Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM

The spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the world′s highest time resolutions for single fluorescent-molecule imaging of 33 (100) µs (multiple single molecules simultaneously) with a single-molecule localization precision of 34 (20) nm for Cy3 (best dye found), and for PALM data acquisition of a view-field of 640x640 pixels at 1 kHz with a single-molecule localization precision of 29 nm for mEos3.2. Both are considered the ultimate rates with available probes. This camera system (1) successfully detected fast hop diffusion of membrane molecules in the plasma membrane, detectable previously only by using less preferable 40-nm gold probes and bright-field microscopy, and (2) enabled PALM imaging of the entire live cell, while revealing meso-scale dynamics and structures, caveolae and paxillin islands in the focal adhesion, proving its usefulness for cell biology research.

NMN treatment reverses unique deep radiomic signature morphology of oocytes from aged mice

One of the earliest physiological consequences of advancing age is the loss of female reproductive potential. This is primarily due to oocyte quality and developmental competence, which is highly sensitive to biological age. We applied deep learning, swarm intelligence and discriminative analysis to images of mouse oocytes taken by common bright field microscopy and were successfully able to identify a highly informative deep radiomic signature (DRS) of oocyte age. This signature distinguished morphological changes in oocytes associated with maternal age with 92% accuracy (AUC~1), reflecting this decline in oocyte quality. We then employed the DRS to evaluate the impact of the treatment of reproductively aged mice with the NAD+ precursor nicotinamide mononucleotide (NMN). The DRS signature classified 60% of oocytes from NMN-treated aged mice as having a 'young' morphology, suggesting that NMN was able to rejuvenate the morphological changes identified by the DRS. These findings indicate that NMN therapy may be able to restore the quality of a sizable subset of oocytes affected by reproductive ageing, and that these oocytes will be able to be distinguished and selected by DRS.

Histologic Examination of a Sea Pig (Scotoplanes sp.) Using Bright Field Light Microscopy

Sea pigs (Scotoplanes spp.) are deep-sea dwelling sea cucumbers of the phylum Echinodermata, class Holothuroidea, and order Elasipodida. Few reports are available on the microscopic anatomy of these deep-sea animals. This study describes the histologic findings of two, wild, male and female Scotoplanes sp. collected from Monterey Bay, California. Microscopic findings were similar to other holothuroids, with a few notable exceptions. Sea pigs were bilaterally symmetrical with six pairs of greatly enlarged tube feet arising from the lateral body wall and oriented ventrally for walking. Neither a rete mirabile nor respiratory tree was identified, and the large tube feet may function in respiration. Dorsal papillae protrude from the bivium and are histologically similar to tube feet with a large, muscular water vascular canal in the center. There were 10 buccal tentacles, the epidermis of which was highly folded. Only a single gonad was present in each animal; both male and female had histologic evidence of active gametogenesis. In the male, a presumed protozoal cyst was identified in the aboral intestinal mucosa, and was histologically similar to previous reports of coccidians. This work provides control histology for future investigations of sea pigs and related animals using bright field microscopy.

Multispectral intravital microscopy for simultaneous bright-field and fluorescence imaging of the microvasculature

Intravital video microscopy permits the observation of microcirculatory blood flow. This often requires fluorescent probes to visualize structures and dynamic processes that cannot be observed with conventional bright-field microscopy. Conventional light microscopes do not allow for simultaneous bright-field and fluorescent imaging. Moreover, in conventional microscopes, only one type of fluorescent label can be observed. This study introduces multispectral intravital video microscopy, which combines bright-field and fluorescence microscopy in a standard light microscope. The technique enables simultaneous real-time observation of fluorescently-labeled structures in relation to their direct physical surroundings. The advancement provides context for the orientation, movement, and function of labeled structures in the microcirculation.

Histologic lesions of experimental infection with Lymantria dispar multicapsid nucleopolyhedrovirus and Lymantria dispar cytoplasmic polyhedrosis virus in European gypsy moth caterpillars (Lymantria dispar dispar)

European gypsy moths ( Lymantria dispar dispar) are an invasive species in North America, and are listed by the International Union for the Conservation of Nature as one of the 100 most destructive invasive species worldwide. They have several known viruses, some of which are used as biological control agents. However, there are no detailed descriptions of many entomopathogenic viral infections, including in European gypsy moths, using bright-field microscopy. In this study, 11 European gypsy moth caterpillars were evaluated histologically: 4 were experimentally infected with Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV; Baculoviridae); 4 were experimentally infected with Lymantria dispar cytoplasmic polyhedrosis virus (LdCPV; Reoviridae); 3 control animals were uninfected. A complete tissue set was evaluated in all animals from all groups using bright-field microscopy, including epidermis, cuticle, striated muscle, tracheae, foregut, midgut, hindgut, Malpighian tubules, hemocytes, fat body, and nervous system. LdMNPV-infected caterpillars had marked karyomegaly and intranuclear viral inclusions in cells of the epidermis, tracheae, fat body, and hemocytes. LdMNPV-infected caterpillars also had hyperplasia and hypertrophy of epidermal and tracheal epithelial cells. LdCPV-infected caterpillars had numerous granular eosinophilic intracytoplasmic viral inclusions in midgut epithelial cells. Both LdMNPV-infected and LdCPV-infected caterpillars had atrophy of fat body adipocytes; this change was more pronounced in LdCPV-infected caterpillars. This work provides the first detailed descriptions of these viral infections in European gypsy moth caterpillars using bright-field light microscopy and provides images of normal histology from control caterpillars.

Recent Trends and Perspectives in Cerebral Organoids Imaging and Analysis

Purpose: Since their first generation in 2013, the use of cerebral organoids has spread exponentially. Today, the amount of generated data is becoming challenging to analyze manually. This review aims to overview the current image acquisition methods and to subsequently identify the needs in image analysis tools for cerebral organoids.Methods: To address this question, we went through all recent articles published on the subject and annotated the protocols, acquisition methods, and algorithms used.Results: Over the investigated period of time, confocal microscopy and bright-field microscopy were the most used acquisition techniques. Cell counting, the most common task, is performed in 20% of the articles and area; around 12% of articles calculate morphological parameters. Image analysis on cerebral organoids is performed in majority using ImageJ software (around 52%) and Matlab language (4%). Treatments remain mostly semi-automatic. We highlight the limitations encountered in image analysis in the cerebral organoid field and suggest possible solutions and implementations to develop.Conclusions: In addition to providing an overview of cerebral organoids cultures and imaging, this work highlights the need to improve the existing image analysis methods for such images and the need for specific analysis tools. These solutions could specifically help to monitor the growth of future standardized cerebral organoids.

Stage-Dependent Topographical and Optical Properties of Plasmodium Falciparum-Infected Red Blood Cells

Efficient malaria treatment is a major healthcare challenge. Addressing this challenge requires in-depth understanding of malaria parasite maturation during the intraerythrocytic cycle. Exploring the structural and functional changes of the parasite through the intraerythrocytic stages and their impact on red blood cells (RBCs) is a cornerstone of antimalarial drug development. In order to precisely trace such changes, we performed a thorough imaging study of RBCs infected by Plasmodium falciparum, by using atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF) supplemented with bright field microscopy for stage assignment. This multifaceted imaging approach allows to reveal structure–function relations via correlations of the parasite maturation with morphological and fluorescence properties of the stages. We established diagnostic patterns characteristic to the parasite stages based on the topographical profile of infected RBCs, which show close correlation with their fluorescence (TIRF) map. Furthermore, we found that hemozoin crystals exhibit a strong optical contrast, possibly due to the quenching of fluorescence. The topographical and optical features provide a tool for locating the hemozoin crystals within the RBCs and following their growth.

Development of an open source program to identify the size of objects using bright-field microscopy - an automated calibration tool

In light microscopy, eyepiece graticules are commonly used to gauge the size of objects at the micron scale. While this is a relatively simple tool to use, not all microscopes possess this feature. Furthermore, calibrating an eyepiece graticule with a stage micrometer can be time-consuming, particularly for inexperienced microscopists. Similarly, calculating the size of individual objects may also take some time. We present an open-source program to determine the size of objects under a microscope using Python and OpenCV. Taking photos of a stage micrometer under a microscope, we identify gradations on the micrometer and calculate the distance between lines on the micrometer in pixels. From this, we can infer the size of objects from bright-field microscopy images. We believe this will improve access to quantitative microscopy techniques and increase the speed at which samples may be analyzed by light microscopy. Future studies may aim to integrate this with machine learning for object identification

Probing roto-translational diffusion of small anisotropic colloidal particles with a bright-field microscope

Soft and biological materials are often composed of elementary constituents exhibiting an incessant roto-translational motion at the microscopic scale. Tracking this motion with a bright-field microscope becomes increasingly challenging when the particle size becomes smaller than the microscope resolution, a case which is frequently encountered. Here we demonstrate squared-gradient differential dynamic microscopy (SG-DDM) as a tool to successfully use bright-field microscopy to extract the roto-translational dynamics of small anisotropic colloidal particles, whose rotational motion cannot be tracked accurately in direct space. We provide analytical justification and experimental demonstration of the method by successful application to an aqueous suspension of peanut-shaped particles. Graphic abstract