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.

Level set simulation of focused ion beam sputtering of a multilayer substrate.

The evolution of a multilayer sample surface during focused ion beam processing was simulated using the level set method and experimentally studied by milling a silicon dioxide layer covering a crystalline silicon substrate. The simulation took into account the redeposition of atoms simultaneously sputtered from both layers of the sample as well as the influence of backscattered ions on the milling process. Monte Carlo simulations were applied to produce tabulated data on the angular distributions of sputtered atoms and backscattered ions. Two sets of test structures including narrow trenches and rectangular boxes with different aspect ratios were experimentally prepared, and their cross sections were visualized in scanning transmission electron microscopy images. The superimposition of the calculated structure profiles onto the images showed a satisfactory agreement between simulation and experimental results. In the case of boxes that were prepared with an asymmetric cross section, the simulation can accurately predict the depth and shape of the structures, but there is some inaccuracy in reproducing the form of the left sidewall of the structure with a large amount of the redeposited material. To further validate the developed simulation approach and gain a better understanding of the sputtering process, the distribution of oxygen atoms in the redeposited layer derived from the numerical data was compared with the corresponding elemental map acquired by energy-dispersive X-ray microanalysis.

On the architecture of starch granules revealed by iodine vapor binding and lintnerization. Part 1: Microscopic examinations.

Structural nature of glucan chains in the amorphous part of granular starch was examined by iodine vapor treatment and lintnerization. Four iodine-stained amylose-containing normal starches and their waxy counterparts were examined under a microscope before, during, and after lintnerization. The presence of amylose retarded the lintnerization rate. The degree of retardation correlated with the structural type of the amylopectin component, suggesting that potato amylopectin (type 4 structure) interacts with amylose in the granules, whereas in barley granules (type 1 structure) the interaction is very weak. The inclusion complexes with iodine were not degraded by the acid treatment. Therefore, the iodine-glucan chain complex formation could be used to study the structural nature of the flexible, amorphous parts of the starch granules. Indeed, at the end of lintnerization, when 20%-30% of the granules remained, substantial amounts of blue-stained complexes were washed out from the granules especially from amylose-containing barley and maize starch, but also from both normal and waxy cassava and potato starch. The complexation with iodine did not affect the rate of lintnerization. This suggested that single helical structures were present during lintnerization also in the absence of iodine and this conformation was the reason for the acid resistance.

mSphere of Influence: The power of observational research.

Katrina Velle is a cell biologist who uses microscopy to study amoebae. In this mSphere of Influence article, she reflects on how a classic paper on Listeria by Tilney and Portnoy made an impact on her by highlighting how much we can learn from simply looking at cells.

Superresolution Expansion Microscopy in Dictyostelium Amoebae.

Expansion microscopy (ExM) is a superresolution technique for fixed specimens that improves resolution of a given microscopy system approximately fourfold. The gain in resolution in ExM is not achieved by improvement of the resolution of the microscope itself but by isotropic expansion of the sample. To achieve this, the sample is cross-linked to an expandable gel matrix that swells approximately fourfold by incubation in water. We have applied the method to Dictyostelium amoebae and discuss the pros and cons of different labeling techniques in combination with pre- and post-expansion staining protocols.

Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy.

Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.

Mapping and spectroscopy of telecom quantum emitters with confocal laser scanning microscopy.

Efficiently coupling single-photon emitters in the telecommunication C-band that are not deterministically positioned to photonic structures requires both spatial and spectral mapping. This study introduces the photoluminescence mapping of telecom C-band self-assembled quantum dots (QDs) by confocal laser scanning microscopy, a technique previously unexplored in this wavelength range which fulfills these two requirements. We consider the effects of distortions inherent to any imaging system but largely disregarded in prior works to derive accurate coordinates from photoluminescence maps. We obtain a position uncertainty below 11 nm for 10\% of the QDs when assuming no distortions, highlighting the potential of the scanning approach. After distortion correction, we found that the previously determined positions are on average shifted by 428 nm from the corrected positions, demonstrating the necessity of this correction for accurate positioning. Then, through error propagation, the position uncertainty for 10\% of the QDs increases to 110 nm.

Assessment of Smear Layer Removal and Penetration Depth of Root Canal Irrigant Using Different Irrigation Activation Systems: A Comparative Study.

AIM: The aim of the current study was to evaluate the penetration depth and smear layer removal of root canal irrigant using various irrigation activation techniques. MATERIALS AND METHODS: In this investigation, sixty single-rooted premolars extracted for orthodontic purposes were chosen. Diamond burs were used to create an access cavity, and #10 K-file was used to determine the patency. About sixty samples were divided into the following three groups (20 samples in each group), group I: Irrigation with conventional needle, group II: Activation of EndoVac system, group III: Passive ultrasonic irrigation (PUI). The efficacy of the smear layer was assessed using a scanning electron microscopy at a ×2000 magnification. One-way ANOVA was used to record and analyze the data. All statistical analyses were performed with a significance level of p < 0.05. RESULTS: At coronal third, the maximum smear layer was removed in group II (1.26 ± 0.02) followed by group III (1.84 ± 0.16) and group I (2.89 ± 0.21). At middle third, smear layer removal was maximum in group I (1.18 ± 0.10) followed by group III (1.72 ± 0.09) and group I (2.66 ± 0.18). At apical third, the more smear layer was removed in group II (1.02 ± 0.01) followed by group III (1.58 ± 0.08) and group I (2.38 ± 0.06). There was a highly significant difference found between the three different irrigation systems at all three levels (p < 0.001). CONCLUSION: In conclusion, every irrigation device that was evaluated was successful in removing the smear layer from the root canal. However, the EndoVac system group removed a greater amount of smear layer compared with PUI and conventional needle group. CLINICAL SIGNIFICANCE: With the goal of promoting cleaning that is beyond the ability of mechanical devices, irrigation is a crucial part of root canal therapy. If an efficient irrigation delivery system is used, the irrigants can reach the working length (WL). This type of distribution system needs to provide a suitable amount of irrigants up to the WL, as well as have enough flow and be effective at debriding the entire canal system. How to cite this article: Pujari MD, Das M, Das A, et al. Assessment of Smear Layer Removal and Penetration Depth of Root Canal Irrigant Using Different Irrigation Activation Systems: A Comparative Study. J Contemp Dent Pract 2024;25(4):331-334.

Effect of different freezing conditions on ice crystal formation behavior and ice-growth inhibition by cryoprotectants.

BACKGROUND: The formation of ice crystals will have adverse effects on aquatic products, and the key to ensure the long-term preservation and better quality preservations of the product is to evaluate the intercellular ice crystal formation to find suitable refrigeration conditions and cryoprotectants. RESULTS: The ice crystal formation was successfully captured by using an inverted microscope cryomicroscopic system equipped with a low-temperature stage, the ice crystals formed under different freezing methods between tuna muscle cells were observed directly, the deformation degree of muscle tissue pores during crystallization was evaluated, and the effect of freeze-thaw times on tuna samples was analyzed. The effects of the use of cryoprotectant such as cellobiose and carboxylated cellulose nanofibers on ice-growth inhibition were investigated, and the reliability of the ice crystal observation results was further verified by the determination of physical properties. The results showed that carboxylated cellulose nanofibers had the best ice-growth inhibition effect, they prevented about 50% cell deformation compared with the control group, and also reduced the minimum size of ice crystal formation. In addition, the addition of cellobiose and sodium tripolyphosphate gave the ice crystals a more uniform size and roundness. CONCLUSION: The experiment proposed a stable and clear observation method for the process of intercellular ice crystal formation, and the accuracy of the observation method was further verified by some physical indicators. This may help in the selection of suitable measurement methods to directly observe ice crystal formation behavior and screen cryoprotectants. © 2024 Society of Chemical Industry.

Mechanism of ceramide synthase inhibition by fumonisin B1.

Ceramide synthases (CerSs) play crucial roles in sphingolipid metabolism and have emerged as promising drug targets for metabolic diseases, cancers, and antifungal therapy. However, the therapeutic targeting of CerSs has been hindered by a limited understanding of their inhibition mechanisms by small molecules. Fumonisin B1 (FB1) has been extensively studied as a potent inhibitor of eukaryotic CerSs. In this study, we characterize the inhibition mechanism of FB1 on yeast CerS (yCerS) and determine the structures of both FB1-bound and N-acyl-FB1-bound yCerS. Through our structural analysis and the observation of N-acylation of FB1 by yCerS, we propose a potential ping-pong catalytic mechanism for FB1 N-acylation by yCerS. Lastly, we demonstrate that FB1 exhibits lower binding affinity for yCerS compared to the C26- coenzyme A (CoA) substrate, suggesting that the potent inhibitory effect of FB1 on yCerS may primarily result from the N-acyl-FB1 catalyzed by yCerS, rather than through direct binding of FB1.

Coaching ribosome biogenesis from the nuclear periphery.

Severe invagination of the nuclear envelope is a hallmark of cancers, aging, neurodegeneration, and infections. However, the outcomes of nuclear invagination remain unclear. This work identified a new function of nuclear invagination: regulating ribosome biogenesis. With expansion microscopy, we observed frequent physical contact between nuclear invaginations and nucleoli. Surprisingly, the higher the invagination curvature, the more ribosomal RNA and pre-ribosomes are made in the contacted nucleolus. By growing cells on nanopillars that generate nuclear invaginations with desired curvatures, we can increase and decrease ribosome biogenesis. Based on this causation, we repressed the ribosome levels in breast cancer and progeria cells by growing cells on low-curvature nanopillars, indicating that overactivated ribosome biogenesis can be rescued by reshaping nuclei. Mechanistically, high-curvature nuclear invaginations reduce heterochromatin and enrich nuclear pore complexes, which promote ribosome biogenesis. We anticipate that our findings will serve as a foundation for further studies on nuclear deformation.

Interorganelle phospholipid communication, a house not so divided.

The presence of membrane-bound organelles with specific functions is one of the main hallmarks of eukaryotic cells. Organelle membranes are composed of specific lipids that govern their function and interorganelle communication. Discoveries in cell biology using imaging and omic technologies have revealed the mechanisms that drive membrane remodeling, organelle contact sites, and metabolite exchange. The interplay between multiple organelles and their interdependence is emerging as the next frontier for discovery using 3D reconstruction of volume electron microscopy (vEM) datasets. We discuss recent findings on the links between organelles that underlie common functions and cellular pathways. Specifically, we focus on the metabolism of ether glycerophospholipids that mediate organelle dynamics and their communication with each other, and the new imaging techniques that are powering these discoveries.

Reconstructing Cancellous Bone From Down-Sampled Optical-Resolution Photoacoustic Microscopy Images With Deep Learning.

OBJECTIVE: Bone diseases deteriorate the microstructure of bone tissue. Optical-resolution photoacoustic microscopy (OR-PAM) enables high spatial resolution of imaging bone tissues. However, the spatiotemporal trade-off limits the application of OR-PAM. The purpose of this study was to improve the quality of OR-PAM images without sacrificing temporal resolution. METHODS: In this study, we proposed the Photoacoustic Dense Attention U-Net (PADA U-Net) model, which was used for reconstructing full-scanning images from under-sampled images. Thereby, this approach breaks the trade-off between imaging speed and spatial resolution. RESULTS: The proposed method was validated on resolution test targets and bovine cancellous bone samples to demonstrate the capability of PADA U-Net in recovering full-scanning images from under-sampled OR-PAM images. With a down-sampling ratio of [4, 1], compared to bilinear interpolation, the Peak Signal-to-Noise Ratio and Structural Similarity Index Measure values (averaged over the test set of bovine cancellous bone) of the PADA U-Net were improved by 2.325 dB and 0.117, respectively. CONCLUSION: The results demonstrate that the PADA U-Net model reconstructed the OR-PAM images well with different levels of sparsity. Our proposed method can further facilitate early diagnosis and treatment of bone diseases using OR-PAM.

The Coking of a Solid Catalyst Rationalized with Combined Raman and Fluorescence Lifetime Microscopy.

The formation of carbon deposits is a major deactivation pathway for solid catalysts. Studying coking on industrially relevant catalysts is, however, often challenging due to the sample heterogeneity. That is especially true for zeolite-containing catalysts where fluorescence often hampers their characterization with Raman spectroscopy. We turned this disadvantage into an advantage and combined Raman and fluorescence (lifetime) microscopy to study the coking behavior of an equilibrium catalyst material used for fluid catalytic cracking of hydrocarbons. The results presented illustrate that this approach can yield new insights in the physicochemical processes occurring within zeolite-containing catalyst particles during their coking process. Ex situ analyses of single catalyst particles revealed considerable intra-sample heterogeneities. The sample-averaged Raman spectra showed a higher degree of graphitization when the sample was exposed to more hexane, while the sample-averaged fluorescence lifetime showed no significant trend. Simultaneous in situ Raman and fluorescence (lifetime) microscopy, used to follow the coking of single particles, gave more insights in the changing fluorescence dynamics. The rise and decline of the average fluorescence lifetime suggested the prolonged presence of smaller coke species that are quenched more and more by adjacent larger polyaromatics acting as Förster-resonance-energy-transfer acceptors.

Quantification of membrane geometry and protein sorting on cell membrane protrusions using fluorescence microscopy.

Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences of membrane proteins.

The effect of hydrostatic pressure on lipid membrane lateral structure.

High pressure is both an environmental challenge to which deep sea biology has to adapt, and a highly sensitive thermodynamic tool that can be used to trigger structural changes in biological molecules and assemblies. Lipid membranes are amongst the most pressure sensitive biological assemblies and pressure can have a large influence on their structure and properties. In this chapter, we will explore the use of high pressure small angle X-ray diffraction and high pressure microscopy to measure and quantify changes in the lateral structure of lipid membranes under both equilibrium high pressure conditions and in response to pressure jumps.

Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity.

Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (ld) and liquid-ordered (lo) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a lo phase enriched in the high-melting lipids and cholesterol and a ld phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated lo/ld phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with lo/ld phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.

The effect of epilepsy and anti-epileptic drugs on the anterior and posterior segment of the eye.

CLINICAL RELEVANCE: Understanding the causes of visual symptoms in epilepsy patients is important for early diagnosis and taking precautions. BACKGROUND: The aim of this study is to evaluate the anterior and posterior segment parameters in patients with generalized tonic-clonic epilepsy (GTCE). METHODS: This retrospective study included 50 eyes of 50 patients with GTCE and 55 eyes of 55 healthy controls. For all participants, detailed ophthalmic examinations were obtained from the files of patients. Anterior segment parameters were measured using corneal topography and non-contact specular microscopy, and posterior segment parameters were measured using swept-source optical coherence tomography. RESULTS: The mean age of the patients with GTCE was 43.3 ± 13.2 years, and in the healthy controls it was 47.6 ± 10.7 years (p = 0.405). In GTCE patients, 34 patients were treated with monotherapy (MT) and 16 patients with polytherapy (PT). Central macular thickness (CMT) was statistically significantly thin in GTCE patients (p = 0.001). The average and four quadrants (superior, inferior, nasal, temporal) retinal nerve fibre layer (RNFL) were thinner in GTCE patients than in the healthy controls, but there was no statistically significant difference (p > 0.05, all). The central corneal thickness was statistically significantly thin in GTCE patients (p = 0.04). Endothelial cell density (ECD), endothelial cell number (ECN), and average cell area (ACA) were statistically significantly lower in GTCE patients than in the healthy controls (p < 0.05, all). Although the CMT, average, and four-quadrants RNFL were thinner in the PT group compared to the MT group, no statistically significant difference was observed (p > 0.05, all). Total high-order aberrations (HOAs) were 0.6 ± 0.4 in the MT group and 0.4 ± 0.1 in the PT group (p = 0.01). ECD, ECN, and ACA measurements were observed to be lower in the PT group compared to the MT group, but no statistically significant difference was detected (p > 0.05, all). CONCLUSION: There could be statistically significant differences between GTCE patients and healthy controls in anterior and posterior segment parameters. This situation may be due to the epilepsy itself or to the antiepileptic drugs.

A scalable, passive sampling method for the quantification of airborne allergens.

Traditionally, airborne concentrations of aeroallergens are sampled in a single location by an active sampler, which requires electricity and regular maintenance. However, aeroallergen concentrations may vary widely over urban and rural environments, requiring a method that is cost-effective and scalable so that many measurements can be made across an air shed. We developed such a method that uses passive sampling and light microscopy for analysis. Inexpensive and easy to operate, passive samplers rely on the gravitational settling of particles onto microscope slides. This determines airborne pollen concentration through: 1) sample collection using a modified Durham sampler, 2) preparation of samples for microscopy and strategic sample imaging, and 3) simplified particle measurements and calculation of pollen concentration following deposition velocity models proposed by Scheppegrell [1] and Wagner and Leith [2]. This method was verified with two sampling campaigns during the ragweed season of 2020 and the tree pollen season of 2021. The concentrations determined with the passive and Burkard sampling methods were found to be well-correlated (r > 0.99, r = 0.87) and precise (%CV = 20 %, 21 %). The validation of passive samplers will enable measurements of aeroallergens over wider spatial scales and help determine where aeroallergen exposure risks are greatest. •An inexpensive and low-cost method was developed to determine airborne pollen counts.•The method was evaluated for its accuracy and reproducibility.•The method can be applied to examine the concentrations and spatial variability of airborne pollen.