Fluorescence Microscopy Techniques Research Articles

Investigating the Biological Efficacy of Albumin-Enriched Platelet-Rich Fibrin (Alb-PRF): A Study on Cytokine Dynamics and Osteoblast Behavior

The development of effective biomaterials for tissue regeneration has led to the exploration of blood derivatives such as leucocyte- and platelet-rich fibrin (L-PRF). A novel variant, Albumin-Enriched Platelet-Rich Fibrin (Alb-PRF), has been introduced to improve structural stability and bioactivity, making it a promising candidate for bone regeneration. This study aimed to evaluate Alb-PRF’s capacity for cytokine and growth factor release, along with its effects on the proliferation, differentiation, and mineralization of human osteoblasts in vitro. Alb-PRF membranes were analyzed using histological, scanning electron microscopy, and fluorescence microscopy techniques. Cytokine and growth factor release was quantified over seven days, and osteoinductive potential was evaluated with MG-63 osteoblast-like cells. Structural analysis showed Alb-PRF as a biphasic, highly cellularized material that releases lower levels of inflammatory cytokines and higher concentrations of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) compared to L-PRF. Alb-PRF exhibited higher early alkaline phosphatase activity and in vitro mineralization (p < 0.05) and significantly increased the OPG/RANKL mRNA ratio (p < 0.05). These results indicate that Alb-PRF has promising potential as a scaffold for bone repair, warranting further in vivo and clinical assessments to confirm its suitability for clinical applications.

LiveLattice: Real-time visualisation of tilted light-sheet microscopy data using a memory-efficient transformation algorithm.

Light-sheet fluorescence microscopy (LSFM), a prominent fluorescence microscopy technique, offers enhanced temporal resolution for imaging biological samples in four dimensions (4D; x, y, z, time). Some of the most recent implementations, including inverted selective plane illumination microscopy (iSPIM) and lattice light-sheet microscopy (LLSM), move the sample substrate at an oblique angle relative to the detection objective's optical axis. Data from such tilted-sample-scan LSFMs require subsequent deskewing and rotation for proper visualisation and analysis. Such data preprocessing operations currently demand substantial memory allocation and pose significant computational challenges for large 4D dataset. The consequence is prolonged data preprocessing time compared to data acquisition time, which limits the ability for live-viewing the data as it is being captured by the microscope. To enable the fast preprocessing of large light-sheet microscopy datasets without significant hardware demand, we have developed WH-Transform, a memory-efficient transformation algorithm for deskewing and rotating the raw dataset, significantly reducing memory usage and the run time by more than 10-fold for large image stacks. Benchmarked against the conventional method and existing software, our approach demonstrates linear runtime compared to the cubic and quadratic runtime of the other approaches. Preprocessing a raw 3D volume of 2 GB (512 × 1536 × 600 pixels) can be accomplished in 3 s using a GPU with 24 GB of memory on a single workstation. Applied to 4D LLSM datasets of human hepatocytes, lung organoid tissue and brain organoid tissue, our method provided rapid and accurate preprocessing within seconds. Importantly, such preprocessing speeds now allow visualisation of the raw microscope data stream in real time, significantly improving the usability of LLSM in biology. In summary, this advancement holds transformative potential for light-sheet microscopy, enabling real-time, on-the-fly data preprocessing, visualisation, and analysis on standard workstations, thereby revolutionising biological imaging applications for LLSM and similar microscopes.

Visualizing and Quantifying Electronic Accessibility in Composite Battery Electrodes using Electrochemical Fluorescent Microscopy

Electronic connections between active material particles and the conductive carbon binder domain govern high-energy commercial Li-ion batteries' rate capability and lifetime (LIB). This work develops an in situ electrochemical fluorescent microscopy (EFM) technique that maps fluorescence intensity to these local electronic connections. Specifically, rapid redox kinetics of an electrofluorophore translates to reaction distributions limited by the electronic accessibility of battery electrode regions and individual active material particles. This technique can visualize hot spots, dead zones, and isolated particles on the electrode surface. EFM characterization of a series of LiNi0.33Mn0.33Co0.33O2 electrodes across processing parameters finds a significant negative correlation between the number of disconnected active particles and the rate capability. This low-cost technique provides quantitative mesoscale characterization of commercial LIB electrodes with fast throughput (<60 s) to facilitate rapid research and development and provide manufacturing quality control.

A comparison of lipid diffusive dynamics in monolayers and bilayers in the context of interleaflet coupling

Cellular membranes are composed of lipids typically organized in a double-leaflet structure. Interactions between these two leaflets – often referred to as interleaflet coupling – play a crucial role in various cellular processes. Despite extensive study, the mechanisms governing such interactions remain incompletely understood. Here, we investigate the effects of interleaflet coupling from a specific point of view, i.e. by comparing diffusive dynamics in bilayers and monolayers, focusing on potential lipid-specific interactions between opposing leaflets. Through quantitative fluorescence microscopy techniques, we characterize lipid diffusion and mean molecular area in monolayers and bilayers composed of different lipids. Our results suggest that the observed decrease in bilayer lipid diffusion compared to monolayers depends on lipid identity. Furthermore, our analysis suggests that lipid acyl chain structure and spatial configuration at the bilayer may strongly influence interleaflet interactions and dynamics in bilayers. These findings provide insights into the role of lipid structure in mediating interleaflet coupling and underscore the need for further experimental investigations to elucidate the underlying mechanisms.

Cell surface morphology mimicking nano-bio platform for immune cell stimulation

Cell surface morphology mimicking nano-bio platform for immune cell stimulation

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part II: What the FLIP, FLAP, FRAP, FRET, and FLIM is Going On?

Abstract The use of fluorescence in imaging has been a pivotal factor in advancing our scientific understanding. As microscopy continues to evolve, the terminology used to describe these techniques becomes increasingly complex, often resulting in a bewildering array of acronyms that resemble alphabet soup. Among the most prominent acronyms are those associated with advanced fluorescence microscopy techniques: Fluorescence Loss in Photobleaching (FLIP), Fluorescence Localization after Photobleaching (FLAP), Fluorescence Recovery after Photobleaching (FRAP), Förster Resonance Energy Transfer (FRET), and Fluorescence Lifetime Imaging Microscopy (FLIM). Each of these methods provides invaluable insights into molecular dynamics and interactions within living cells and tissues. This article, Part 2 of the Alphabet Soup of Microscopy series, aims to clarify these widely used fluorescence microscopy techniques and to illuminate their contributions to our understanding of cellular processes.

Unique root hydraulic and mechanical properties support the resilience of grapevines adapted to the Atacama Desert.

Cortical lacunae caused by drought, especially observed in hybrids originating from Vitis rupestris, disrupt the connection between roots and soil. Yet, the physiological processes behind lacuna formation during drought and its consistency across Vitis species remain unclear. Here, we used a root pressure probe to investigate fine root hydraulic and mechanical properties, in the arid-adapted R-65 and drought-susceptible 101-14Mgt cultivars. We then performed P-V curves, root sap osmolality, and electrolyte leakage (EL) and used fluorescent light microscopy techniques. Only 101-14Mgt showed lacunae formation during drought due to its stiffer cortical tissue, unlike R-65. Lacunae resulted in a notable decline in root hydraulic conductivity during severe drought, with increased EL and root sap osmolality, indicating potential cellular damage. R-65 displayed different and xerophyte-like characteristics featuring a higher turgor loss point and decreased root capacitance, essential for maintaining root structural integrity in arid conditions. Our findings highlight lacuna formation is impacted by root tissue elasticity possibly linked to specific Vitis species favoring deeper rooting. In arid-adapted grapevines, hydraulic regulators such as reduced turgor loss point, and root capacitance could contribute to enhanced drought tolerance.

Revealing enhanced dilution effect of conjugated polymers in partially miscible blends

AbstractRecent experiments have shown that hole traps could be suppressed in polymer light‐emitting diodes under current stress by diluting the light‐emitting conjugated polymers within an “inert” large‐bandgap host material. However, it is unclear why there is an enhanced dilution effect in partially miscible blends rather than fully miscible blends, as intuition would suggest that better miscibility leads to better dilution. In this work, we propose a cascade analysis by combining multiple fluorescence microscopic techniques and all‐atom molecular dynamics simulations to study the solid‐to‐solid dilution of poly[2‐methoxy‐5‐(2‐ethylhexyloxy)‐1,4‐phenylenevinylene] (MEH‐PPV) in MEH‐PPV/polystyrene (PS) blends and MEH‐PPV/poly(vinylcarbazole) (PVK) blends. By varying the molecular weights of PS and PVK, we can regulate their miscibility with MEH‐PPV. The results corroborate that the dilution effect is enhanced in partially miscible blends rather than fully miscible ones. This is because, in partially miscible blends undergoing phase separation, the concentration of MEH‐PPV is notably decreased in the phase occupying the majority of the volume, leading to an overall greater dilution effect than in fully miscible blends. Moreover, MEH‐PPV could adopt the more extended conformation in the fully miscible blend, causing a shorter intermolecular distance to further undermine the dilution effect. These findings explain the seemingly counterintuitive more effective dilution effect observed in the recently reported partially miscible blends and provide guidance for further enhancing the performance of future generations of polymer light‐emitting diodes.

A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium.

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyze their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

Benefits and Limitations of Encapsulating Platinum Electrocatalysts with Niobium Oxide

The development of energy infrastructure based on renewable sources is vital to reduce our reliance on fossil fuels. Technologies such as hydrogen fuel cells and water electrolyzers are able to bridge the gap between the intermittency issues associated with green energy sources such as wind and solar, and the demand for reliable electricity by being able to convert between electrical and chemical energy. Nanostructured and mesoporous platinum (Pt) prepared by electrodeposition or sputter coating have been shown to have increased performance in hydrogen fuel cells compared to the industry standard of Pt nanoparticles supported on carbon.1,2 Over the lifetime of a fuel cell Pt nanostructures and nanoparticles are, however, known to restructure and lose their surface area by mechanisms such as Ostwald ripening, dissolution, and coalescence. Previously, thin film Pt catalysts have been shown to be stabilized by a nanoscale film of niobium oxide.3 Herein, nanoscale thin films of niobium oxide are investigated as a stabilizing material through use by encapsulating nanostructured Pt catalysts.Nanostructured Pt catalysts were electrodeposited onto Pt-coated Si wafer substrates. Subsequently, niobium oxide films with thicknesses of 0.5 nm, 3 nm, and 4.5 nm were deposited onto the Pt catalyst using atomic layer deposition. X-ray fluorescence and conductive atomic force microscopy techniques were used to confirm the presence and coverage of the niobium oxide layer on the Pt catalyst. The performance of the encapsulated and un-encapsulated Pt catalysts as oxygen reduction reaction catalysts over the course of up to a thousand degradation cycles were evaluated using electrochemical techniques. The morphology of the catalyst before and after the degradation testing was also assessed using scanning electron microscopy techniques. Electrochemical impedance spectroscopy indicated that increasing the thickness of the niobium oxide layer increased the resistance of the catalyst towards the oxygen reduction reaction. The combined results of the analyses performed by electrochemical techniques and electron microscopy measurements revealed a range of film thicknesses for the niobium oxide that successfully stabilized the electrochemically active surface area and surface morphology of the Pt catalyst. Paul, M. T. Y.; Gates, B. D. Mesoporous Platinum Prepared by Electrodeposition for Ultralow Loading Proton Exchange Membrane Fuel Cells. Sci Rep 2019, 9, 4161.Debe, M. K. Tutorial on the Fundamental Characteristics and Practical Properties of Nanostructured Thin Film (NSTF) Catalysts. Electrochem. Soc. 2013, 160, F522.Eastcott, J.; Gates, B. D. Nanoscale Thin Films of Niobium Oxide on Platinum Surfaces: Creating a Platform for Optimizing Material Composition and Electrochemical Stability. Can. J. Chem. 2017, 96, 260–266.

The DNA-PAINT palette: a comprehensive performance analysis of fluorescent dyes

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution fluorescence microscopy technique that achieves single-molecule ‘blinking’ by transient DNA hybridization. Despite blinking kinetics being largely independent of fluorescent dye choice, the dye employed substantially affects measurement quality. Thus far, there has been no systematic overview of dye performance for DNA-PAINT. Here we defined four key parameters characterizing performance: brightness, signal-to-background ratio, DNA-PAINT docking site damage and off-target signal. We then analyzed 18 fluorescent dyes in three spectral regions and examined them both in DNA origami nanostructures, establishing a reference standard, and in a cellular environment, targeting the nuclear pore complex protein Nup96. Finally, having identified several well-performing dyes for each excitation wavelength, we conducted simultaneous three-color DNA-PAINT combined with Exchange-PAINT to image six protein targets in neurons at ~16 nm resolution in less than 2 h. We thus provide guidelines for DNA-PAINT dye selection and evaluation and an overview of performances of commonly used dyes.

Investigating the applicability of storage stability test for waste plastic modified bitumen: Morphological analyses

Plastics have proven as effective modifiers for asphalt binders; however, significant knowledge gaps exist around appropriateness of standard method of storage stability test and phase separation mechanism. These gaps are even more critical when using waste plastics containing polymeric cross-contamination. This study addresses these gaps through the application of asphalt tests, fluorescence microscopy (FM) and image processing techniques. The FM and image processing results showed that there is a relationship between elevated plastic swelling rates and plastic dispersion in bitumen matrix. Quantitative analysis of the images indicated that LDPE and LLDPE modified binders exhibited the lowest level of phase separation (5 %), followed by HDPE modified binder (13 %), and PP modified binder showed the highest level (20 %). Furthermore, cross-contamination of LDPE, LLDPE, and HDPE with PP notably worsened the storage stability of the resulting modified binders by approximately 10 %. The findings from FM and image processing indicated that the softening point test conducted through the storage stability tube test is insufficient for assessing the storage stability of plastic modified binders. To further validate this point, asphalt performance was examined for asphalt mixtures made with the binders before and after storage stability test. The findings demonstrated a substantial influence of phase separation of binder on key properties of the resulting asphalt mixture.

Stimulated Raman-induced beam focusing

Stimulated Raman scattering, employing a pump and a Stokes beam, exhibits itself through both the Raman loss observed in the pump beam and the Raman gain in the Stokes beam. This phenomenon finds application in spectroscopy for chemical analyses and microscopy for label-free bioimaging studies. Recent efforts have been made to implement super-resolution Raman microscopy using a doughnut-shaped pump, Stokes, or depletion beam. In this study, it is shown that the amplitude and phase of the pump or Stokes beam undergo significant modulation through the stimulated Raman process when they are configured as one of the higher-order Laguerre-Gauss modes, achieved using appropriate spiral phase plates or spatial light modulators. The resulting intensity distributions of the pump and Stokes beams are determined by a superposition of multiple Laguerre-Gauss modes that are coupled through nonlinear Raman gain and loss processes. Calculation results are used to elucidate the limitations associated with super-resolution coherent Raman imaging with a toroidal pump or Stokes beam. This stands in contrast with the stimulated emission depletion fluorescence microscopy technique, which has no fundamental limit in the spatial resolution enhancement.

Microplastics contamination in the most popular brands of Iranian sausages and evaluation of its human exposure

Microplastics (MPs) pollution represents a nascent environmental contaminant that has recently infiltrated human life and the food chain. The primary objective of this study was to investigate the presence of MPs in different brands of Iranian sausages. Qualitative and quantitative analyses of MPs particles were conducted using stereo- and fluorescent microscopy, FT-IR (Fourier-transform infrared spectroscopy), and SEM-EDS (Scanning electron microscopy-energy dispersive X-ray spectroscopy) techniques. Samples were collected from the most commonly consumed sausage brands in Iranian markets. The findings showed that the various sausage brands contained an average abundance of 25.7 ± 21.68 (range 10–70) and 55.45 ± 45.5 (range 10–175) particles/kg based on optical and fluorescent microscopy analyses, respectively. Predominantly, MPs were identified in fiber form (77–89 %), with a smaller proportion present in fragmented form (11–23 %). Polymer analysis using FT-IR identified polyethylene (PE) and polystyrene (PS) as the primary constituents. Furthermore, the estimated annual intake (EAI) of MPs was calculated at 804 and 3517 particles/kg bw/year for adults and children, respectively, based on optical microscopy observations. In comparison, fluorescent microscopy indicated an intake of 1734 and 7589 particles/kg bw/year for the respective age groups. These results emphasize the potential of MPs contamination to penetrate into different food products including sausages through processing routes, which can threaten human health.

Fluorescence microscopy for brain activity imaging: one-photon microscopy and its application to pharmacological research

The development of genetically-encoded fluorescent probes for the detection of intracellular calcium ions and various neurotransmitters has progressed significantly in recent years, and there is a growing need for techniques that rapidly and efficiently image these signals in the living brain for pharmacological studies of the central nervous system. In this article, we discuss one-photon fluorescence microscopy techniques used for brain activity imaging, particularly wide-field imaging and head-mounted miniaturized microscopy, and introduce their basic principles, recent advances, and applications in pharmacological research. Wide-field calcium imaging is suitable for mesoscopic observation of cortical activity during behavioral tasks in head-fixed awake mice, while head-mounted miniaturized microscopes can be attached to the animal's head to image brain activity associated with naturalistic behaviors such as social behavior and sleep. One-photon microscopy allows for the development of a simple and cost-effective imaging system using an affordable excitation light source such as a light-emitting diode. Its excitation light illuminates the entire field of view simultaneously, making it easy to perform high-speed imaging using a high-sensitivity camera. In contrast, the short wavelength of the excitation light limits the field of observation to areas on or near the brain surface due to its strong light scattering. Moreover, the out-of-focus fluorescence makes it difficult to obtain images with a high signal-to-noise ratio and spatial resolution. The use of one-photon microscopy in brain activity imaging has been limited compared to two-photon microscopy, but its advantages have recently been revisited. Therefore, this technique is expected to become a useful method for pharmacologists to visualize the activity of the living brain.

Deep learning-based localization algorithms on fluorescence human brain 3D reconstruction: a comparative study using stereology as a reference

3D reconstruction of human brain volumes at high resolution is now possible thanks to advancements in tissue clearing methods and fluorescence microscopy techniques. Analyzing the massive data produced with these approaches requires automatic methods able to perform fast and accurate cell counting and localization. Recent advances in deep learning have enabled the development of various tools for cell segmentation. However, accurate quantification of neurons in the human brain presents specific challenges, such as high pixel intensity variability, autofluorescence, non-specific fluorescence and very large size of data. In this paper, we provide a thorough empirical evaluation of three techniques based on deep learning (StarDist, CellPose and BCFind-v2, an updated version of BCFind) using a recently introduced three-dimensional stereological design as a reference for large-scale insights. As a representative problem in human brain analysis, we focus on a 4-cm3\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$4~\ ext {-cm}^3$$\\end{document} portion of the Broca’s area. We aim at helping users in selecting appropriate techniques depending on their research objectives. To this end, we compare methods along various dimensions of analysis, including correctness of the predicted density and localization, computational efficiency, and human annotation effort. Our results suggest that deep learning approaches are very effective, have a high throughput providing each cell 3D location, and obtain results comparable to the estimates of the adopted stereological design.

Evaluation of Ilex guayusa and Piper marginatum Extract Cytotoxicity on Human Dental Pulp Mesenchymal Stem Cells.

Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp. To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use. Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program. The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability. The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.

Three-Dimensional Imaging of Bioinspired Lipidic Mesophases Using Multicolored Light-Emitting Carbon Nanodots.

Recent progress in the design of carbon nanostructures exhibiting strong multiphoton-excited emission opens new pathways to explore the self-organization of lipids found in living organisms. Phospholipid-based lyotropic myelin figures (MFs) are promising materials as simplified models of biomembranes due to their structural resemblance to a multilamellar sheath insulating the axon. This study demonstrates the possibility of selective labeling of MFs by strongly emitting multicolor phloroglucinol-derived carbon nanodots (PG CNDs). Such dopants are efficiently excited by visible and near-infrared light; therefore, one- and two-photon fluorescence microscopies are incorporated to gain 3D insights into the MFs. Combining nondestructive fluorescence microscopy and spectroscopy techniques along with polarized light microscopy gives details on the stability and morphology of lipidic mesophases. Our findings suggest that PG CNDs can be a viable and simple alternative to conventional fluorescent lipid stains to image biologically relevant phospholipid-based structures.

LiveLattice: Real-time visualization of tilted light-sheet microscopy data using a memory-efficient transformation algorithm.

Light-sheet fluorescence microscopy (LSFM), a prominent fluorescence microscopy technique, offers enhanced temporal resolution for imaging biological samples in four dimensions (4D; x, y, z, time). Some of the most recent implementations, including inverted selective plane illumination microscopy (iSPIM) and lattice light-sheet microscopy (LLSM), rely on a tilting of the sample plane with respect to the light sheet of 30-45 degrees to ease sample preparation. Data from such tilted-sample-plane LSFMs require subsequent deskewing and rotation for proper visualization and analysis. Such transformations currently demand substantial memory allocation. This poses computational challenges, especially with large datasets. The consequence is long processing times compared to data acquisition times, which currently limits the ability for live-viewing the data as it is being captured by the microscope. To enable the fast preprocessing of large light-sheet microscopy datasets without significant hardware demand, we have developed WH-Transform, a novel GPU-accelerated memory-efficient algorithm that integrates deskewing and rotation into a single transformation, significantly reducing memory requirements and reducing the preprocessing run time by at least 10-fold for large image stacks. Benchmarked against conventional methods and existing software, our approach demonstrates linear scalability. Processing large 3D stacks of up to 15 GB is now possible within one minute using a single GPU with 24 GB of memory. Applied to 4D LLSM datasets of human hepatocytes, human lung organoid tissue, and human brain organoid tissue, our method outperforms alternatives, providing rapid, accurate preprocessing within seconds. Importantly, such processing speeds now allow visualization of the raw microscope data stream in real time, significantly improving the usability of LLSM in biology. In summary, this advancement holds transformative potential for light-sheet microscopy, enabling real-time, on-the-fly data processing, visualization, and analysis on standard workstations, thereby revolutionizing biological imaging applications for LLSM, SPIM and similar light microscopes.

Elastic contact between rough surfaces: Bridging the gap between theory and experiment

Friction is generated where solids touch, thus quantitative understanding of the microscopic contact area is crucial to predict macroscopic friction. All surfaces are rough, and typically small-scale roughness rather than macroscopic geometry sets the contact area. Rough contact theories have been developed that predict the contact area, using the surface topography and the mechanical properties of the solids as input. However, the validity of these theories remains under debate. Systematic comparison between theory and experiment has been lacking, due to the experimental difficulty to access the contact area, especially at small length scales where idealised assumptions of contact models may not hold. Here, we use a state-of-the-art fluorescence microscopy technique to directly access the contact area of a glass-glass contact with nanometric out-of-plane resolution. We systematically vary the roughness of the contact and carefully measure the surface topography across length scales. Comparison of high-resolution contact experiments to different contact theories enables us to unambigously conclude that only elastic Persson theory quantitatively describes experimental observations.