Conventional Epifluorescence Microscopy Research Articles

Molecular histopathology of matrix proteins through autofluorescence super-resolution microscopy

Extracellular matrix diseases like fibrosis are elusive to diagnose early on, to avoid complete loss of organ function or even cancer progression, making early diagnosis crucial. Imaging the matrix densities of proteins like collagen in fixed tissue sections with suitable stains and labels is a standard for diagnosis and staging. However, fine changes in matrix density are difficult to realize by conventional histological staining and microscopy as the matrix fibrils are finer than the resolving capacity of these microscopes. The dyes further blur the outline of the matrix and add a background that bottlenecks high-precision early diagnosis of matrix diseases. Here we demonstrate the multiple signal classification method-MUSICAL-otherwise a computational super-resolution microscopy technique to precisely estimate matrix density in fixed tissue sections using fibril autofluorescence with image stacks acquired on a conventional epifluorescence microscope. We validated the diagnostic and staging performance of the method in extracted collagen fibrils, mouse skin during repair, and pre-cancers in human oral mucosa. The method enables early high-precision label-free diagnosis of matrix-associated fibrotic diseases without needing additional infrastructure or rigorous clinical training.

Opto-combinatorial indexing enables high-content transcriptomics by linking cell images and transcriptomes.

We introduce a simple integrated analysis method that links cellular phenotypic behaviour with single-cell RNA sequencing (scRNA-seq) by utilizing a combination of optical indices from cells and hydrogel beads. With our method, the combinations, referred to as joint colour codes, enable the link via matching the optical combinations measured by conventional epi-fluorescence microscopy with the concatenated DNA molecular barcodes created by cell-hydrogel bead pairs and sequenced by next-generation sequencing. We validated our approach by demonstrating an accurate link between the cell image and scRNA-seq with mixed species experiments, longitudinal cell tagging by electroporation and lipofection, and gene expression analysis. Furthermore, we extended our approach to multiplexed chemical transcriptomics, which enabled us to identify distinct phenotypic behaviours in HeLa cells treated with various concentrations of paclitaxel, and determine the corresponding gene regulation associated with the formation of a multipolar spindle.

Multiplex immunofluorescence staining of coverslip-mounted paraffin-embedded tissue sections.

Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

Rcell2: Microscopy‐Based Cytometry in R

This article describes a method for quantifying various cellular features (e.g., volume, curvature, total and sub-cellular fluorescence localization) of individual cells from sets of microscope images, and for tracking them over time-course microscopy experiments. One purposely defocused transmission image (sometimes referred to as bright-field or BF) is used to segment the image and locate each cell. Fluorescence images (one for each of the color channels or z-stacks to be analyzed) may be acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses a set of R packages called rcell2. Relative to the original release of Rcell (Bush etal., 2012), the updated version bundles, into a single software suite, the image-processing capabilities of Cell-ID, offers new data analysis tools for cytometry, and relies on the widely used data analysis and visualization tools of the statistical programming framework R. © 2023 Wiley Periodicals LLC. Basic Protocol: Extracting quantitative information from single cells Support Protocol 1: Obtaining and installing Cell-ID and R Support Protocol 2: Preparing cells for imaging.

Measuring the viscoelastic behavior of dilute polymer solutions using high-speed statistical particle microrheology

The viscoelastic behavior of polymer solutions is commonly measured using oscillating shear rheometry, however, the accuracy of such methods is limited by the oscillating frequency of the equipment and since the relaxation time of the dilute polymer solutions is short, this requires measurement at very high frequencies. Microrheology has been proposed to overcome this technical challenge. Yet the equipment for resolving the statistics of particle displacements in microrheology is expensive. In this work, we measured the viscoelastic behavior of Methocel solutions at various concentrations using a conventional epi-fluorescence microscope coupled to a high-speed intensified camera. Statistical Particle Tracking is used in analyzing the mean-squared displacement of the dispersive particles. Relaxation times ranging from 0.76 – 9.00 ms and viscoelastic moduli, G′ between 11.34 and 3.39 are reported for Methocel solutions of concentrations between 0.063 – 0.5%.

An easy brain thick‐section‐clearing protocol to observe antigens in the brains of neurological disease mouse models by conventional epifluorescence and confocal laser scanning microscopy

We developed a brain thick-section-clearing (BTS-C) procedure to observe coronal sections of the entire mouse brain with conventional epifluorescence microscopy (EFM). After clearing with the BTS procedure, the white light transmittance of the 1-mm-thick mouse brain sections was more than 96%. The distribution patterns of amyloid plaques or α-synuclein in 1-mm-thick brain sections of five different mouse strains cleared with this procedure were easily observed by EFM. In addition, the detailed distribution of antigens at higher magnifications was revealed by observing the same slides with a confocal laser scanning microscope (CLSM). In conclusion, we have shown that the BTS protocol can replace laborious and time-consuming semi-thin-sectioning procedures, and the cleared 1-mm-thick sections can be examined by EFM and CLSM to elucidate the distribution patterns of antigens.

Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging.

Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.

Laser-free super-resolution microscopy.

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope set-up and a mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localization microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as 'blinking'), detected and localized. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that stimulated emission depletion and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Zn2+-Depletion Enhances Lysosome Fission in Cultured Rat Embryonic Cortical Neurons Revealed by a Modified Epifluorescence Microscopic Technique.

Lysosomes are integration hubs for several signaling pathways, such as autophagy and endocytosis, and also crucial stores of ions, including Zn2+. Lysosomal dysfunction caused by changes in their morphology by fusion and fission processes can result in several pathological disorders. However, the role of Zn2+ in modulating the morphology of lysosomes is unclear. The resolution of conventional epifluorescence microscopy restricts accurate observation of morphological changes of subcellular fluorescence punctum. In this study, we used a modified epifluorescence microscopy to identify the center of a punctum from a series of z-stack images and calculate the morphological changes. We stained primary cultured rat embryonic cortical neurons with FluoZin3, a Zn2+-sensitive fluorescent dye, and Lysotracker, a lysosome-specific marker, to visualize the distribution of Zn2+-enriched vesicles and lysosomes, respectively. Our results revealed that treating neurons with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, a cell-permeable Zn2+ chelator, shrank Zn2+-enriched vesicles and lysosomes by up to 25% in an hour. Pretreating the neurons with YM201636, a blocker of lysosome fission, could suppress this shrinkage. These results demonstrate the usefulness of the modified epifluorescence microscopy for investigating the homeostasis of intracellular organelles and related disorders.

New 1,3-Disubstituted Benzo[h]Isoquinoline Cyclen-Based Ligand Platform: Synthesis, Eu3+ Multiphoton Sensitization and Imaging Applications.

The development of lanthanide-based luminescent probes with a long emission lifetime has the potential to revolutionize imaging-based diagnostic techniques. By a rational design strategy taking advantage of computational predictions, a novel, water-soluble Eu3+ complex from a cyclen-based ligand bearing 1,3-disubstituted benzo[h]isoquinoline arms was realized. The ligand has been obtained overcoming the lack of reactivity of position 3 of the isoquinoline moiety. Notably, steric hindrance of the heteroaromatic chromophore allowed selective and stoichiometry-controlled insertion of two or three antennas on the cyclen platform without any protection strategy. The complex bears a fourth heptanoic arm for easy conjugation to biomolecules. This new chromophore allowed the sensitization of the metal center either with one or two photons excitation. The suitability as a luminescent bioprobe was validated by imaging BMI1 oncomarker in lung carcinoma cells following an established immunofluorescence approach. The use of a conventional epifluorescence microscope equipped with a linear structured illumination module disclosed a simple and inexpensive way to image confocally Ln-bioprobes by single photon excitation in the 350–400 nm window, where ordinary confocal systems have no excitation sources.

Folic acid functionalized gold nanoclusters for enabling targeted fluorescence imaging of human ovarian cancer cells

Photoluminescent gold nanoclusters have attracted an extensive research interest in bioimaging and therapeutics due to several distinctive advantages such as high fluorescent photostability, good dispersibility, low toxicity and large Stokes shift. However, a better understanding of the correlation between optical properties in various environments and their uptake by specific cancer cells is still needed. Herein, we developed bovine serum albumin stabilized gold nanoclusters (BSA-AuNCs) with an intrinsic tunable photoluminescence emission in the first biological window. The as-synthetized BSA-AuNCs agents consists in protein polymerized-chains dopped with AuNCs with an average size of 2–3 nm and were found to exhibit relevant properties as high photostability, temperature-dependent and excitation induced tunable red photoluminescence. The photostable BSA-AuNCs were functionalized with folic acid (FA-BSA-AuNCs) in order to achieve for the first time an active targeting of NIH:OVCAR-3 human ovarian adenocarcinoma cells, via AuNCs, towards bioimaging applications. After confirming their biocompatibility up to a concentration of 40 mg/ml, the improved cellular uptake and staining ability of FA-BSA-AuNCs compared to the BSA-AuNCs was validated by conventional wide-field epi-fluorescence microscopy, while the intracellular localization was monitored by confocal fluorescence lifetime imaging microscopy (FLIM). Considering their valuable intrinsic photoluminescent properties, the synthesized FA-BSA-AuNCs hold great promise for direct application in cellular imaging as efficient contrast agents towards early cancer diagnosis and image-guided therapy of cancer.

Inside Cover: Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

AbstractA type of compact and cost‐effective light‐sheet imaging device, termed sub‐voxel‐resolving light‐sheet add‐on module (SLAM), is developed to cooperate with conventional 2D epifluorescence microscope, allowing high‐contrast, resolution‐improved 3D imaging of various biological samples at high throughput.Further details can be found in the article by Fang Zhao, Yicong Yang, Yi Li, et al. (e201960243). image

Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis.

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

Single-Molecule Detection with Lightguiding Nanowires: Determination of Protein Concentration and Diffusivity in Supported Lipid Bilayers.

Determining the surface concentration and diffusivity of cell-membrane-bound molecules is central to the understanding of numerous important biochemical processes taking place at cell membranes. Here we use the high aspect ratio and lightguiding properties of semiconductor nanowires (NWs) to detect the presence of single freely diffusing proteins bound to a lipid bilayer covering the NW surface. Simultaneous observation of light-emission dynamics of hundreds of individual NWs occurring on the time scale of only a few seconds is interpreted using analytical models and employed to determine both surface concentration and diffusivity of cholera toxin subunit B (CTxB) bound to GM1 gangliosides in supported lipid bilayer (SLB) at surface concentrations down to below one CTxB per μm2. In particular, a decrease in diffusivity was observed with increasing GM1 content in the SLB, suggesting increasing multivalent binding of CTxB to GM1. The lightguiding capability of the NWs makes the method compatible with conventional epifluorescence microscopy, and it is shown to work well for both photostable and photosensitive dyes. These features make the concept an interesting complement to existing techniques for studying the diffusivity of low-abundance cell-membrane-bound molecules, expanding the rapidly growing use of semiconductor NWs in various bioanalytical sensor applications and live cell studies.

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately tento twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Multiphoton structured thin-plane imaging with a single optical path.

Optical sectioning has become an indispensable technique for high-speed volumetric imaging in the past decade. Here we present a novel optical-sectioning method that produces a thin plane of illumination by exploiting the spatial and temporal properties of multiphoton excitation. Critically, the illumination and detection share the same optical path, as in a conventional epi-fluorescence microscope configuration. Therefore, the imaged sample can be prepared as for standard fluorescence microscopy. Our method also leads to a laterally structured illumination pattern, and this feature can be utilized in structured illumination microscopy to further enhance the imaging performance. We show an example of such an approach, which achieves axial resolution finer than confocal microscopy. We also demonstrate the potential of the new method for biological applications by performing three-dimensional imaging of living Caenorhabditis elegans.

X10 expansion microscopy enables 25-nm resolution on conventional microscopes.

Expansion microscopy is a recently introduced imaging technique that achieves super‐resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20–30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000‐fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25–30 nm on conventional epifluorescence microscopes. X10 provides multi‐color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high‐quality super‐resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.

Ultrasensitive optical imaging with lanthanide lumiphores.

In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identify three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcome these limitations with a new lanthanide imaging modality, trans-reflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Cyclic Immunofluorescence (CycIF), A Highly Multiplexed Method for Single-cell Imaging.

Cyclic Immunofluorescence (CycIF) is a public-domain method for performing highly multiplexed immunofluorescence imaging using a conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up to 30 channels by sequential 4- to 6-channel imaging followed by fluorophore inactivation. Three variant methods are described, the most generally useful of which involves staining fixed cells with antibodies directly conjugated to Alexa Fluor dyes and imaging in four colors, inactivating fluorophores using a mild base in the presence of hydrogen peroxide and light, and then performing another round of staining and imaging. Cell morphology is preserved through multiple rounds of CycIF, and signal-to-noise ratios appear to increase. Unlike antibody-stripping methods, CycIF is gentle and optimized for monolayers of cultured cells. A second protocol involves indirect immunofluorescence and a third enables chemical inactivation of genetically encoded fluorescent proteins, allowing multiplex immunofluorescence to be combined with live-cell analysis of cells expressing fluorescent reporter proteins. © 2016 by John Wiley & Sons, Inc.