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.

Axially swept open-top light-sheet microscopy for densely labeled clinical specimens.

Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.

1 Million Segmented Red Blood Cells With 240 K Classified in 9 Shapes and 47 K Patches of 25 Manual Blood Smears.

Around 20% of complete blood count samples necessitate visual review using light microscopes or digital pathology scanners. There is currently no technological alternative to the visual examination of red blood cells (RBCs) morphology/shapes. True/non-artifact teardrop-shaped RBCs and schistocytes/fragmented RBCs are commonly associated with serious medical conditions that could be fatal, increased ovalocytes are associated with almost all types of anemias. 25 distinct blood smears, each from a different patient, were manually prepared, stained, and then sorted into four groups. Each group underwent imaging using different cameras integrated into light microscopes with 40X microscopic lenses resulting in total 47 K + field images/patches. Two hematologists processed cell-by-cell to provide one million + segmented RBCs with their XYWH coordinates and classified 240 K + RBCs into nine shapes. This dataset (Elsafty_RBCs_for_AI) enables the development/testing of deep learning-based (DL) automation of RBCs morphology/shapes examination, including specific normalization of blood smear stains (different from histopathology stains), detection/counting, segmentation, and classification. Two codes are provided (Elsafty_Codes_for_AI), one for semi-automated image processing and another for training/testing of a DL-based image classifier.

Comparison of microscopic and xpert MTB diagnoses of presumptive mycobacteria tuberculosis infection: retrospective analysis of routine diagnosis at Cape Coast Teaching Hospital.

INTRODUCTION: Tuberculosis is a global health problem that causes 1. 4 million deaths every year. It has been estimated that sputum smear-negative diagnosis but culture-positive pulmonary TB diagnosis contribute to 12.6% of pulmonary TB transmission. TB diagnosis by smear microscopy smear has a minimum detection limit (LOD) of 5,000 to 10,000 bacilli per milliliter (CFU/ml) of sputum result in missed cases and false positives. However, GeneXpert technology, with a LOD of 131-250 CFU/ml in sputum samples and its implementation is believe to facilitate early detection TB and drug-resistant TB case. Since 2013, Ghana health Service (GHS) introduce GeneXpert MTB/RIF diagnostic in all regional hospitals in Ghana, however no assessment of performance between microscopy and GeneXpert TB diagnosis cross the health facilities has been reported. The study compared the results of routine diagnoses of TB by microscopy and Xpert MTB from 2016 to 2020 at the Cape Coast Teaching Hospital (CCTH). METHODS: The study compared routine microscopic and GeneXpert TB diagnosis results at the Cape Coast Teaching Hospital (CCTH) from 2016 to 2020 retrospectively. Briefly, sputum specimens were collected into 20 mL sterile screw-capped containers for each case of suspected TB infection and processed within 24 h. The samples were decontaminated using the NALC-NaOH method with the final NaOH concentration of 1%. The supernatants were discarded after the centrifuge and the remaining pellets dissolved in 1-1.5 ml of phosphate buffer saline (PBS) and used for diagnosis. A fixed smears were Ziehl-Neelsen acid-fast stain and observed under microscope and the remainings were used for GeneXpert MTB/RIF diagnosis. The data were analyze using GraphPad Prism. RESULTS: 50.11% (48.48-51.38%) were females with an odd ratio (95% CI) of 1.004 (0.944-1.069) more likely to report to the TB clinic for suspected TB diagnosis. The smear-positive cases for the first sputum were 6.6% (5.98-7.25%), and the second sputum was 6.07% (5.45-6.73%). The Xpert MTB-RIF diagnosis detected 2.93% (10/341) (1.42-5.33%) in the first and 5.44% (16/294) (3.14-8.69%) in the second smear-negative TB samples. The prevalence of Xpert MTB-RIF across smear positive showed that males had 56.87% (178/313) and 56.15% (137/244) and females had 43.13% (135/313) and 43.85% (107/244) for the first and second sputum. Also, false negative smears were 0.18% (10/5607) for smear 1 and 0.31% (16/5126) for smear 2. CONCLUSION: In conclusion, the study highlights the higher sensitivity of the GeneXpert assay compared to traditional smear microscopy for detecting MTB. The GeneXpert assay identified 10 and 16 positive MTB from smear 1 and smear 2 samples which were microscopic negative.

[Development of Two-Photon Super-Resolution Microscopy].

Two-photon excitation microscopy enables in vivo deep-tissue imaging within organisms. This technique is based on two-photon excitation, a nonlinear optical process that uses near-infrared light for excitation, resulting in high tissue permeability. Notably, two-photon excitation occurs only near the focal plane; therefore, minimally invasive tomographic images can be obtained. Owing to these features, two-photon excitation microscopy is currently widely used in medical and life-science research, particularly in the domain of neuroscience for in vivo visualization of deep tissues. However, the use of long-wavelength excitation light in two-photon excitation microscopy has resulted in a larger focused spot size and relatively low spatial resolution, which is a limitation of this technique for further applications. Recent studies have described super-resolution microscopy techniques applied to two-photon excitation microscopy in an attempt to observe living organisms "as they are in their natural state" with high spatial resolution. We have also addressed this topic using an optical approach (two-photon stimulated emission depletion microscopy) and an image analysis approach (two-photon super-resolution radial fluctuation). Here, we describe these approaches together with a discussion of our recent accomplishments.

Using lipid binding proteins and advanced microscopy to study lipid domains.

Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.

Precise Serial Microregistration Enables Quantitative Microscopy Imaging Tracking of Human Skin Cells In Vivo.

We developed an automated microregistration method that enables repeated in vivo skin microscopy imaging of the same tissue microlocation and specific cells over a long period of days and weeks with unprecedented precision. Applying this method in conjunction with an in vivo multimodality multiphoton microscope, the behavior of human skin cells such as cell proliferation, melanin upward migration, blood flow dynamics, and epidermal thickness adaptation can be recorded over time, facilitating quantitative cellular dynamics analysis. We demonstrated the usefulness of this method in a skin biology study by successfully monitoring skin cellular responses for a period of two weeks following an acute exposure to ultraviolet light.

Gastrointestinal parasites of Wolffsohn's viscacha (Chinchillidae: Lagidium wolffsohni), an endemic rodent species from the wild Patagonia.

Parasites are ubiquitous in wildlife populations and have a profound impact on population dynamics. Interest in parasites of wildlife has increased significantly in recent years, particularly in those with relevant conservation status. Patagonia is one of the wildest and remote areas of the world. The Wolffsohn's viscacha lives in a small mountainous area of Patagonia. Until now, little is known about the biology and ecology of this species. The aim of this research was to study the gastrointestinal parasite diversity in this rodent from a coprological survey. A total of 125 fecal samples from 25 colonies were examined. Each sample was rehydrated, homogenized, and analyzed using three parasitological techniques: spontaneous sedimentation, Mini-FLOTAC, and centrifugation-flotation in sucrose-saturated solution, followed by examination under optical microscopy. The samples, eggs, and oocysts of parasites were described, measured, and photographed. All colonies were positive for at least one parasite species. A total of 10 parasitic species were identified: Viscachataenia sp., possibly V. quadrata, Monoecocestus sp., an unidentified anoplocephalid, Heteroxynema sp., possibly H. (Cavioxyura) viscaciae, Helminthoxys sp., possibly H. effilatus, an unidentified strongylid-type egg, Trichuris sp., two morphologies of unidentified coccidians and Eimeria sp. This is the first exhaustive study of gastrointestinal parasites in L. wolffsohni and a large number of eggs and oocysts of parasites were found. Our results highlight the use of noninvasive techniques for the study of parasites of wildlife hosts; as in the case of this rodent with a remote habitat, which makes sampling difficult. The results of our study provide baseline information on gastrointestinal parasite infections in this species.

Endohelminth diversity in the invasive Clarias gariepinus (Burchell, 1822) from two freshwater lakes: Naivasha and Ol'Bolossat, Kenya.

Fish parasitology contributes to our understanding of the potential risks posed by diverse groups of parasitic organisms on fish stocks in either wild and culture systems. This study was conducted in May 2023 and aimed at assessing the diversity of endohelminths in the invasive North African catfish Clarias gariepinus (Burchell, 1822) obtained from two freshwater lakes, Naivasha and Ol'Bolossat, in Kenya. Parasitological examination of 66 and 35 fish samples collected from the two lakes respectively was achieved using light and scanning electron microscopy methods. Results revealed endohelminth diversity broadly classified as four digeneans, two nematodes, and one cestode. Seven taxa of endohelminths were found in C. gariepinus samples, but only four of these taxa could be identified up to the species level. Six of the taxa (Diplostomum sp., Tylodelphys mashonense, Plagiorchioidea sp., Paracamallanus cyathopharynx, Contracaecum sp., and Tetracampos ciliotheca) were common in samples from the two lakes. Glossidium pedatum only occurred in samples from Lake Ol'Bolossat. Parasite prevalence ranged from 8.6 (T. mashonense) to 100% (Diplostomum sp., T. ciliotheca, and Contracaecum sp.) and mean intensity from 1.4 (T. mashonense) to 16.9 (Diplostomum sp.). The diversity and richness indices were comparatively higher in fish samples from Lake Ol'Bolossat and attributed to the occurrence of G. pedatum in the Ol'Bolossat. However, parasitic infestation of fish samples from the two lakes depicted close similarity, both in diversity and prevalence. These findings form an important baseline data for further follow-up studies, and they suggest the need for further molecular analyses to fully describe three of the taxa only identified up to the genus level.

Detection of demodex mites in papulopustular rosacea using microscopic examination and polymerase chain reaction: a comparative case-control study.

Demodex mite proliferation is frequently involved in the pathogenesis of rosacea. The gold standard for Demodex identification is microscopic examination on a standardized skin surface biopsy. However, this method of sampling can be distressing and painful, especially when performed on hairy sites. In this case-control study, we compared the sensitivity of PCR and microscopic examination in diagnosing a Demodex infestation. Moreover, we investigated the possible correlations between the presence of Demodex mites and clinical characteristics. In total, 20 patients affected by papulopustular rosacea and 10 controls were included. At both microscopic examination and PCR, patients with rosacea presented a greater prevalence of positive samples than controls at the scalp and at the face. Microscopy had sensitivity of 50% at the face and of 46.7% at the scalp. PCR had sensitivity of 93.75% at the face and of 86.7% at the scalp. The positivity of PCR was associated to a higher frequency of facial papules and pustules. Patients with positivity at the face had a more frequent positivity at the scalp. The scalp could represent a reservoir for the Demodex mites, and should be investigated by sensitive and painless methods. PCR performed on painlessly collected samples should be further investigated.

Multimodal Imaging Using Raman Spectroscopy and FTIR in a Single Analytical Instrument with a Microscope (Infrared Raman Microscopy AIRsight, Shimadzu): Opportunities and Applications.

Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy are powerful analytical techniques widely used separately in different fields of study. Integrating these two powerful spectroscopic techniques into one device represents a groundbreaking advance in multimodal imaging. This new combination which merges the molecular vibrational information from Raman spectroscopy with the ability of FTIR to study polar bonds, creates a unique and complete analytical tool. Through a detailed examination of the microscope's operation and case studies, this article illustrates how this integrated analytical instrument can provide more thorough and accurate analysis than traditional methods, potentially revolutionising analytical sample characterisation. This article aims to present the features and possible uses of a unified instrument merging FTIR and Raman spectroscopy for multimodal imaging. It particularly focuses on the technological progress and collaborative benefits of these two spectroscopic techniques within the microscope system. By emphasising this approach's unique benefits and improved analytical capabilities, the authors aim to illustrate its applicability in diverse scientific and industrial sectors.

Trackoscope: A low-cost, open, autonomous tracking microscope for long-term observations of microscale organisms.

Cells and microorganisms are motile, yet the stationary nature of conventional microscopes impedes comprehensive, long-term behavioral and biomechanical analysis. The limitations are twofold: a narrow focus permits high-resolution imaging but sacrifices the broader context of organism behavior, while a wider focus compromises microscopic detail. This trade-off is especially problematic when investigating rapidly motile ciliates, which often have to be confined to small volumes between coverslips affecting their natural behavior. To address this challenge, we introduce Trackoscope, a 2-axis autonomous tracking microscope designed to follow swimming organisms ranging from 10µm to 2mm across a 325cm2 area (equivalent to an A5 sheet) for extended durations-ranging from hours to days-at high resolution. Utilizing Trackoscope, we captured a diverse array of behaviors, from the air-water swimming locomotion of Amoeba to bacterial hunting dynamics in Actinosphaerium, walking gait in Tardigrada, and binary fission in motile Blepharisma. Trackoscope is a cost-effective solution well-suited for diverse settings, from high school labs to resource-constrained research environments. Its capability to capture diverse behaviors in larger, more realistic ecosystems extends our understanding of the physics of living systems. The low-cost, open architecture democratizes scientific discovery, offering a dynamic window into the lives of previously inaccessible small aquatic organisms.

Validation of digital image slides for diagnosis in cervico-vaginal cytology.

OBJECTIVE: To test the diagnostic concordance between microscopic (MI) and digital (DG) observation of cervico-vaginal (CV) cytology in a validation study of the technique. METHODS: Five cytotechnologists (CT) reviewed 888 routine CV cytology cases from the Cervical Pathology Unit of our center over a 2-week period of time. The cases were first observed by MI and at the end of the day the cases were observed by DG. STATISTICAL ANALYSIS USED: Agreement calculated using the Kappa index. RESULTS: Most of the diagnoses corresponded to benign (64%) or inflammatory conditions (14%) and 24% corresponded to the intraepithelial lesion or malignancy (ILM) category. The overall kappa coefficient of concordance was strong (0.87). Among the different CTs it was almost perfect in two, strong in two and moderate in one. In 18 cases (10%) there were discrepancies between techniques in the category of ILM. In 10 (56%) cases there was an overdiagnosis in DG and in 8 (44%) an overdiagnosis in MI. Only in two cases, the diagnostic discrepancy exceeded one degree of difference between lesions, and they were ASCUS or AGUS for DG and CIN 2 for MI. CONCLUSIONS: In this validation test in which routine cases during a two-week period have been used, observing the cases with both techniques on the same day, we have obtained a strong degree of concordance. The discordances obtained have not been considered relevant.

Single-beam digital holographic reconstruction: a phase-support enhanced complex wavefront on phase-only function for twin-image elimination.

Significance: In in-line digital holographic microscopy (DHM), twin-image artifacts pose a significant challenge, and reduction or complete elimination is essential for object reconstruction. Aim: To facilitate object reconstruction using a single hologram, significantly reduce inaccuracies, and avoid iterative processing, a digital holographic reconstruction algorithm called phase-support constraint on phase-only function (PCOF) is presented. Approach: In-line DHM simulations and tabletop experiments employing the sliding-window approach are used to compute the arithmetic mean and variance of the phase values in the reconstructed image. A support constraint mask, through variance thresholding, effectively enabled twin-image artifacts. Results: Quantitative evaluations using metrics such as mean squared error, peak signal-to-noise ratio, and mean structural similarity index show PCOF's superior capability in eliminating twin-image artifacts and achieving high-fidelity reconstructions compared with conventional methods such as angular spectrum and iterative phase retrieval methods. Conclusions: PCOF stands as a promising approach to in-line digital holographic reconstruction, offering a robust solution to mitigate twin-image artifacts and enhance the fidelity of reconstructed objects.

Multimodal Image Fusion Workflow Incorporating MALDI Imaging Mass Spectrometry and Microscopy for the Study of Small Pharmaceutical Compounds.

Multimodal imaging analyses of dosed tissue samples can provide more comprehensive insights into the effects of a therapeutically active compound on a target tissue compared to single-modal imaging. For example, simultaneous spatial mapping of pharmaceutical compounds and endogenous macromolecule receptors is difficult to achieve in a single imaging experiment. Herein, we present a multimodal workflow combining imaging mass spectrometry with immunohistochemistry (IHC) fluorescence imaging and brightfield microscopy imaging. Imaging mass spectrometry enables direct mapping of pharmaceutical compounds and metabolites, IHC fluorescence imaging can visualize large proteins, and brightfield microscopy imaging provides tissue morphology information. Single-cell resolution images are generally difficult to acquire using imaging mass spectrometry but are readily acquired with IHC fluorescence and brightfield microscopy imaging. Spatial sharpening of mass spectrometry images would thus allow for higher fidelity coregistration with other higher-resolution microscopy images. Imaging mass spectrometry spatial resolution can be predicted to a finer value via a computational image fusion workflow, which models the relationship between the intensity values in the mass spectrometry image and the features of a high-spatial resolution microscopy image. As a proof of concept, our multimodal workflow was applied to brain tissue extracted from a Sprague-Dawley rat dosed with a kratom alkaloid, corynantheidine. Four candidate mathematical models, including linear regression, partial least-squares regression, random forest regression, and two-dimensional convolutional neural network (2-D CNN), were tested. The random forest and 2-D CNN models most accurately predicted the intensity values at each pixel as well as the overall patterns of the mass spectrometry images, while also providing the best spatial resolution enhancements. Herein, image fusion enabled predicted mass spectrometry images of corynantheidine, GABA, and glutamine to approximately 2.5 µm spatial resolutions, a significant improvement compared to the original images acquired at 25 µm spatial resolution. The predicted mass spectrometry images were then coregistered with an H&E image and IHC fluorescence image of the µ-opioid receptor to assess colocalization of corynantheidine with brain cells. Our study also provides insights into the different evaluation parameters to consider when utilizing image fusion for biological applications.

Perspective Article: Space-time dynamics of genome replication studied with super-resolved microscopy.

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.

Comparative Optics of the Surgical Microscope and Rigid Endoscopes in Neurosurgery.

This chapter is intended to provide a brief overview of the optics of surgical microscopes and rigid endoscopes, with the aim of providing the reader with the principles dictating the nature of surgical visualization when either of the visual control systems is used. It is not by any means geared toward elaborating on the detailed optical physics of these systems, which is beyond the scope and objective of this chapter.