reference standard for the microscopic diagnosis of S. stercoralis infection thereby facilitating individual case detection, mapping of the disease and proper evaluation of treatment responses. METHODS/PRINCIPAL FINDINGS: A community based cross sectional study was conducted at Zenzelima, Bahir Dar Zuria Ethiopia. A total of 437 stool samples were collected and analyzed by the following procedures: conventional Baermann (CB), modified Baermann (MB), and modified Baermann with charcoal pre-incubation (MBCI). The diagnostic sensitivity and Negative Predictive Value (NPV) of each technique was calculated using the combination of all the three techniques as a composite reference standard. Our result indicated that larvae of S. stercoralis were detected in 151 (34.6%) stool samples. The prevalence of S. stercoralis infection based on the three diagnostic methods was 9.6%, 8.0%, and 31.3% by CB, MB, and MBCI respectively. The sensitivity and NPV for CB, MB, and MBCI were 26.7% and 70.8%, 22.1% and 69.6%, and 87.0% and 93.2%, respectively. The MBCI showed significant difference (P- value = <0.001) in the sensitivity and NPV values when compared with CB and MB values. The agreement between CB, MB, and MBCI with the composite reference standard was 31.8%, 26.7%, 89.6%, respectively. CONCLUSION/SIGNIFICANCE: Our results suggest the superior performance of MBCI. It is relatively easy to implement, simple to perform and comparatively cheaper. The CB is by far the commonly used method in routine diagnostic although this technique significantly underestimates the true burden of the disease and thereby contributing to the exclusion of S. stercoralis from the control strategies. Therefore, MBCI is recommended as a routine microscopy-based diagnostic test for S. stercoralis infection, particularly in settings where molecular procedures are not available.

Validation of Whole Slide Imaging for primary surgical pathology diagnosis of prostate biopsies.

CONTEXT: Whole slide imaging (WSI) is an important component of digital pathology which includes digitization of glass slides and their storage as digital images. Implementation of WSI for primary surgical pathology diagnosis is evolving, following various studies which have evaluated the feasibility of WSI technology for primary diagnosis. AIMS, SETTINGS AND DESIGN: The present study was a single-center, observational study which included evaluation by three pathologists and aimed at assessing concordance on specialty-specific diagnosis and comparison of time taken for diagnosis on WSI and conventional light microscopy (CLM). MATERIALS AND METHODS: Seventy prostate core biopsy slides (reported between January 2016 and December 2016) were scanned using Pannoramic MIDI II scanner, 3DHISTECH, Budapest, Hungary, at 20× and 40×. Sixty slides were used for validation study following training with 10 slides. STATISTICAL ANALYSIS USED: Intraobserver concordance for diagnosis between the two platforms of evaluation was analyzed using Cohen's κ statistics and intraclass correlation coefficient (ICC); observation time for diagnosis was compared by Wilcoxon signed-rank test. RESULTS: Interpretation on WSI using 20× and 40× was comparable with no major discordance. A high level of intraobserver agreement was observed between CLM and WSI for all three observers, both for primary diagnosis (κ = 0.9) and Grade group (κ = 0.7-0.8) in cases of prostatic adenocarcinoma. The major discordance rate between CLM and WSI was 3.3%-8.3%, which reflected the expertise of the observers. The time spent for diagnosis using WSI was variable for the three pathologists. CONCLUSION: WSI is comparable to CLM and can be safely incorporated for primary histological diagnosis of prostate core biopsies.