Wide-field Microscopy Research Articles

Coordinate-based neural representations for computational adaptive optics in widefield microscopy

Coordinate-based neural representations for computational adaptive optics in widefield microscopy

High throughput compressive fluorescence lifetime imaging with a silicon photomultiplier detector

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique for studying biological processes. There exists a growing interest in developing strategies to enhance throughput and reduce acquisition time of FLIM systems, which commonly employ laser scanning excitation and time-correlated single-photon counting (TCSPC) detection. In this work, we propose a wide-field FLIM microscope based on compressive sensing and high photon rate detection (beyond pile-up limit) based on a high-efficiency silicon photomultiplier detector as a single-pixel camera. We experimentally validate the capabilities of this design achieving 20 frames per second FLIM images on free-moving green algae sample.

Click chemical ligation-enabled digital particle counting for multiplexed microRNA analysis

Digital counting assays, that quantify targets by counting individual signal entities, provide a promising way for the sensitive analysis of biomarkers even at the single-molecule level. Considering the requirements of complex enzyme-catalyzed amplification techniques and specialized instruments in traditional digital counting biosensors, herein, a simple digital counting platform for microRNA (miRNA) analysis is developed by employing the miRNA-templated click chemical ligation to hinge ultrabright quantum dot-doped nanoparticles (QDNPs) on the bottom of microplate well. Compared with the traditional short miRNA-mediated sandwich hybridization mechanism, the click chemistry-mediated ligation featured enhanced stability, achieving higher sensitivity by directly counting the number of QDNPs with a common wide-field fluorescence microscope. Furthermore, enzyme-free cycling click ligation strategy is adopted to push the detection limit of miRNA down to a low level of 8 fM. What is more, taking advantages of the tunable emission wavelength and narrow emission spectra of fluorescent nanoparticles, the platform enables simultaneous detection of multiplex miRNA targets without cross interference. Benefiting from the simple operation, high sensitivity, and good generality, miRNA analysis in complex samples is successfully achieved. This method not only pioneers a new route for digital counting assays but also holds great potential in miRNA-related biological researches.

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale ("mesoscopic") imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. We aim to develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm light-emitting diodes positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. We demonstrate the performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake, head-fixed mice with a curved "crystal skull" window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. Our widefield microscope design with a single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Comparison of polystyrene and hydrogel microcarriers for optical imaging of adherent cells.

The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition. Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells. Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto, three-dimensional, high-resolution visualization of cells cultured on microcarriers. The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography. The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes.

Revealing the Existence of Long-Range Liquid-Liquid Interfacial Potential in Phase-Transfer Processes.

By employing fluorescence wide-field microscopy and a nanoparticle-based phase transfer catalyst (PTC), consisting of a fluorescent silica nanoparticle functionalized with trioctylpropylammonium bromide, we demonstrate that in the presence of NaOH, single nanoparticles display subdiffusive motion along the axis normal to an aqueous liquid-organic liquid interface. This is because of an extended interfacial potential with a shallow well (∼1 kBT) that stretches a few μm into the organic phase, in contrast to previous molecular dynamics studies that reported narrow interfaces on the order of ∼1 nm. Spontaneous interfacial emulsification induced by NaOH results in the propagation of water-in-oil nanoemulsions into the organic solvent that creates an equilibrium hybrid-solvent composition that solvates the PTC. A greater mobility and longer residence time of the PTC at the potential well enhance the interfacial phase transfer process and catalytic efficiency.

Extended depth-of-field wide-field fluorescence microscopy with a micro-mirror array lens system for versatile cellular examination.

We present a versatile extended depth-of-field (EDOF) wide-field fluorescence microscopy using a new, to the best of our knowledge, active device, micro-mirror array lens system (MALS) for calibration-free and orientation-insensitive EDOF imaging. The MALS changed the focal plane during image acquisition, and the system could be operated in any orientation. Two EDOF imaging modes of high-speed accumulation and low-speed surface sectioning were implemented. The performance was demonstrated in non-contact imaging of conjunctival goblet cells in live mice and depth-resolved cellular examination of ex-vivo human cancer specimens. MALS-based EDOF microscopy has potential for versatile cellular examination.

Multimodal analysis of traction forces and the temperature dynamics of living cells with a diamond-embedded substrate.

Cells and tissues are constantly exposed to chemical and physical signals that regulate physiological and pathological processes. This study explores the integration of two biophysical methods: traction force microscopy (TFM) and optically detected magnetic resonance (ODMR) to concurrently assess cellular traction forces and the local relative temperature. We present a novel elastic substrate with embedded nitrogen-vacancy microdiamonds that facilitate ODMR-TFM measurements. Optimization efforts focused on minimizing sample illumination and experiment duration to mitigate biological perturbations. Our hybrid ODMR-TFM technique yields TFM maps and achieves approximately 1 K precision in relative temperature measurements. Our setup employs a simple wide-field fluorescence microscope with standard components, demonstrating the feasibility of the proposed technique in life science laboratories. By elucidating the physical aspects of cellular behavior beyond the existing methods, this approach opens avenues for a deeper understanding of cellular processes and may inspire the development of diverse biomedical applications.

Single image Fourier ring correlation

We address resolution assessment for (light super-resolution) microscopy imaging. In modalities where imaging is not diffraction limited, correlation between two noise independent images is the standard way to infer the resolution. Here we take away the need for two noise independent images by computationally splitting one image acquisition into two noise independent realizations. This procedure generates two Poisson noise distributed images if the input is Poissonian distributed. As most modern cameras are shot-noise limited this procedure is directly applicable. However, also in the presence of readout noise we can compute the resolution faithfully via a correction factor. We evaluate our method on simulations and experimental data of widefield microscopy, STED microscopy, rescan confocal microscopy, image scanning microscopy, conventional confocal microscopy, and transmission electron microscopy. In all situations we find that using one image instead of two results in the same computed image resolution.

Pixel-wise programmability enables dynamic high-SNR cameras for high-speed microscopy

High-speed wide-field fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio at high frame rates, limiting their ability to detect faint fluorescent events. Here, we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high signal-to-noise ratio. In high-speed voltage imaging experiments, our image sensor significantly increases the output signal-to-noise ratio compared to a low-noise scientific CMOS camera (~2–3 folds). This signal-to-noise ratio gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions.

Efficient high-resolution fluorescence projection imaging over an extended depth of field through optical hardware and deep learning optimizations.

Optical microscopy has witnessed notable advancements but has also become more costly and complex. Conventional wide field microscopy (WFM) has low resolution and shallow depth-of-field (DOF), which limits its applications in practical biological experiments. Recently, confocal and light sheet microscopy become major workhorses for biology that incorporate high-precision scanning to perform imaging within an extended DOF but at the sacrifice of expense, complexity, and imaging speed. Here, we propose deep focus microscopy, an efficient framework optimized both in hardware and algorithm to address the tradeoff between resolution and DOF. Our deep focus microscopy achieves large-DOF and high-resolution projection imaging by integrating a deep focus network (DFnet) into light field microscopy (LFM) setups. Based on our constructed dataset, deep focus microscopy features a significantly enhanced spatial resolution of ∼260 nm, an extended DOF of over 30 µm, and broad generalization across diverse sample structures. It also reduces the computational costs by four orders of magnitude compared to conventional LFM technologies. We demonstrate the excellent performance of deep focus microscopy in vivo, including long-term observations of cell division and migrasome formation in zebrafish embryos and mouse livers at high resolution without background contamination.

Optical redox imaging to screen synthetic hydrogels for stem cell-derived cardiomyocyte differentiation and maturation.

Heart disease is the leading cause of death in the United States, yet research is limited by the inability to culture primary cardiac cells. Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) are a promising solution for drug screening and disease modeling. Induced pluripotent stem cell-derived CM (iPSC-CM) differentiation and maturation studies typically use heterogeneous substrates for growth and destructive verification methods. Reproducible, tunable substrates and touch-free monitoring are needed to identify ideal conditions to produce homogenous, functional CMs. We generated synthetic polyethylene glycol-based hydrogels for iPSC-CM differentiation and maturation. Peptide concentrations, combinations, and gel stiffness were tuned independently. Label-free optical redox imaging (ORI) was performed on a widefield microscope in a 96-well screen of gel formulations. We performed live-cell imaging throughout differentiation and early to late maturation to identify key metabolic shifts. Label-free ORI confirmed the expected metabolic shifts toward oxidative phosphorylation throughout the differentiation and maturation processes of iPSC-CMs on synthetic hydrogels. Furthermore, ORI distinguished high and low differentiation efficiency cell batches in the cardiac progenitor stage. We established a workflow for medium throughput screening of synthetic hydrogel conditions with the ability to perform repeated live-cell measurements and confirm expected metabolic shifts. These methods have implications for reproducible iPSC-CM generation in biomanufacturing.

Nanopore Blockade Sensors for Quantitative Analysis Using an Optical Nanopore Assay.

Nanopore sensing is a popular biosensing strategy that is being explored for the quantitative analysis of biomarkers. With low concentrations of analytes, nanopore sensors face challenges related to slow response times and selectivity. Here, we demonstrate an approach to rapidly detect species at ultralow concentrations using an optical nanopore blockade sensor for quantitative detection of the protein vascular endothelial growth factor (VEGF). This sensor relies on monitoring fluorescent polystyrene nanoparticles blocking nanopores in a nanopore array of 676 nanopores. The fluorescent signal is read out using a wide-field fluorescence microscope. Nonspecific blockade events are then distinguished from specific blockade events based on the ability to pull the particles out of the pore using an applied electric field. This allows the detection of VEGF at sub-picomolar concentration in less than 15 min.

DNA choreography: correlating mobility and organization of DNA across different resolutions from loops to chromosomes

The dynamics of DNA in the cell nucleus plays a role in cellular processes and fates but the interplay of DNA mobility with the hierarchical levels of DNA organization is still underexplored. Here, we made use of DNA replication to directly label genomic DNA in an unbiased genome-wide manner. This was followed by live-cell time-lapse microscopy of the labeled DNA combining imaging at different resolutions levels simultaneously and allowing one to trace DNA motion across organization levels within the same cells. Quantification of the labeled DNA segments at different microscopic resolution levels revealed sizes comparable to the ones reported for DNA loops using 3D super-resolution microscopy, topologically associated domains (TAD) using 3D widefield microscopy, and also entire chromosomes. By employing advanced chromatin tracking and image registration, we discovered that DNA exhibited higher mobility at the individual loop level compared to the TAD level and even less at the chromosome level. Additionally, our findings indicate that chromatin movement, regardless of the resolution, slowed down during the S phase of the cell cycle compared to the G1/G2 phases. Furthermore, we found that a fraction of DNA loops and TADs exhibited directed movement with the majority depicting constrained movement. Our data also indicated spatial mobility differences with DNA loops and TADs at the nuclear periphery and the nuclear interior exhibiting lower velocity and radius of gyration than the intermediate locations. On the basis of these insights, we propose that there is a link between DNA mobility and its organizational structure including spatial distribution, which impacts cellular processes.

Zero-shot learning enables instant denoising and super-resolution in optical fluorescence microscopy.

Computational super-resolution methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary super-resolution imaging conditions, in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional wide-field microscopy, confocal microscopy, two-photon microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy, which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans.

Impact of temporal resolution in single particle tracking analysis

Temporal resolution is a key parameter in the observation of dynamic processes, as in the case of single molecules motions visualized in real time in two-dimensions by wide field (fluorescence) microscopy, but a systematic investigation of its effects in all the single particle tracking analysis steps is still lacking. Here we present tools to quantify its impact on the estimation of diffusivity and of its distribution using one of the most popular tracking software for biological applications on simulated data and movies. We found important shifts and different widths for diffusivity distributions, depending on the interplay of temporal sampling conditions with various parameters, such as simulated diffusivity, density of spots, signal-to-noise ratio, lengths of trajectories, and kind of boundaries in the simulation. We examined conditions starting from the ones of experiments on the fluorescently labelled receptor p75NTR, a relatively fast-diffusing membrane receptor (diffusivity around 0.5–1 µm2/s), visualized by TIRF microscopy on the basal membrane of living cells. From the analysis of the simulations, we identified the best conditions in cases similar to these ones; considering also the experiments, we could confirm a range of values of temporal resolution suitable for obtaining reliable diffusivity results. The procedure we present can be exploited in different single particle/molecule tracking applications to find an optimal temporal resolution.

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures.

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and embedding conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

Delivery of Mesenchymal Stem Cells during Hypothermic Machine Perfusion in a Translational Kidney Perfusion Study.

In transplantation, hypothermic machine perfusion (HMP) has been shown to be superior to static cold storage (SCS) in terms of functional outcomes. Ex vivo machine perfusion offers the possibility to deliver drugs or other active substances, such as Mesenchymal Stem Cells (MSCs), directly into an organ without affecting the recipient. MSCs are multipotent, self-renewing cells with tissue-repair capacities, and their application to ameliorate ischemia- reperfusion injury (IRI) is being investigated in several preclinical and clinical studies. The aim of this study was to introduce MSCs into a translational model of hypothermic machine perfusion and to test the efficiency and feasibility of this method. Methods: three rodent kidneys, six porcine kidneys and three human kidneys underwent HMP with 1-5 × 106 labelled MSCs within respective perfusates. Only porcine kidneys were compared to a control group of 6 kidneys undergoing HMP without MSCs, followed by mimicked reperfusion with whole blood at 37 °C for 2 h for all 12 kidneys. Reperfusion perfusate samples were analyzed for levels of NGAL and IL-β by ELISA. Functional parameters, including urinary output, oxygen consumption and creatinine clearance, were compared and found to be similar between the MSC treatment group and the control group in the porcine model. IL-1β levels were higher in perfusate and urine samples in the MSC group, with a median of 285.3 ng/mL (IQR 224.3-407.8 ng/mL) vs. 209.2 ng/mL (IQR 174.9-220.1), p = 0.51 and 105.3 ng/mL (IQR 71.03-164.7 ng/mL) vs. 307.7 ng/mL (IQR 190.9-349.6 ng/mL), p = 0.16, respectively. MSCs could be traced within the kidneys in all models using widefield microscopy after HMP. The application of Mesenchymal Stem Cells in an ex vivo hypothermic machine perfusion setting is feasible, and MSCs can be delivered into the kidney grafts during HMP. Functional parameters during mimicked reperfusion were not altered in treated kidney grafts. Changes in levels of IL-1β suggest that MSCs might have an effect on the kidney grafts, and whether this leads to a positive or a negative outcome on IRI in transplantation needs to be determined in further experiments.

A Simple Doublet Lens Design for Mid-Infrared Imaging.

Wide-field mid-infrared (MIR) hyperspectral imaging offers a promising approach for studying heterogeneous chemical systems due to its ability to independently characterize the molecular properties of different regions of a sample. However, applications of wide-field MIR microscopy are limited to spatial resolutions no better than ∼1 μm. While methods exist to overcome the classical diffraction limit of ∼λ/2, chromatic aberration from transmissive imaging reduces the achievable resolution. Here we describe the design and implementation of a simple MIR achromatic lens combination that we believe will aid in the development of resolution-enhanced wide-field MIR hyperspectral optical and chemical absorption imaging. We also examine the use of this doublet lens to image through polystyrene microspheres, an emerging and simple means for enhancing spatial resolution.

Advancing insights into in vivo meningeal lymphatic vessels with stereoscopic wide-field photoacoustic microscopy

Meningeal lymphatic vessels (mLVs) play a pivotal role in regulating metabolic waste from cerebrospinal fluid (CSF). However, the current limitations in field of view and resolution of existing imaging techniques impede understanding the stereoscopic morphology and dynamic behavior of mLVs in vivo. Here, we utilized dual-contrast functional photoacoustic microscopy to achieve wide-field intravital imaging of the lymphatic system, including mLVs and glymphatic pathways. The stereoscopic photoacoustic microscopy based on opto-acoustic confocal features has a depth imaging capability of 3.75 mm, facilitating differentiation between mLVs on the meninges and glymphatic pathways within the brain parenchyma. Subsequently, using this imaging technique, we were able to visualize the dynamic drainage of mLVs and identify a peak drainage period occurring around 20–40 min after injection, along with determining the flow direction from CSF to lymph nodes. Inspiringly, in the Alzheimer’s disease (AD) mouse model, we observed that AD mice exhibit a ~ 70% reduction in drainage volume of mLVs compared to wild-type mice. With the development of AD, there is be continued decline in mLVs drainage volume. This finding clearly demonstrates that the AD mouse model has impaired CSF drainage. Our study opens up a horizon for understanding the brain’s drainage mechanism and dissecting mLVs-associated neurological disorders.