Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

Full spectrum fluorescence lifetime imaging with 0.5 nm spectral and 50 ps temporal resolution.

The use of optical techniques to interrogate wide ranging samples from semiconductors to biological tissue for rapid analysis and diagnostics has gained wide adoption over the past decades. The desire to collect ever more spatially, spectrally and temporally detailed optical signatures for sample characterization has specifically driven a sharp rise in new optical microscopy technologies. Here we present a high-speed optical scanning microscope capable of capturing time resolved images across 512 spectral and 32 time channels in a single acquisition with the potential for ~0.2 frames per second (256 × 256 image pixels). Each pixel in the resulting images contains a detailed data cube for the study of diverse time resolved light driven phenomena. This is enabled by integration of system control electronics and on-chip processing which overcomes the challenges presented by high data volume and low imaging speed, often bottlenecks in previous systems.

Confocal hyperspectral microscopic imager for the detection and classification of individual microalgae.

We propose a confocal hyperspectral microscopic imager (CHMI) that can measure both transmission and fluorescent spectra of individual microalgae, as well as obtain classical transmission images and corresponding fluorescent hyperspectral images with a high signal-to-noise ratio. Thus, the system can realize precise identification, classification, and location of microalgae in a free or symbiosis state. The CHMI works in a staring state, with two imaging modes, a confocal fluorescence hyperspectral imaging (CFHI) mode and a transmission hyperspectral imaging (THI) mode. The imaging modes share the main light path, and thus obtained fluorescence and transmission hyperspectral images have point-to-point correspondence. In the CFHI mode, a confocal technology to eliminate image blurring caused by interference of axial points is included. The CHMI has excellent performance with spectral and spatial resolutions of 3 nm and 2 µm, respectively (using a 10× microscope objective magnification). To demonstrate the capacity and versatility of the CHMI, we report on demonstration experiments on four species of microalgae in free form as well as three species of jellyfish with symbiotic microalgae. In the microalgae species classification experiments, transmission and fluorescence spectra collected by the CHMI were preprocessed using principal component analysis (PCA), and a support vector machine (SVM) model or deep learning was then used for classification. The accuracy of the SVM model and deep learning method to distinguish one species of individual microalgae from another was found to be 96.25% and 98.34%, respectively. Also, the ability of the CHMI to analyze the concentration, species, and distribution differences of symbiotic microalgae in symbionts is furthermore demonstrated.

Extended range and aberration-free autofocusing via remote focusing and sequence-dependent learning.

Rapid autofocusing over long distances is critical for tracking 3D topological variations and sample motion in real time. Taking advantage of a deformable mirror and Shack-Hartmann wavefront sensor, remote focusing can permit fast axial scanning with simultaneous correction of system-induced aberrations. Here, we report an autofocusing technique that combines remote focusing with sequence-dependent learning via a bidirectional long short term memory network. A 120 µm autofocusing range was achieved in a compact reflectance confocal microscope both in air and in refractive-index-mismatched media, with similar performance under arbitrary-thickness liquid layers up to 1 mm. The technique was validated on sample types not used for network training, as well as for tracking of continuous axial motion. These results demonstrate that the proposed technique is suitable for real-time aberration-free autofocusing over a large axial range, and provides unique advantages for biomedical, holographic and other related applications.

Mid-infrared multispectral confocal microscope using off-axis parabolic mirrors to study epiretinal membranes.

Mid-infrared (mid-IR) multispectral microscopy, especially operating at the wavelength of 5-11 µm, is an effective tool for detecting, identifying, and quantifying the structure and composition of biological tissues. Compared with that based on the optical lens, the mid-infrared microscope composed of off-axis parabolic (OAP) mirrors is low cost, simple, and suitable for longer range of wavelength without chromatic aberrations, while keeping the optical transmission efficiency. Here we report a compact and versatile mid-infrared multispectral confocal microscope based on off-axis parabolic mirrors. We also perform numerical calculations based on the vectorial diffraction theory on OAP mirrors and analyze the typical aberrations and misalignment of the OAP-based optical system. Finally, we perform multispectral imaging of the epiretinal membrane of the human eyes with the spectrum selected according to its resonance absorption peak. The system is designed to perform multispectral or even hyperspectral imaging to identify and predict potential disease.

An origami paper-based analytical device for DNA damage analysis.

Detection and characterization of DNA damage plays a critical role in genotoxicity testing, drug screening, and environmental health. We developed a fully integrated origami paper-based analytical device (oPAD) for measuring DNA damage. This simple device allows on-paper cell lysis, DNA extraction, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction and signal readout with simple operation steps, enabling rapid (within 30 min) and high throughput assessment of multiple DNA damages induced by exogenous chemical agents.

Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies.

Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.

Protocol for assessing real-time changes in mitochondrial morphology, fission and fusion events in live cells using confocal microscopy.

Changes in mitochondrial size, shape, and subcellular position, a process collectively known as mitochondrial dynamics, are exploited for various cancer traits. Modulation of subcellular mitochondrial trafficking and accumulation at the cortical cytoskeleton has been linked to the machinery of cell movements, fueling cell invasion and metastatic spreading. Here, we detail a technique to track changes in mitochondrial volume using a commercial CellLight™ Mitochondria-RFP/GFP reporter and live confocal microscopy. This allows a real-time study of mitochondrial dynamics in live cells. For complete details on the use and execution of this protocol, please refer to Bertolini et al. (2020).

A ball microscope for viewing the entire surface of amphibian embryos.

Waves on the surface of developing eggs/embryos need to be viewed from all sides of their 3D tissue. The ball microscope will enable tracking of cellular waves and determine their interactions with the cells on the surface. Nine microscopes are arrayed in a spherical formation around an imaging stage to create whole surface images of objects anywhere from 0.5 mm3 to 60 mm3 in size. The 3D printed ball-based microscope is made using nine, Opti-Tekscope OT-HD Digital USB Microscope Camera Magnifiers. Eight of the microscope cameras fit into the ball at 90° angles to each other and one bottom microscope is used for a base to hold the stage. The base will support a customised cuvette to hold the embryo in water. The microscopes are the size of a pen (13 cm long and 1 cm in diameter) and each have a ring light around their diameter for self illumination. The nine microscopes can be attached to a microcontroller for time-lapse automated imaging. This microscope will be compared to other microscopes developed for the same purpose. The microscope can be used for time lapse imaging of the surface of small 3D objects and can be used to view Axolotl salamander embryo development as the Axolotl embryos are 2 mm in diameter. Other amphibian eggs can also be imaged using this technique.

High-speed laser-scanning biological microscopy using FACED.

Laser scanning is used in advanced biological microscopy to deliver superior imaging contrast, resolution and sensitivity. However, it is challenging to scale up the scanning speed required for interrogating a large and heterogeneous population of biological specimens or capturing highly dynamic biological processes at high spatiotemporal resolution. Bypassing the speed limitation of traditional mechanical methods, free-space angular-chirp-enhanced delay (FACED) is an all-optical, passive and reconfigurable laser-scanning approach that has been successfully applied in different microscopy modalities at an ultrafast line-scan rate of 1-80 MHz. Optimal FACED imaging performance requires optimized experimental design and implementation to enable specific high-speed applications. In this protocol, we aim to disseminate information allowing FACED to be applied to a broader range of imaging modalities. We provide (i) a comprehensive guide and design specifications for the FACED hardware; (ii) step-by-step optical implementations of the FACED module including the key custom components; and (iii) the overall image acquisition and reconstruction pipeline. We illustrate two practical imaging configurations: multimodal FACED imaging flow cytometry (bright-field, fluorescence and second-harmonic generation) and kHz 2D two-photon fluorescence microscopy. Users with basic experience in optical microscope operation and software engineering should be able to complete the setup of the FACED imaging hardware and software in ~2-3 months.

Live-cell imaging of microglial interactions with radial glia in transgenic embryonic mouse brains using slice culture.

Microglial dynamics and interactions with nearby radial glia can be visualized in real time in embryonic mouse brain tissue using time-lapse imaging in slice culture. This live-cell imaging protocol can be used to study the morphology and activities of a number of cell types across a variety of brain regions and developmental time points. The advantage of this brain slice culture model is that it allows for the visualization of cellular interactions and movements in real time, especially across embryogenesis. For complete details on the use and execution of this protocol, please refer to Rosin et al. (2021).

Pupil function design for multifocal confocal, STED, and isoSTED microscopy.

Point scanning super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy are powerful tools to observe biological samples at sub-diffraction limited resolution in three dimensions. However, scanning the sample with only a single beam limits the imaging speed in these microscopes. Here, we propose a concept to increase this speed by introducing highly flexible multifocal illumination and detection. We introduce phase patterns in the objectives' pupil planes to create arrays of foci in the sample plane with negligible loss of laser power. High uniformity of these foci's intensities is achieved by iteratively applying a weighted Gerchberg-Saxton phase retrieval algorithm. We characterize the performance of this iterative approach numerically and present simulation results that demonstrate the high quality of the focus arrays for future implementations in laser-scanning STED and isoSTED microscopes. The same approach can also be applied in diffraction-limited confocal laser scanning microscopy.

A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities.

Cellular responses to DNA double-strand breaks (DSBs) not only promote genomic integrity in healthy tissues, but also largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. A growing body of evidence suggests that activation of the mechanisms that detect, signal and repair DSBs may depend on the complexity of the initiating DNA lesions. Studies focusing on this, as well as on many other radiobiological questions, require reliable methods to induce DSBs of varying complexity, and to visualize the ensuing cellular responses. Accelerated particles of different energies and masses are exceptionally well suited for this task, due to the nature of their physical interactions with the intracellular environment, but visualizing cellular responses to particle-induced damage - especially in their early stages - at particle accelerator facilities, remains challenging. Here we describe a straightforward approach for real-time imaging of early response to particle-induced DNA damage. We rely on a transportable setup with an inverted fluorescence confocal microscope, tilted at a small angle relative to the particle beam, such that cells can be irradiated and imaged without any microscope or beamline modifications. Using this setup, we image and analyze the accumulation of fluorescently-tagged MDC1, RNF168 and 53BP1-key factors involved in DSB signalling-at DNA lesions induced by 254 MeV α-particles. Our results provide a demonstration of technical feasibility and reveal asynchronous initiation of accumulation of these proteins at different individual DSBs.

Comparison of the effects of operating microscopes with light emitting diode and halogen light source on the eye: a rabbit study.

PURPOSE: To evaluate the potential toxicity of operation microscopes with halogen and light emitting diode (LED) light source on the rabbit eyes. MATERIALS AND METHODS: Thirty-two eyes of 16 male New Zealand pigmented rabbits were involved in the study. The rabbits were divided into two groups according to the type of light source applied. Only one eye of each rabbit was exposed to illumination light, unexposed fellow eyes served as the control group. Experimental groups included group 1 exposed to halogen light for 2 h and evaluated 1 day and 1 week after the illumination, group 2 exposed to LED light for two hours and evaluated 1 day and 1 week after the illumination. On the first and seventh days after exposing the light, we evaluated the rabbit corneas using in vivo confocal microscopy (IVCM). At the end of the seventh day, the Hematoxylin-eosin staining and TUNEL staining were performed to investigate the presence of apoptosis in the retina and retina pigment epithelium. RESULTS: Early IVCM findings revealed corneal epithelial cell ovalization and indistinct intercellular borders in the halogen light group. We also observed more increase in the keratocyte density index (23.7% vs 14.1%, p = 0.001, respectively) and the Bowman reflectivity index (12.4% vs 4.1%, p = 0.001, respectively) at first day of the light exposure in halogen light group compared to LED light group. However, late IVCM indicated that these findings disappeared one week later. No apoptosis was observed in the corneal and retinal layers in early and late examination groups. CONCLUSION: The present experimental study demonstrated that both halogen and LED lights, which were commonly used for microscopic eye surgery, had no sustained adverse effect on the cornea and retina of the rabbits; however, halogen light had a temporary adverse effect on corneal epithelium and stroma, which resolved within 1 week.

Update of Mitochondrial Network Analysis by Imaging: Proof of Technique in Schizophrenia.

Mitochondria, similar to living cells and organelles, have a negative membrane potential, which ranges between (-108) and (150) mV as compared to (-70) and (-90) mV of the plasma membrane. Therefore, permeable lipophilic cations tend to accumulate in the mitochondria. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of two different dyes for labeling mitochondrial membrane potential (Δψm) in live cells. One is the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active mitochondria) with increasing mitochondrial membrane potential (Δψm). The other is MitoTracker® Orange, a mitochondrion-selective probe which passively diffuses across the plasma membrane and accumulates in active mitochondria depending on their Δψm. We show that in addition to changes in Δψm, these specific dyes can be used to follow alterations in mitochondrial distribution and mitochondrial network connectivity. We suggest that JC-1 is a preferable probe to compare between different cell types and cell state, as a red to green ratio of fluorescence intensities is used for analysis. This ratio depends only on the mitochondrial membrane potential and not on other cellular and/or mitochondrial-dependent or independent factors that may alter, for example, due to treatment or disease state. However, in cells labeled either with green or red fluorescence protein, JC-1 cannot be used. Therefore, other dyes are preferable. We demonstrate various applications of JC-1 and MitoTracker Orange staining to study mitochondrial abnormalities in different cell types derived from schizophrenia patients and healthy subjects.

Real-time multi-angle projection imaging of biological dynamics.

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.

Pinhole Closure Improves Spatial Resolution in Confocal Scanning Microscopy.

Confocal microscopy is a simple, super-resolution technique, which does not produce a marked increase in resolution compared to other advanced techniques, such as super-resolution nanoscopy. Here, we present a simple protocol to acquire "slightly, but easily resolved" images by pinhole closure (<1 airy unit) in a conventional confocal scanning microscope equipped with an avalanche photodiode, a detector with high sensitivity. We use murine neuroblastoma Neuro2a cells to demonstrate the image resolution obtained via this protocol without the use of any special software to enhance image quality.

Subcellular three-dimensional imaging deep through multicellular thick samples by structured illumination microscopy and adaptive optics.

Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 150 nm lateral and 570 nm axial resolution at a depth of 80 µm through Caenorhabditis elegans. We demonstrate that Adaptive Optics improves the three-dimensional resolution, especially along the axial direction, and reduces artifacts, successfully realizing 3D-Structured Illumination Microscopy in a variety of biological samples.