Bright-field Light Microscopy Research Articles

Confocal interferometric scattering microscopy reveals 3D nanoscopic structure and dynamics in live cells

Bright-field light microscopy and related phase-sensitive techniques play an important role in life sciences because they provide facile and label-free insights into biological specimens. However, lack of three-dimensional imaging and low sensitivity to nanoscopic features hamper their application in many high-end quantitative studies. Here, we demonstrate that interferometric scattering (iSCAT) microscopy operated in the confocal mode provides unique label-free solutions for live-cell studies. We reveal the nanometric topography of the nuclear envelope, quantify the dynamics of the endoplasmic reticulum, detect single microtubules, and map nanoscopic diffusion of clathrin-coated pits undergoing endocytosis. Furthermore, we introduce the combination of confocal and wide-field iSCAT modalities for simultaneous imaging of cellular structures and high-speed tracking of nanoscopic entities such as single SARS-CoV-2 virions. We benchmark our findings against simultaneously acquired fluorescence images. Confocal iSCAT can be readily implemented as an additional contrast mechanism in existing laser scanning microscopes. The method is ideally suited for live studies on primary cells that face labeling challenges and for very long measurements beyond photobleaching times.

Morfologia e morfometria de Paratanaisia bragai () Freitas, 1959 (Digenea, Eucotylidae) através da microscopia de luz e microscopiaeletrônica de varredura

Paratanaisia bragai is a digenetic trematode that reaches sexual maturity in the kidney collecting ducts of domestic and wild birds, while the snails Subulina octona and Leptinaria unilamellata serve as its intermediate hosts in Brazil. The present study analyzed the morphology and morphometry of P. bragai. Adult specimens of the parasite were collected from naturally infected Columba livia kidneys, fixed and prepared for observation via bright field and differential interference contrast light microscopy and scanning electron microscopy. The parasite has an elongated and flattened body, with a subterminal oral sucker located at the anterior end of the body, as observed by all techniques used. Staining the parasite with hematoxylin-eosin enabled observation of the pharynx, located posteriorly to the oral sucker, the vitelline glands, which are extra-cecal and extend anteriorly to the pre-ovarian region and later to the median region of the body, and intestinal caeca parallel to the vitelline glands. The presence and functionality of the acetabulum are controversial points in the literature, but it was observed in all specimens analyzed by scanning electron microscopy, with a major diameter of 38.36 ± 6.96 (28.77 - 45.39) and minor diameter of 31.59 ± 7.04 (21.75 - 38.16). Close to the acetabulum, scales were observed in the integument of the parasite. Scales with (1 - 5) blade divisions were identified. In the genital pore, it was possible to see the everted cirrus with rosette shape. The excretory pore (first morphometric record) is dorsal and subterminal, with major diameter of 12.27 ± 9.16 (5.79 - 18.75) and minor diameter of 3.95 ± 1.49 (2.89 - 5.00).

Discrete localization patterns of PIP5Kγ and PLCβ3 working sequentially in phosphoinositide-cycle within mouse sensory neuron somata.

It is known that phosphatidylinositol phosphate 5 kinase (PIP5K) γ and phospholipase C (PLC) β3, working sequentially in the phosphoinositide cycle, are localized in dorsal root ganglion (DRG) somata and are involved in the regulation of pain and related sensations. However, the sites of their involvement have remained to be clarified. In the present study, immunoreactivity for PLCβ3 was distinct only in the central process of mouse DRG, but not in its peripheral process, in contrast to distinct PIP5Kγ-immunoreactivity in both peripheral and central DRG processes. No nerve terminals showing immunoreactivity for PLCβ3 were detected in any peripheral sensory fields, similar to PIP5Kγ-immunoreactivity. In DRG somata, PIP5Kγ-immunoreactivity was rather confined to the neurolemma in which dots and threads were discerned in 3D bright field light microscopy. This feature well corresponded to its discontinuous localization along the plasma membranes in immuno-electron microscopy. In contrast, PLCβ3-immunoreactivity occurred diffusely throughout the somata, but did not take distinct appearance of immunoreaction on neurolemma or plasma membranes, unlike PIP5Kγ-immunoreactivity. In addition, satellite glial cells were immunonegative for PLCβ3, but immunopositive for PIP5Kγ. The involvement of PLCβ3 in regulation of pain and related sensations is thus suggested to be mainly exerted at levels of the DRG soma and its upstream, but to be less significant in the peripheral sensory fields, similar to PIP5Kγ. The possibility is also suggested that PIP, PIP5Kγ-target, is localized heterogeneously, but PIP2, PLCβ3-target, is localized homogenously over the plane of the neuronal plasma membranes. RESEARCH HIGHLIGHTS: PIP5Kγ, different from PLCβ3, was localized heterogeneously on neuronal membranes, and this difference was demonstrated in 3D-bright field immuno-light and electron microscopy. Either PIP5Kγ or PLCβ3 was not detected in peripheral nerve terminals.

Application of Microscopy in the Quality Control of Licorice Roots: Comparative Anatomy of the Roots and Rhizomes of Five Species of Glycyrrhiza.

The raw materials of “licorice root” in the commerce consist of roots and/or rhizomes (stolons) of different species of Glycyrrhiza. Licorice products and raw materials are frequently mislabeled and often have mixed, misidentified, or unidentified species and parts. This paper provides a detailed comparative analysis of the morpho-anatomies of the rhizomes and roots of five species of Glycyrrhiza, namely G. glabra, G. uralensis, G. echinata, G. inflata, and G. lepidota, by bright-field light microscopy and scanning electron microscopy. The studied species showed some similarities in their basic anatomical features due to the fact that they are phylogenetically closely related and belong to the same genus. However, differences in microscopic features such as the thickness of cork and medullary rays, pore frequency, and size of the vessels were observed. The rhizomes can readily be distinguished by the presence of a distinct pith. The roots lack a well-defined pith and instead have primary xylem in the center.

Unravelling mechanisms of protein and lipid oxidation in mayonnaise at multiple length scales

In mayonnaise, lipid and protein oxidation are closely related and the interplay between them is critical for understanding the chemical shelf-life stability of mayonnaise. This is in particular the case for comprehending the role of low-density lipoprotein (LDL) particles acting as a main emulsifier. Here, we monitored oxidation and the concomitant aggregation of LDLs by bright-field light microscopy and cryogenic transmission electron microscopy. We further probed the formation of protein radicals and protein oxidation by imaging the accumulation of a water-soluble fluorescent spin trap and protein autofluorescence. The effect of variation of pH and addition of EDTA on the accumulation of the spin trap validated that protein radicals were induced by lipid radicals. Our data suggests two main pathways of oxidative protein radical formation in LDL particles: (1) at the droplet interface, induced by lipid free radicals formed in oil droplets, and (2) in the continuous phase induced by an independent LDL-specific mechanism.

Micro-morpho-anatomical transitions at various stages of in vitro development of Crinum malabaricum Lekhak and Yadav: A critically endangered medicinal plant.

Crinum malabaricum Lekhak & Yadav is a recently discovered and critically endangered aquatic bulbous plant of the family Amaryllidaceae. It gained attention as a wild source of the acetylcholinesterase inhibiting alkaloid 'galanthamine' used to treat Alzheimer and Parkinson diseases. The bulbs of this plant contain the highest amount of galanthamine among Crinum species. In vitro regeneration systems were developed to produce quality uniform plantlets of C. malabaricum. Bright field light microscopy was used to analyse micro-morpho-anatomical developments taking place in the leaves and roots during in vitro, ex vitro and in vivo transitions of plantlets. Leaves and roots of plants raised in vitro possessed a higher degree of microscopic structural anomalies, such as underdeveloped epicuticular wax deposition, immature and non-functional stomata, more aquiferous parenchyma with a reduced lumen. Roots developed in vitro were characterized by extremely large, uneven cortical cells and reduced intercellular spaces. The vascular tissues were under-developed and only primary vascular tissues were observed. As a result of ex vitro acclimation, there was a significant acceleration in the improvement of tissue systems in leaves and roots. Such plantlets can tolerate elevated temperatures and light under in vivo conditions. Thus, the microscopic evaluation of the structural trajectory in different stages of plantlet development provides an understanding of the acclimation process and structural adaptations, which could help enhance survival of in vitro raised plantlets under ex vitro and in vivo conditions.

Cell segmentation from telecentric bright-field transmitted light microscopy images using a Residual Attention U-Net: A case study on HeLa line

Living cell segmentation from bright-field light microscopy images is challenging due to the image complexity and temporal changes in the living cells. Recently developed deep learning (DL)-based methods became popular in medical and microscopy image segmentation tasks due to their success and promising outcomes. The main objective of this paper is to develop a deep learning, U-Net-based method to segment the living cells of the HeLa line in bright-field transmitted light microscopy. To find the most suitable architecture for our datasets, a residual attention U-Net was proposed and compared with an attention and a simple U-Net architecture.The attention mechanism highlights the remarkable features and suppresses activations in the irrelevant image regions. The residual mechanism overcomes with vanishing gradient problem. The Mean-IoU score for our datasets reaches 0.9505, 0.9524, and 0.9530 for the simple, attention, and residual attention U-Net, respectively. The most accurate semantic segmentation results was achieved in the Mean-IoU and Dice metrics by applying the residual and attention mechanisms together. The watershed method applied to this best – Residual Attention – semantic segmentation result gave the segmentation with the specific information for each cell.

Evaluation of In Situ Antiaging Activity in Saccharomyces cerevisiae BY611 Yeast Cells Treated with Polyalthia longifolia Leaf Methanolic Extract (PLME) Using Different Microscopic Approaches: A Morphology-Based Evaluation.

Polyalthia longifolia is known for its anti-oxidative properties, which might contribute to the antiaging action. Hence, the current research was conducted to evaluate the antiaging activity of P. longifolia leaf methanolic extract (PLME) in a yeast model based on morphology using microscopic approaches. Saccharomyces cerevisiae BY611 strain yeast cells were treated with 1.00 mg/mL of PLME. The antiaging activity was assessed by determining the replicative lifespan, total lifespan, vacuole morphology by light microscopy, extra-morphology by scanning (SEM), and intra-morphology by transmission (TEM) electron microscopy. The findings demonstrated that PLME treatment significantly accelerated the replicative and total lifespan of the yeast cells. PLME treatment also delays the formation of large apoptotic-like type 3 yeast cell vacuoles. The untreated yeast cells demonstrated aging morphology via SEM analysis, such as shrinking, regional invaginations, and wrinkled cell surface. The TEM analysis revealed the quintessential aging intracellular morphology such as swollen, wrinkled, or damaged vacuole formation of the circular endoplasmic reticulum, a rupture in the nuclear membrane, fragmentation of the nucleus, and complete damaged cytoplasm. Decisively, the present study revealed the vital role of PLME in the induction of antiaging activity in a yeast model using three microscopic approaches—SEM, TEM, and bright-field light microscope.

Synergistic Effect of Quercetin Magnetite Nanoparticles and Targeted Radiotherapy in Treatment of Breast Cancer.

Quercetin is a potent cancer therapeutic agent present in fruits and vegetables. The pharmaceutical uses of quercetin are limited due to many problems associated with low solubility, bioavailability, permeability, and instability. In addition, the high doses of quercetin show toxic effects in clinical and experimental studies. Therefore, a new strategy is warranted to overcome these problems without the use of toxic doses. The iron oxide nanoparticles can be used as a drug delivery system. This study aimed to prepare quercetin-conjugated magnetite nanoparticles (QMNPs) using biological simple nanoprecipitation and mediated by fungus Aspergillus oryzae. Also, we initiated in vitro and in vivo studies to determine whether QMNPs might sensitize breast cancer to radiotherapy treatment. The structural, morphological, and magnetic properties of the prepared nanoparticles were studied. The results indicated that QMNPs were spherical in shape and 40 nm in diameter. The in vitro studies showed that the incubation of MCF-7, HePG-2, and A459 cancer cells with QMNPs for 24 h effectively inhibited the growth of cancer cell lines in a concentration-dependent manner with IC50 values of 11, 77.5, and104 nmol/mL, respectively. The combination of QMNPs with irradiation (IR) potently blocked MCF-7 cancer cell proliferation and showed significant changes in the morphology of these cells as observed by bright-field inverted light microscopy. Focusing on the long-term toxicity of QMNPs (20 ml/kg), the assessment of hematological, hepatic, and renal markers indicated no toxic effect. Besides, QMNPs inhibited tumor growth and potently enhanced the lateral radiotherapy treatment in N-methyl-N-nitrosourea (MNU)-induced breast cancer in female white albino rats. These anticancer and radiosensitizing activities were ascribed to cytotoxicity, cell cycle arrest, immunomodulation, and efficiency through induction of apoptosis. In a conclusion, these observations suggest that the QMNPs combined with LRT could act as a potential targeted therapy in breast cancer.

Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

Spectroscopic Approach to Correction and Visualisation of Bright-Field Light Transmission Microscopy Biological Data

The most realistic information about a transparent sample such as a live cell can be obtained using bright-field light microscopy. Under high-intensity pulsing LED illumination, we captured a primary 12-bit-per-channel (bpc) response from an observed sample using a bright-field microscope equipped with a high-resolution (4872 × 3248) image sensor. In order to suppress data distortions originating from the light interactions with elements in the optical path, poor sensor reproduction (geometrical defects of the camera sensor and some peculiarities of sensor sensitivity), we propose a spectroscopic approach for the correction of these uncompressed 12 bpc data by simultaneous calibration of all parts of the experimental arrangement. Moreover, the final intensities of the corrected images are proportional to the photon fluxes detected by a camera sensor. It can be visualized in 8 bpc intensity depth after the Least Information Loss compression.

Morpho-anatomical characterization of Eryngium yuccifolium ('Rattlesnake-master').

This paper provides detailed morpho-anatomical and histochemical characteristics of the leaves, peduncles, stems, and roots of Eryngium yuccifolium Michx. (Apiaceae) by bright-field light and scanning electron microscopy. Noteworthy morpho-anatomical features include the monocot-like leaves with parallel venation and distant marginal bristles, amphistomatic leaves with paracytic or anisocytic stomata and heterogenous isobilateral mesophyll, the presence of epicuticular wax crystalloids on both leaf surfaces, calcium oxalate druses and secretory canals in all the studied plant parts, presence of hypodermis, stem and peduncle with longitudinal ribs containing collenchyma, presence of aerenchyma in the peduncle and root, and absence of sclerenchyma in all the plant parts analyzed. These characteristics would aid in the taxonomy, species identification as well as quality control of the botanical.

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.

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.

Characterization of Oleogels Based on Waxes and Their Hydrolyzates

AbstractIn this paper, the structuring of liquid oils, also known as oleogelation, is systematically investigated for the first time using a quasi‐quaternary mixing system approach. Native waxes with different quantities of wax esters (WE), n‐alkanes (hydrocarbons (HC)), fatty acids (FA), and fatty alcohols (FaOH) are applied in mixtures with hydrolyzed waxes to systematically change the composition. Hydrolyzed waxes contain high levels of FA and FaOH. The model systems are investigated on microscopic level (brightfield light microscopy (BFM), cryogenic scanning electron microscopy (cryo‐SEM)) as well as on their macroscopic properties (rheology, gel hardness) and calorimetric behavior (differential scanning calorimetry (DSC)). It is found that sunflower wax (SFW)‐based gels (12% structurant) become less hard on any admixture. Beeswax (BW)‐based gels show significant increases in hardness when 25% and 50% (w/w) hydrolyzate are admixed. This could be related to stepwise crystallization. Further analysis reveals that the dissolution/melting behavior of the wax ester mixtures can be surprisingly well described as ideal solubility of a single pseudocomponent. The approach to unravel the individual contributions of the different species present in waxes is successful and marks a first step to better understand the systematic of wax functionality as oleogelators.Practical Application: The substitution of hardstock fats in structured oil phases is of interest for two reasons. The improved nutritional profile oleogels offer are beneficial for public health while the elimination of palm oil based ingredients appears to be a general public desire. Among the technical solutions for non‐TAG oil structuring waxes are very promising. This is primarily due to their availability, prior consumption, potentially low cost for functionality. Currently waxes are technically and scientifically wrongly treated as single components. In order to better utilize the potential of waxes and design future sourcing strategies it is necessary to understand the wax functionality at a compositional/molecular level. This contribution marks the first step into this direction by considering classes of molecules with respect to their contribution to functionality. This understanding is considered as a key for future compositional design.

Fungicides With Contrasting Mode of Action Differentially Affect Hyphal Healing Mechanism in Gigaspora sp. and Rhizophagus irregularis.

Fungicides are widely used in conventional agriculture to control fungal diseases, but may also affect non-target microorganisms such as arbuscular mycorrhizal (AM) fungi. These root symbionts develop extended mycelial networks within the soil via mechanisms such as anastomosis that indistinctly concerns intact and damaged hyphae, the latter being named hyphal healing mechanism (HHM). The HHM differs between Glomeraceae and Gigasporaceae. However, the effects of fungicides on this mechanism in unknown. Here, the impact of azoxystrobin, pencycuron, flutolanil, and fenpropimorph at 0.02 and 2 mg L–1 were tested in vitro on the HHM of Gigaspora sp. MUCL 52331 and Rhizophagus irregularis MUCL 41833, and repair events visualized carefully under a dissecting bright-field light microscope. Azoxystrobin was the more detrimental for both AM fungi at 2 mg L–1, while fenpropimorph impacted only R. irregularis (stimulating at low and inhibiting at high concentration). Conversely, flutolanil and pencycuron did not impact any of the two AM fungi. The mechanisms involved remains to be elucidated, but perturbation in the still-to-be firmly demonstrated spitzenkörper or in sterols content as well as a process of hormesis are possible avenues that deserve to be explored in view of a rationale management of chemicals to control fungal pathogens without harming the beneficial AM fungi.

Optical multi-channel interrogation instrument for bacterial colony characterization

A single instrument that includes multiple optical channels was developed to simultaneously measure various optical and associated biophysical characteristics of a bacterial colony. The multi-channel device can provide five distinct optical features without the need to transfer the sample to multiple locations or instruments. The available measurement channels are bright-field light microscopy, 3-D colony-morphology map, 2-D spatial optical-density distribution, spectral forward-scattering pattern, and spectral optical density. The series of multiple morphological interrogations is beneficial in understanding the bio-optical features of a bacterial colony and the correlations among them, resulting in an enhanced power of phenotypic bacterial discrimination. To enable a one-shot interrogation, a confocal laser scanning module was built as an add-on to an upright microscope. Three different-wavelength diode lasers were used for the spectral analysis, and high-speed pin photodiodes and CMOS sensors were utilized as detectors to measure the spectral OD and light-scatter pattern. The proposed instrument and algorithms were evaluated with four bacterial genera, Escherichia coli, Listeria innocua, Salmonella Typhimurium, and Staphylococcus aureus; their resulting data provided a more complete picture of the optical characterization of bacterial colonies.

Quasi-spectral characterization of intracellular regions in bright-field light microscopy images

Investigation of cell structure is hardly imaginable without bright-field microscopy. Numerous modifications such as depth-wise scanning or videoenhancement make this method being state-of-the-art. This raises a question what maximal information can be extracted from ordinary (but well acquired) bright-field images in a model-free way. Here we introduce a method of a physically correct extraction of features for each pixel when these features resemble a transparency spectrum. The method is compatible with existent ordinary bright-field microscopes and requires mathematically sophisticated data processing. Unsupervised clustering of the spectra yields reasonable semantic segmentation of unstained living cells without any a priori information about their structures. Despite the lack of reference data (to prove strictly that the proposed feature vectors coincide with transparency), we believe that this method is the right approach to an intracellular (semi)quantitative and qualitative chemical analysis.

Fundamentals of Microscopy.

The light (or optical) microscope is the icon of science. The aphorism "seeing is believing" is often quoted in scientific papers involving microscopy. Unlike many scientific instruments, the light microscope will deliver an image however badly it is set up. Fluorescence microscopy is a widely used research tool across all disciplines of biological and biomedical science. Most universities and research institutions have microscopes, including confocal microscopes. This introductory paper in a series detailing advanced light microscopy techniques explains the foundations of both electron and light microscopy for biologists and life scientists working with the mouse. An explanation is given of how an image is formed. A description is given of how to set up a light microscope, whether it be a brightfield light microscope on the laboratory bench, a widefield fluorescence microscope, or a confocal microscope. These explanations are accompanied by operational protocols. A full explanation on how to set up and adjust a microscope according to the principles of Köhler illumination is given. The importance of Nyquist sampling is discussed. Guidelines are given on how to choose the best microscope to image the particular sample or slide preparation that you are working with. These are the basic principles of microscopy that a researcher must have an understanding of when operating core bioimaging facility instruments, in order to collect high-quality images. © 2020 The Authors. Basic Protocol 1: Setting up Köhler illumination for a brightfield microscope Basic Protocol 2: Aligning the fluorescence bulb and setting up Köhler illumination for a widefield fluorescence microscope Basic Protocol 3: Generic protocol for operating a confocal microscope.

Sarcomere Lengths Become More Uniform Over Time in Intact Muscle-Tendon Unit During Isometric Contractions

The seemingly uniform striation pattern of skeletal muscles, quantified in terms of sarcomere lengths (SLs), is inherently non-uniform across all hierarchical levels. The SL non-uniformity theory has been used to explain the force creep in isometric contractions, force depression following shortening of activated muscle, and residual force enhancement following lengthening of activated muscle. Our understanding of sarcomere contraction dynamics has been derived primarily from in vitro experiments using regular bright-field light microscopy or laser diffraction techniques to measure striation/diffraction patterns in isolated muscle fibers or myofibrils. However, the collagenous extracellular matrices present around the muscle fibers, as well as the complex architecture in the whole muscles may lead to different contraction dynamics of sarcomeres than seen in the in vitro studies. Here, we used multi-photon excitation microscopy to visualize in situ individual sarcomeres in intact muscle tendon units (MTUs) of mouse tibialis anterior (TA), and quantified the temporal changes of SL distribution as a function of SLs in relaxed and maximally activated muscles for quasi-steady state, fixed-end isometric conditions. The corresponding muscle forces were simultaneously measured using a force transducer. We found that SL non-uniformity, quantified by the coefficient of variation (CV) of SLs, decreased at a rate of 1.9–3.1%/s in the activated muscles, but remained constant in the relaxed muscles. The force loss during the quasi-steady state likely did not play a role in the decrease of SL non-uniformity, as similar force losses were found in the activated and relaxed muscles, but the CV of SLs in the relaxed muscles underwent negligible change over time. We conclude that sarcomeres in the mid-belly of maximally contracting whole muscles constantly re-organize their lengths into a more uniform pattern over time. The molecular mechanisms accounting for SL non-uniformity appear to differ in active and passive muscles, and need further elucidation, as do the functional implications of the SL non-uniformity.