Perspectives on label-free microscopy of heterogeneous and dynamic biological systems.

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Structured illumination microscopy for cancer identification in diagnostic breast biopsies.

Breast cancer is the second most common cancer diagnosed in women in the US with almost 280,000 new cases anticipated in 2023. Currently, on-site pathology for location guidance is not available during the collection of breast biopsies or during surgical intervention procedures. This shortcoming contributes to repeat biopsy and re-excision procedures, increasing the cost and patient discomfort during the cancer management process. Both procedures could benefit from on-site feedback, but current clinical on-site evaluation techniques are not commonly used on breast tissue because they are destructive and inaccurate. Ex-vivo microscopy is an emerging field aimed at creating histology-analogous images from non- or minimally-processed tissues, and is a promising tool for addressing this pain point in clinical cancer management. We investigated the ability structured illumination microscopy (SIM) to generate images from freshly-obtained breast tissues for structure identification and cancer identification at a speed compatible with potential on-site clinical implementation. We imaged 47 biopsies from patients undergoing a guided breast biopsy procedure using a customized SIM system and a dual-color fluorescent hematoxylin & eosin (H&E) analog. These biopsies had an average size of 0.92 cm2 (minimum 0.1, maximum 4.2) and had an average imaging time of 7:29 (minimum 0:22, maximum 37:44). After imaging, breast biopsies were submitted for standard histopathological processing and review. A board-certified pathologist returned a binary diagnostic accuracy of 96% when compared to diagnoses from gold-standard histology slides, and key tissue features including stroma, vessels, ducts, and lobules were identified from the resulting images.

Aspergillus hubkae, a Novel Species Isolated from a Patient with Probable Invasive Pulmonary Aspergillosis.

A 50-year-old man, previously diagnosed with pulmonary tuberculosis and lung cavities, presented with symptoms including fever, shortness of breath, and cough. A pulmonary CT scan revealed multiple cavities, consolidation and tree-in-bud in the upper lungs. Further investigation through direct examination of bronchoalveolar lavage fluid showed septate hyphae with dichotomous acute branching. Subsequent isolation and morphological analysis identified the fungus as belonging to Aspergillus section Nigri. The patient was diagnosed with probable invasive pulmonary aspergillosis and successfully treated with a three-month oral voriconazole therapy. Phylogenetic analysis based on partial ß-tubulin, calmodulin and RNA polymerase second largest subunit sequences revealed that the isolate represents a putative new species related to Aspergillus brasiliensis, and is named Aspergillus hubkae here. Antifungal susceptibility testing demonstrated that the isolate is resistant to itraconazole but susceptible to voriconazole. This phenotypic and genetic characterization of A. hubkae, along with the associated case report, will serve as a valuable resource for future diagnoses of infections caused by this species. It will also contribute to more precise and effective patient management strategies in similar clinical scenarios.

From ultrasound to microscopy: Actualities in thyroid investigation in cattle.

Thyroid ultrasonography examination is widely used in human and small animal medicine. However, it has rarely been applied in cattle. The aim of this study was to determine whether the measurements of the thyroid gland by ultrasound examination correlate to those taken during post-mortem examination. A sample of 22 cows and 23 calves was selected for thyroid gland evaluation. An ultrasound scan was performed ante-mortem, followed by euthanasia (for medical reasons) or slaughtered in the food chain and the dissection of the thyroid gland was therefore performed. Post-mortem, the gland was weighed and its dimensions and volume measured. The volume and weight measurements were compared with the predicted ones on US using the formulas available in the literature. Finally, histological examination was performed on thyroid glands. The dimensions of the thyroid gland measured by ultrasonography were significantly different (p<0.05) from those observed post-mortem, except for lobe lengths in calves (p>0.1). However, in calves, there was no systematic bias between the ultrasound and post-mortem examination of the thyroid gland, which were concordant (with an average error of 18%). Cystic lesions were observed on ultrasound in 9/22 cows and could be found on histological examination in 7 of these. Other lesions, such as follicular hypoplasia and hyperplasia, were seen on histological examination but not on ultrasound. Although the ultrasound measurements did not significantly correlate with those taken post-mortem, this examination may allow to differentiate non-standard thyroids in the case of hyperplastic goiter, as demonstrated in other species. This study also describes and illustrates interesting lesions of the thyroid gland in cattle. These findings are innovative in the description of the use of thyroid ultrasound in cattle, although further studies are needed to allow deeper conclusions.

Non-telecentric photoacoustic microscopy for multi-scale imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) excels in precisely imaging a biological tissue based on absorption contrast. However, existing OR-PAMs are confined by fixed compromises between spatial resolution and field of view (FOV), preventing the integration of large FOV and local high-resolution within one system. Here, we present a non-telecentric OR-PAM (nTC-PAM) that empowers efficient adaptation of FOV and spatial resolution to match the multi-scale requirement of diverse biological imaging. Our method allows for a large-scale transformation in FOV and even surpassing the nominal FOV of the objective with minimal marginal degradation of the lateral resolution. We demonstrate the advantage of nTC-PAM through multi-scale imaging of the leaf phantom, mouse ear, and cortex. The results reveal that nTC-PAM can switch the FOV and spatial resolution to meet the requirements of different biological tissues, such as large-scale imaging of the whole cerebral cortex and high-resolution imaging of microvascular structures in local brain regions.

Simultaneous deep ultraviolet transmission and scattering microscopy for virtual histology.

In recent years, the emergence of a variety of novel optical microscopy techniques has enabled the generation of virtual optical stains of unlabeled tissue specimens, which have the potential to transform existing clinical histopathology workflows. In this work, we present a simultaneous deep ultraviolet transmission and scattering microscopy system that can produce virtual histology images that show concordance to conventional gold-standard histological processing techniques. The results of this work demonstrate the system's diagnostic potential for characterizing unlabeled thin tissue sections and streamlining histological workflows.

Comparison of malaria diagnostic methods for detection of asymptomatic Plasmodium infections among pregnant women in northwest Ethiopia.

BACKGROUND: Malaria in pregnancy remains a major public health problem in the globe, especially in sub-Saharan Africa. In malaria endemic areas, most pregnant women remain asymptomatic, but malaria could still cause complications on the mother and her offspring; as well as serve as reservoirs to transmit infection. Despite these effects, no attention is given to the diagnosis of asymptomatic Plasmodium infections (APIs) using highly sensitive and specific laboratory diagnostic tools in Ethiopia. Therefore, the goal of this study was to compare the performance of Rapid Diagnostic Test (RDT), microscopy and real-time polymerase chain reaction (RT-PCR) to detect APIs among pregnant women. METHODS: A health facility based cross -sectional study was conducted among pregnant women attending antenatal care at Fendeka town health facilities Jawi district, northwest Ethiopia from February to March, 2019. A total of 166 participants were enrolled by using convenient sampling technique. Socio-demographic features were collected using a semi structured questionnaire. Dried blood spot (DBS) samples were collected for molecular analysis. Asymptomatic Plasmodium infection on pregnant women was diagnosed using RDT, microscopy and RT-PCR. Descriptive statistics were used to determine the prevalence of APIs. Method comparison was performed, and Cohen's kappa coefficient (k) was used to determine the degree of agreement among the diagnostic methods. Parasite densities were also calculated. RESULTS: The prevalence of API was 9.6%, 11.4% and 18.7% using RDT, microscopy and RT-PCR, respectively. The overall proportion of API was 19.3%. Sensitivity of the RDT was 83.3% as compared with microscopy. Rapid Diagnostic Test and microscopy also showed sensitivity of 50% and 60%, respectively, as compared with RT-PCR. The mean parasite density was 3213 parasites/µl for P falciparum and 1140 parasites/µl of blood for P. vivax. CONCLUSION: Prevalence of API in the study area was high. Both RDT and microscopy had lower sensitivity when compared with RT-PCR. Therefore, routine laboratory diagnosis of API among pregnant women should be given attention and done with better sensitive and specific laboratory diagnostic tools.

Comparative evaluation of the diagnostic accuracies of four different malaria rapid diagnostic test kits available in Ghana.

Malaria rapid diagnostic test (mRDT) kit is one of the techniques for diagnosing malaria. Due to its inherent advantages over the microscopy technique, several brands of the kit have flooded malaria endemic countries, without prior in-country evaluation. Two of such mRDT kits are Oscar (India) and Standard Q (Korea Republic). In this study, the performance of Oscar and Standard Q mRDT kits were compared to First Response (India) and CareStart (USA) mRDTs, which have been evaluated and deployed for use approved by the Ministry of Health (MOH). In this comparative study, whole blood samples were collected from patients suspected of malaria. Plasmodium falciparum was detected in each sample using nested polymerase chain reaction (nPCR), microscopy and the four mRDTs. The sensitivities, specificities, accuracies, positive and negative predictive values and accuracies of the mRDTs were determined using nPCR as a reference technique. Kappa statistic was used to determine the level of agreement among the techniques. Two hundred (200) blood samples were analyzed in this study. The overall detection rates of P. falciparum by microscopy, First Response, CareStart, Oscar-PfHRP2, Standard Q mRDT kits and nPCR were 31.5%, 34.5%, 33.5%, 32%, 31% and 43% (x2 = 6.1, p = 0.046), respectively. The accuracies of CareStart and First Response were comparable (90.5% vs. 89.5%). Further, comparing their sensitivities, Oscar-PfHRP2 was 74.4% (95% confidence interval (CI): 63.9-83.2) while that of Standard Q was 72.1% (95% CI: 61.4-81.2), with comparable accuracies (Oscar-PfHRP2-89% and Standard Q -88%). Apart from First Response that was 98.3% specific, the others were 100% specific. Kappa test revealed perfect diagnostic agreement (κ = 0.90-0.98) among the four mRDTs. That notwithstanding, Oscar-PfHRP2 agreed better with CareStart (κ = 0.94) and First Response (κ = 0.92) compared to the agreement between Standard Q and, CareStart (κ = 0.92) and First Response (κ = 0.90). Taken together, the diagnostic performance of the four mRDT kits were statistically similar. That notwithstanding, new mRDT kits should be evaluated prior to deployment for use.

Hyperspectral dark-field microscopy of human breast lumpectomy samples for tumor margin detection in breast-conserving surgery.

Significance: Hyperspectral dark-field microscopy (HSDFM) and data cube analysis algorithms demonstrate successful detection and classification of various tissue types, including carcinoma regions in human post-lumpectomy breast tissues excised during breast-conserving surgeries. Aim: We expand the application of HSDFM to the classification of tissue types and tumor subtypes in pre-histopathology human breast lumpectomy samples. Approach: Breast tissues excised during breast-conserving surgeries were imaged by the HSDFM and analyzed. The performance of the HSDFM is evaluated by comparing the backscattering intensity spectra of polystyrene microbead solutions with the Monte Carlo simulation of the experimental data. For classification algorithms, two analysis approaches, a supervised technique based on the spectral angle mapper (SAM) algorithm and an unsupervised technique based on the K-means algorithm are applied to classify various tissue types including carcinoma subtypes. In the supervised technique, the SAM algorithm with manually extracted endmembers guided by H&E annotations is used as reference spectra, allowing for segmentation maps with classified tissue types including carcinoma subtypes. Results: The manually extracted endmembers of known tissue types and their corresponding threshold spectral correlation angles for classification make a good reference library that validates endmembers computed by the unsupervised K-means algorithm. The unsupervised K-means algorithm, with no a priori information, produces abundance maps with dominant endmembers of various tissue types, including carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma. The two carcinomas' unique endmembers produced by the two methods agree with each other within <2% residual error margin. Conclusions: Our report demonstrates a robust procedure for the validation of an unsupervised algorithm with the essential set of parameters based on the ground truth, histopathological information. We have demonstrated that a trained library of the histopathology-guided endmembers and associated threshold spectral correlation angles computed against well-defined reference data cubes serve such parameters. Two classification algorithms, supervised and unsupervised algorithms, are employed to identify regions with carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma present in the tissues. The two carcinomas' unique endmembers used by the two methods agree to <2% residual error margin. This library of high quality and collected under an environment with no ambient background may be instrumental to develop or validate more advanced unsupervised data cube analysis algorithms, such as effective neural networks for efficient subtype classification.

Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?

The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.

Unresolved haemosporidia of the Australian skink, Egernia stokesii.

The Australian skink Egernia stokesii had been recognised as a host of two species of Plasmodium, Plasmodium mackerrasae and P. circularis; nevertheless, molecular data are available for only a single haemosporidian species of this host. Its sequences are labelled as "Plasmodium sp." or "Plasmodium mackerrasae", but morphological characteristics of this isolate are unavailable. Phylogenetic analyses of these sequences placed them into the clade of the genus Haemocystidium. In this study, blood samples of six E. stokesii were analysed by both, molecular and microscopic methods to clarify the haemosporidia of this lizard. Application of these approaches offered discordant results. Whereas sequence analysis clustered our isolates with lizard species of Haemocystidium, morphology of blood stages is more akin to Plasmodium than Haemocystidium. However, limited sampling, indistinguishable nuclei/merozoites and risk of possible hidden presence of mixed infection prevent reliable species identification of detected parasites or their description as new species of Haemocystidium.

Unveiling Trichosporon austroamericanum sp. nov.: A Novel Emerging Opportunistic Basidiomycetous Yeast Species.

During an epidemiological survey, a potential novel species within the basidiomycetous yeast genus Trichosporon was observed. The clinical strain was obtained from a urine sample taken from a Brazilian kidney transplant recipient. The strain was molecularly identified using the intergenic spacer (IGS1) ribosomal DNA locus and a subsequent phylogenetic analysis showed that multiple strains that were previously reported by other studies shared an identical IGS1-genotype most closely related to that of Trichosporon inkin. However, none of these studies provided an in-depth characterization of the involved strains to describe it as a new taxon. Here, we present the novel clinically relevant yeast for which we propose the name Trichosporon austroamericanum sp. nov. (holotype CBS H-24937). T. austroamericanum can be distinguished from other siblings in the genus Trichosporon using morphological, physiological, and phylogenetic characters.

Visualization of intracellular ice formation and growth in mouse oocytes.

BACKGROUND: Characterization of intracellular ice formation (IIF) in oocytes during the freezing and thawing processes will contribute to optimizing their cryopreservation. However, the observation of the ice formation process in oocytes is limited by the spatiotemporal resolution of the cryomicroscope systems. OBJECTIVE: To observe the intracellular icing of oocytes during cooling and rewarming, and to study the mechanism of formation and growth of intracellular ice in oocytes. MATERIALS AND METHODS: Mouse oocytes were frozen at different cooling rates to induce intracellular ice formation using a cryomicroscopy system consisting of a microscope equipped with a cryogenic cold stage, an automatic cooling system, a temperature control system, and a high-speed camera. The growth patterns of intracellular ice in oocytes were analyzed from the images recorded. Finally, the growth rate of intracellular ice formation in oocytes was calculated using an automatic intracellular ice tracking method. RESULTS: The IIF temperature decreased gradually with the increase in cooling rate. Initiation sites of IIF could be classified into three categories: marginal type, internal type and coexisting type. There was a strong predominance for ice crystal initiation site in the oocytes, with up to 80% of the initiation sites located in the marginal region. The intracellular ice growth modes of darkening and twitching cells were characterized by "spreading" and "clustering", respectively. In addition, twitching cells started to recrystallize during rewarming, while darkening cells did not. The instantaneous maximal growth rate of ice crystals in twitching cells was about 10 times higher than that in darkening cells. CONCLUSION: By visualising the growth of ice crystals in mouse oocytes during cooling and rewarming, we obtained valuable information on the kinetics of ice formation and melting in these cells. This information can help us understand how ice formation and melting affect the viability and quality of oocytes after cryopreservation. Doi.org/10.54680/fr24310110412.

Using Dimensionality Reduction to Visualize Phenotypic Changes in High-Throughput Microscopy.

High-throughput microscopy has enabled screening of cell phenotypes at unprecedented scale. Systematic identification of cell phenotype changes (such as cell morphology and protein localization changes) is a major analysis goal. Because cell phenotypes are high-dimensional, unbiased approaches to detect and visualize the changes in phenotypes are still needed. Here, we suggest that changes in cellular phenotype can be visualized in reduced dimensionality representations of the image feature space. We describe a freely available analysis pipeline to visualize changes in protein localization in feature spaces obtained from deep learning. As an example, we use the pipeline to identify changes in subcellular localization after the yeast GFP collection was treated with hydroxyurea.

Art under the Microscope.

In March 2018, Greece issued five commemorative stamps that show a beautiful mélange of art and science. Images are, however, genuine views captured through light microscopy of stained human tissue during histopathological examination. Many will remember having drawn such diagrams in their histology and pathology journals during their medical school years. The microscopic images are photographed beautifully by Dr Maria Lambropaulou. She is an Associate Professor of Histology-embryology at the Medical Department of the Democritus University of Thrace, Greece.

Laser speckle rheological microscopy reveals wideband viscoelastic spectra of biological tissues.

Viscoelastic transformation of tissue drives aberrant cellular functions and is an early biomarker of disease pathogenesis. Tissues scale a range of viscoelastic moduli, from biofluids to bone. Moreover, viscoelastic behavior is governed by the frequency at which tissue is probed, yielding distinct viscous and elastic responses modulated over a wide frequency band. Existing tools do not quantify wideband viscoelastic spectra in tissues, leaving a vast knowledge gap. We present wideband laser speckle rheological microscopy (WB-SHEAR) that reveals elastic and viscous response over sub-megahertz frequencies previously not investigated in tissue. WB-SHEAR uses an optical, noncontact approach to quantify wideband viscoelastic spectra in specimens spanning a range of moduli from low-viscosity fibrin to highly elastic bone. Via laser scanning, micromechanical imaging is enabled to access wideband viscoelastic spectra in heterogeneous tumor specimens with high spatial resolution (25 micrometers). The ability to interrogate the viscoelastic landscape of diverse biospecimens could transform our understanding of mechanobiological processes in various diseases.

Tools and methods for high-throughput single-cell imaging with the mother machine.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning-based segmentation, 'what you put is what you get' (WYPIWYG) - that is, pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother machine-based high-throughput imaging and analysis methods in their research.