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Collagen, the main load-bearing component in tissue, is present in all animals and forms a variety of networks from the fibrils, fibers, bundles, and lamellae into which it self-assembles. The collagen microstructure is different among tissue types, and the different microstructures give rise to tissue-specific mechanical properties. Therefore, methods for visualizing collagen fibers and their orientation are essential for understanding the biomechanical properties of tissue. Our aim in this review is to provide the basis for understanding the methodology of polarized light imaging methods and how they can be used to characterize collagen microstructure. We begin with a description of collagen microstructure and its relationship to tissue biomechanics, a basic formalism of polarized light, and how collagen interacts with polarized light. We then describe polarized light microscopy and its various forms, particularly instant polarized light microscopy, then polarization-sensitive optical coherence tomography, and last, polarization-resolved second-harmonic generation microscopy. We describe methods for imaging collagen microstructure with polarized light from methods to high-resolution volumetric imaging of tissue sections. We intend to help those interested in using polarized light to image and understand the relationship between collagen microstructure and biomechanics.

Background Light microscopy is often used for malaria diagnosis in the field. However, it is time-consuming and quality of the results depends heavily on the skill of microscopists. Automating malaria light microscopy is a promising solution, but it still remains a challenge and an active area of research. Current tools are often expensive and involve sophisticated hardware components, which makes it hard to deploy them in resource-limited areas. Results We designed an Android mobile application called Malaria Screener, which makes smartphones an affordable yet effective solution for automated malaria light microscopy. The mobile app utilizes high-resolution cameras and computing power of modern smartphones to screen both thin and thick blood smear images for P. falciparum parasites. Malaria Screener combines image acquisition, smear image analysis, and result visualization in its slide screening process, and is equipped with a database to provide easy access to the acquired data. Conclusion Malaria Screener makes the screening process faster, more consistent, and less dependent on human expertise. The app is modular, allowing other research groups to integrate their methods and models for image processing and machine learning, while acquiring and analyzing their data.

Polarized light microscopy (PLM) is a common but critical method for pharmaceutical crystallinity characterization, which has been widely introduced for research purposes or drug testing and is recommended by many pharmacopeias around the world. To date, crystallinity characterization of pharmaceutical solids is restricted to laboratories due to the relatively bulky design of the conventional PLM system, while little attention has been paid to on-site, portable, and low-cost applications. Herein, we developed a smartphone-based polarized microscope with an ultra-miniaturization design (“hand-held” scale) for these purposes. The compact system consists of an optical lens, two polarizers, and a tailor-made platform to hold the smartphone. Analytical performance parameters including resolution, imaging quality of interference color, and imaging reproducibility were measured. In a first approach, we illustrated the suitability of the device for pharmaceutical crystallinity characterization and obtained high-quality birefringence images comparable to a conventional PLM system, and we also showed the great promise of the device for on-site characterization with high flexibility. In a second approach, we employed the device as a proof of concept for a wider application ranging from liquid crystal to environmental pollutants or tissues from plants. As such, this smartphone-based hand-held polarized light microscope shows great potential in helping pharmacists both for research purposes and on-site drug testing, not to mention its broad application prospects in many other fields. Graphical Abstract

Wastewater treatment plants (WWTPs) are one of the most important ways of releasing microplastics (MPs) into the environment. In this study, the size, number, color, and shape of MPs during the wastewater treatment process were investigated in six WWTPs with different processes, which include WWTPs A and B (activated sludge process, ASP), WWTP C and D (aerated lagoon, AL), WWTP E (sequencing batch reactor process, SBR), and WWTP F (stabilization pond, SP). The MP particles were detected by the polarized light microscopy. In all six WWTPs, the clear color was observed as the dominant color in the effluent. Among the forms of MPs, fibers had the highest removal efficiency in WWTPs A (97.3%), B (99.2%), C (95.5%), and D (94.3%). In both WWTPs E and F, the highest removal rate of MP shapes was related to films (96.1%) and granules (86.1%), respectively. MPs with size 25–125 μm had the highest amount (0.39 to 4.08 MP/L) in the effluent of WWTPs compared to larger sizes. With respect to the type of the wastewater treatment process, the number of MPs during the treatment process decreased from 3.75–25.31 to 0.51–6.28MP/L. Based on the results of this study, ASP with a removal rate of 91.87% had the highest efficiency compared to other processes. However, daily 4.95 × 104 to 1.49 × 108 MP enter the environment via the effluent of these WWTPs. The study recommends reducing the use of MPs and plasticizers in widely used products as much as possible and replacing them with nature-friendly materials.

Background Calcium oxalate (CaOx) crystals are commonly found in many plant species. These crystals vary in distribution and morphology and to elucidate their role in plants multiple methods have been applied. Raman imaging and polarized light microscopy (PLM) easily visualize the crystals within plant tissues, but both methods are limited in spatial resolution by the diffraction of light. To unravel the distinctive shape and morphology of CaOx crystals down to the nanoscale and how they are embedded within cells, high resolution scanning electron microscopy is needed. To grasp the full potential of multiple methods in CaOx studies, a novel and easy-to-build correlative sampling approach is presented on different nut species (pecan ( Carya illinoinensis ), Turkish hazel ( Corylus colurna ) and black walnut ( Juglans nigra )), including soft tissues (young developmental stages) as well as hard tissues (mature nutshells). Result Young seed coat tissues as well as mature nutshells included distinct morphological CaOx features, like druses and prismatic crystals. By Raman imaging the chemical composition of all investigated crystals was verified as calcium oxalate monohydrate (COM) and Raman band intensity changed according to crystal plane orientation with respect to incident laser polarisation. Calcium oxalate dihydrate (COD) was only found in the young C. illinoinensis seed coat and was restricted to a few pixels adjacent to cell walls. These thin cell walls were identified as pectin-rich, while in the mature nutshells the crystals were surrounded by thicker and highly lignified cell walls. The Raman and light microscopy results were correlated with SEM images, which gave additional information on crystal surface structure and/or internal porosity on the nanoscale. Conclusion The presented correlative approach preserved the structural integrity of crystals and cellular structures during cutting and transferring between microscopes. Analysing exactly the same sample (position) by Raman, polarized light microscopy and SEM opens the view on the distribution within tissues and cells as well as the molecular structure of the crystals and adjacent cell structures. Such a comprehensive in-situ characterization paves the way for a better understanding of mineralization processes of different minerals in all kinds of biological tissues. Graphical abstract

Determining egg quality is the foremost challenge in assisted reproductive technology (ART). Although extensive advances have been made in multiple areas of ART over the last 40 years, oocyte quality assessment tools have not much evolved beyond standard morphological observation. The oocyte not only delivers half of the nuclear genetic material and all of the mitochondrial DNA to an embryo but also provides complete developmental support during embryonic growth. Oocyte mitochondrial numbers far exceed those of any somatic cell, yet little work has been done to evaluate the mitochondrial bioenergetics of an oocyte. Current standard oocyte assessment in in vitro fertilization (IVF) centers include the observation of oocytes and their surrounding cell complex (cumulus cells) via stereomicroscope or inverted microscope, which is largely primitive. Additional oocyte assessments include polar body grading and polarized light meiotic spindle imaging. However, the evidence regarding the aforementioned methods of oocyte quality assessment and IVF outcomes is contradictory and non-reproducible. High-resolution microscopy techniques have also been implemented in animal and human models with promising outcomes. The current era of oocyte imaging continues to evolve with discoveries in artificial intelligence models of oocyte morphology selection albeit at a slow rate. In this review, the past, current, and future oocyte imaging techniques will be examined with the goal of drawing attention to the gap which limits our ability to assess oocytes in real time. The implications of improved oocyte imaging techniques on patients undergoing IVF will be discussed as well as the need to develop point of care oocyte assessment testing in IVF labs. Summary Sentence Non-invasive oocyte imaging techniques in assisted reproductive technologies are scarce and underdeveloped. The future of oocyte imaging methods should be low cost and safe with high-resolution technology. Graphical Abstract

Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at −140 °C.

Woody cells, such as tracheids, fibers, vessels, rays etc., have unique structural characteristics such as nano-scale ultrastructure represented by multilayers, microfibril angle (MFA), micro-scale anatomical properties and spatial arrangement. Simultaneous evaluation of the above indices is very important for their adequate quantification and extracting the effects of external stimuli from them. However, it is difficult in general to achieve the above only by traditional methodologies. To overcome the above point, a new methodological framework combining polarization optical microscopy, fluorescence microscopy, and image segmentation is proposed. The framework was tested to a model softwood species, Chamaecyparis obtusa for characterizing intra-annual transition of MFA and tracheid morphology in a radial file unit. According our result, this framework successfully traced the both characteristics tracheid by tracheid and revealed the high correlation (| r | > 0.5) between S 2 microfibril angles and tracheidal morphology (lumen radial diameter, tangential wall thickness and cell wall occupancy). In addition, radial file based evaluation firstly revealed their complex transitional behavior in transition and latewood. The proposed framework has great potential as one of the unique tools to provide detailed insights into heterogeneity of intra and inter-cells in the wide field of view through the simultaneous evaluation of cells’ ultrastructure and morphological properties.

Significance: The cerebrovasculature has become increasingly recognized as a major player in overall brain health and many brain disorders. Although there have been several landmark studies to understand details of these crucially important structures in an anatomically defined area, brain-wide examination of the whole cerebrovasculature, including microvessels, has been challenging. However, emerging techniques, including tissue processing and three-dimensional (3D) microscopy imaging, enable neuroscientists to examine the total vasculature in the entire mouse brain. Aim: Here, we aim to highlight advances in these high-resolution 3D mapping methods including block-face imaging and light sheet fluorescent microscopy. Approach: We summarize latest mapping tools to understand detailed anatomical arrangement of the cerebrovascular network and the organizing principles of the neurovascular unit (NVU) as a whole. Results: We discuss biological insights gained from studies using these imaging methods and how these tools can be used to advance our understanding of the cerebrovascular network and related cell types in the entire brain. Conclusions: This review article will help to understand recent advance in high-resolution NVU mapping in mice and provide perspective on future studies.

This study evaluates the quality of 30 biomass pellets sold for residential use in Poland. It provides data on their physical, chemical, and petrographic properties and compares them to existing standards and the information provided by the fuel producers. The results reveal considerable variations in the quality of the pellets and show that some of the purchased samples are not within the DINplus and/or ENplus certification thresholds. Among all 30 purchased samples, only one passes the quality thresholds set by the PL-US BIO, a newly established quality certification in Poland that combines quality assessment following DINplus with optical microscopy analysis. The primary issues causing a decrease in pellet quality include elevated ash and fines content, compromised mechanical durability, too low ash melting temperature, and additions of undesired additions like bark, inorganic matter, and petroleum products. Our research highlights the need for improved fuel quality control measures, and transparent and accurate product labeling, as well as the need for a comprehensive and publicly available national database of solid biomass fuel producers and fuels sold. These are essential steps toward increasing customers’ awareness and trust, encouraging them to embrace biomass fuels as reliable and sustainable sources of energy.