Conventional Fluorescence Microscopy Research Articles

Acoustofluidic scanning fluorescence nanoscopy with a large field of view

Large-field nanoscale fluorescence imaging is invaluable for many applications, such as imaging subcellular structures, visualizing protein interactions, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy requires a trade-off between resolution and field of view due to the nature of the optics used to form the image. To overcome this barrier, we developed an acoustofluidic scanning fluorescence nanoscope that simultaneously achieves superior resolution, a large field of view, and strong fluorescent signals. The acoustofluidic scanning fluorescence nanoscope utilizes the superresolution capabilities of microspheres that are controlled by a programmable acoustofluidic device for rapid fluorescence enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope resolves structures that cannot be resolved with conventional fluorescence microscopes with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution realized with enhanced fluorescent signals and the large field of view achieved via acoustofluidic scanning fluorescence nanoscopy provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients.

Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O2-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed "MMIR" (multimodal infrared), incorporate an NIR O2-sensitive metalloporphyrin (PtTPTBPF) and deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing for oxygen gradient quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated the nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest, and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroid size, and formation method influenced spheroid oxygenation. Some spheroids produced from HCT116 and dental pulp stem cells exhibited "inverted" oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered "normal" gradient of hypoxia toward the core caused by diffusion. Further microscopy analysis of spheroids with an "inverted" gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H indicated the formation of a glycolytic core and localization of OxPhos-active cells at the periphery. Collectively, we demonstrate a strong potential of NIR-emitting ratiometric nanosensors for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models.

Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization

Bacterial biofilms form when bacteria attach to surfaces and generate an extracellular matrix that embeds and stabilizes a growing community. Detailed visualization and quantitative analysis of biofilm architecture by optical microscopy are limited by the law of diffraction. Expansion Microscopy (ExM) is a novel Super-Resolution technique where specimens are physically enlarged by a factor of ∼4, prior to observation by conventional fluorescence microscopy. ExM requires homogenization of rigid constituents of biological components by enzymatic digestion. We developed an ExM approach capable of expanding 48-h old Proteus mirabilis biofilms 4.3-fold (termed PmbExM), close to the theoretic maximum expansion factor without gross shape distortions. Our protocol, based on lytic and glycoside-hydrolase enzymatic treatments, degrades rigid components in bacteria and extracellular matrix. Our results prove PmbExM to be a versatile and easy-to-use Super-Resolution approach for enabling studies of P. mirabilis biofilm architecture, assembly, and even intracellular features, such as DNA organization.

Expansion and Light-Sheet Microscopy for Nanoscale 3D Imaging.

Expansion Microscopy (ExM) and Light-Sheet Fluorescence Microscopy (LSFM) are forefront imaging techniques that enable high-resolution visualization of biological specimens. ExM enhances nanoscale investigation using conventional fluorescence microscopes, while LSFM offers rapid, minimally invasive imaging over large volumes. This review explores the joint advancements of ExM and LSFM, focusing on the excellent performance of the integrated modality obtained from the combination of the two, which is refer to as ExLSFM. In doing so, the chemical processes required for ExM, the tailored optical setup of LSFM for examining expanded samples, and the adjustments in sample preparation for accurate data collection are emphasized. It is delve into various specimen types studied using this integrated method and assess its potential for future applications. The goal of this literature review is to enrich the comprehension of ExM and LSFM, encouraging their wider use and ongoing development, looking forward to the upcoming challenges, and anticipating innovations in these imaging techniques.

Retrospective validation of MetaSystems' deep-learning-based digital microscopy platform with assistance compared to manual fluorescence microscopy for detection of mycobacteria.

This manuscript presents a full validation of MetaSystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module using a probability threshold of 96% including accuracy, precision studies, and evaluation of limit of AFB detection on respiratory samples when the technology is used with assistance. This study is complementary to the conversation started by Tomasello et al. on the use of image analysis artificial intelligence software in routine mycobacterial diagnostic activities within the context of high-throughput laboratories with low incidence of tuberculosis.

High-throughput light sheet imaging of adult and larval C. elegans Parkinson's disease model using a low-cost optofluidic device and a fluorescent microscope.

Recent advancements at the interface of microfluidics technology and light sheet fluorescence microscopy have opened the door for high-throughput and high-content investigation of C. elegans disease models. In this paper, we report on the development of a simple, miniaturized, and low-cost optofluidic platform that can be added to a conventional inverted fluorescent microscope for continuous light sheet imaging of transgenic worm populations with high lateral and axial resolutions of 1.1 µm and 2.4 µm, respectively. The optofluidic device is made entirely of PDMS with integrated optics for light sheet generation. Laser excitation is delivered to the device via a low-cost free space laser, and cross-sections of worm populations are imaged as they pass continuously through a channel. Results show the platform can image NW1229 whole worms with pan-neural fluorescent expression at a throughput of >20 worms per minute at L3 and young adult (YA) stages. As a benchmark test, we show that the low-cost device can quantify the reduced neuronal expressions of L3 and YA NW1229 worms when exposed to 500 µM 6-OHDA neurodegenerative agent. Following the benchmark validation, we utilized the platform in a novel application for imaging human alpha-synuclein reporter in populations of Parkinson's transgenic model (ERS100). Results show the ability of the low-cost platform to reliably detect and quantify the anomalous neural phenotypic changes in ERS100 populations at L3 and YA stages with high spatial resolution. The findings of this study show the potential of our low-cost optofluidic add-on platform to equip conventional fluorescent microscopes with light sheet capability for quantitative phenotypic studies of transgenic C. elegans at high resolution and throughput.

High-throughput assessment of cellular senescence.

Cellular senescence is a molecular process that is activated in response to a large variety of distinct stress signals. Mechanistically, cellular senescence is characterized by an arrest in cell cycle accompanied by phenotypic adaptations and physiological alterations including changes in the secretory profile of senescent cells termed the senescence-associated secretory phenotype (SASP). Here we describe a detailed, automation- compatible method for the detection of senescence-associated beta galactosidase (SA-β-gal) activity as a hallmark of cellular senescence using a conventional fluorescent microscope equipped with a transmitted light module. Moreover, we outline a protocol for the automated analysis of cellular senescence using convolutional neural networks (CNNs) and mathematical morphology. In sum, we provide a toolset for the high throughput assessment of cellular senescence based on light microscopy and automated image analysis.

Following the cellular itinerary of renal aquaporin-2 shuttling with 4.5x expansion microscopy.

The shuttling of renal collecting duct aquaporin-2 (AQP2) between intracellular vesicles and the apical plasma membrane is paramount for regulation of renal water reabsorption. The binding of the circulating antidiuretic hormone arginine vasopressin (AVP) to the basolateral AVP receptor increases intracellular cAMP, which ultimately leads to AQP2 plasma membrane accumulation via a dual effect on AQP2 vesicle fusion with the apical plasma membrane and reduced AQP2 endocytosis. This AQP2 plasma membrane accumulation increases water reabsorption and consequently urine concentration. Conventional fluorescent microscopy provides a lateral resolution of ∼250 nm, which is insufficient to resolve the AQP2-containing endosomes/vesicles. Therefore, detailed information regarding the AQP2 vesicular population is still lacking. Newly established 4.5x Expansion Microscopy (ExM) can increase resolution to 60-70 nm. Using 4.5x ExM, we detected AQP2 vesicles/endosomes as small as 79 nm considering an average expansion factor of 4.3 for endosomes. Using different markers of the endosomal system provided detailed information of the cellular AQP2 itinerary upon changes in endogenous cAMP levels. Before cAMP elevation, AQP2 colocalized with early and recycling, but not late endosomes. Forskolin-induced cAMP increase was characterized by AQP2 insertion into the plasma membrane and AQP2 withdrawal from large perinuclear endosomes as well as some localization to lysosomal compartments. Forskolin washout promoted AQP2 endocytosis where AQP2 localized to not only early and recycling endosomes but also late endosomes and lysosomes indicating increased AQP2 degradation. Thus, our results show that 4.5 ExM is an attractive approach to obtain detailed information regarding AQP2 shuttling.NEW & NOTEWORTHY Renal aquaporin-2 (AQP2) imaged by expansion microscopy provides unprecedented 3-D information regarding the AQP2 itinerary in response to changes in cellular cAMP.

IU-ExM: nanoscopy of organelles and tissues with iterative ultrastructure expansion microscopy

Expansion microscopy (ExM) is a highly effective technique for super-resolution fluorescence microscopy that enables imaging of biological samples beyond the diffraction limit with conventional fluorescence microscopes. Despite the development of several enhanced protocols, ExM has not yet demonstrated the ability to achieve the precision of nanoscopy techniques such as Single Molecule Localization Microscopy (SMLM). Here, to address this limitation, we have developed an iterative ultrastructure expansion microscopy (iU-ExM) approach that achieves SMLM-level resolution. With iU-ExM, it is now possible to visualize the molecular architecture of gold-standard samples, such as the eight-fold symmetry of nuclear pores or the molecular organization of the conoid in Apicomplexa. With its wide-ranging applications, from isolated organelles to cells and tissue, iU-ExM opens new super-resolution avenues for scientists studying biological structures and functions.

Multiplane HiLo microscopy with speckle illumination and non-local means denoising.

HiLo microscopy synthesizes an optically sectioned image from two images, one obtained with uniform and another with patterned illumination, such as laser speckle. Speckle-based HiLo has the advantage of being robust to aberrations but is susceptible to residual speckle noise that is difficult to control. We present a computational method to reduce this residual noise without undermining resolution. In addition, we improve the versatility of HiLo microscopy by enabling simultaneous multiplane imaging (here nine planes). Our goal is to perform fast, high-contrast, multiplane imaging with a conventional camera-based fluorescence microscope. Multiplane HiLo imaging is achieved with the use of a single camera and z-splitter prism. Speckle noise reduction is based on the application of a non-local means (NLM) denoising method to perform ensemble averaging of speckle grains. We demonstrate the capabilities of multiplane HiLo with NLM denoising both with synthesized data and by imaging cardiac and brain activity in zebrafish larvae at 40Hz frame rates. Multiplane HiLo microscopy aided by NLM denoising provides a simple tool for fast optically sectioned volumetric imaging that can be of general utility for fluorescence imaging applications.

Comparison of minimal detectable protoporphyrin IX concentrations with a loupe device and conventional 5-ALA fluorescence microscopy: an experimental study.

The 5-aminolevulinic acid (5-ALA) fluorescence technique is now widely applied for intraoperative visualization of specific central nervous system (CNS) tumors. Previous technical implementations of this technique have relied on specifically modified surgical microscopes to visualize intratumoral fluorescent protoporphyrin (PpIX). While this approach evidently allows for reliable intraoperative tumor visualization, it requires the availability of specifically modified surgical microscopes and their use even in cases where the operating neurosurgeon would prefer to use surgical loupes. Recently, a novel loupe device was introduced that is also capable of visualizing 5-ALA fluorescence. The aim of this study was therefore to compare the detected PpIX concentrations between the conventional fluorescence microscope and the novel loupe device. We used fluorescence phantoms of different PpIX concentrations for comparison between a conventional fluorescence microscope and the novel loupe device. For this purpose, we created fluorescence images using the excitation light sources of the conventional fluorescence microscope and the loupe device with both available background illumination modes (low and high). Subsequently, the minimal detectable PpIX concentrations according to each technique were determined by five independent neurosurgeons. Using the conventional fluorescence microscope, the median minimal detectable PpIX concentration was (range: 0.15 to ). By the loupe device, the median minimal detectable PpIX concentration was (range: 0.10 to ) and (range: 0.07 to ) for the high- and low-modes, respectively. Altogether, the minimal detectable PpIX concentrations were significantly lower using the loupe device compared to the conventional fluorescence microscope (). Our data indicate that the novel loupe device is able to visualize 5-ALA fluorescence with high sensitivity and thus might serve as a powerful tool for visualization of specific CNS tumors in the future.

Expansion Microscopy of Ciliary Proteins.

Label-retention expansion microscopy (LR-ExM) is a sample preparation technique, which embeds the cells or tissues in a swellable hydrogel and expands the sample so that one can achieve a high resolution with any conventional fluorescence microscopes. Fluorescence loss during polymerization and protein denaturation have been a major limitation of standard expansion microscopy. To minimize fluorescence loss, LR-ExM uses trifunctional anchors, which can survive from polymerization and denaturation, and then introduce fluorophores after expansion. By using LR-ExM, one can study the structure of primary cilia at molecular-scale resolution with a much higher signal-to-noise ratio, compared with previously introduced expansion microscopy methods. In this chapter, we describe a detailed procedure showing how LR-ExM is used to study ciliary proteins.

Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Analysis of cortical cell polarity by imaging flow cytometry.

Metastasis is the main cause of cancer-related death and therapies specifically targeting metastasis are highly needed. Cortical cell polarity (CCP) is a prometastatic property of circulating tumor cells affecting their ability to exit blood vessels and form new metastases that constitute a promising point of attack to prevent metastasis. However, conventional fluorescence microscopy on single cells and manual quantification of CCP are time-consuming and unsuitable for screening regulators. In this study, we developed an imaging flow cytometry-based method for high-throughput screening of factors affecting CCP in melanoma cells. The artificial intelligence-supported analysis method we developed is highly reproducible, accurate, and orders of magnitude faster than manual quantification. Additionally, this method is flexible and can be adapted to include additional cellular parameters. In a small-scale pilot experiment using polarity-, cytoskeleton-, or membrane-affecting drugs, we demonstrate that our workflow provides a straightforward and efficient approach for screening factors affecting CCP in cells in suspension and provide insights into the specific function of these drugs in this cellular system. The method and workflow presented here will facilitate large-scale studies to reveal novel cell-intrinsic as well as systemic factors controlling CCP during metastasis.

Optimization of highly inclined illumination for diffraction-limited and super-resolution microscopy.

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and of how this impacts on the image quality complicates the setting of HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the effects of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 µm). Based on this, we present a simple optical solution that exploits a rectangular slit to reduce the inclined beam thickness down to 2.6 µm while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

Structured illumination microscopy with a phase-modulated spinning disk for optical sectioning.

Among various super-resolution microscopic techniques, structured illumination microscopy (SIM) stands out for live-cell imaging because of its higher imaging speed. However, conventional SIM lacks optical sectioning capability. Here we demonstrate a new, to the best of our knowledge, approach using a phase-modulated spinning disk (PMSD) that enhances the optical sectioning capability of SIM. The PMSD consists of a pinhole array for confocal imaging and a transparent polymer layer for light phase modulation. The light phase modulation was designed to cancel the zeroth-order diffracted beam and create a sharp lattice illumination pattern using the interference of four first-order diffracted beams. In the detection optical path, the PMSD serves as a spatial filter to physically reject about 80% of the out-of-focus signals, an approach that allows for real-time optical reconstruction of super-resolved images with enhanced contrast. Furthermore, the simplicity of the design makes it easy to upgrade a conventional fluorescence microscope to a PMSD SIM system.

Recent Applications of Deconvolution Microscopy in Medicine.

Deconvolution microscopy is a computational image-processing technique used in conjunction with fluorescence microscopy to increase the resolution and contrast of three-dimensional images. Fluorescence microscopy is a widely used technique in biology and medicine that involves labeling specific molecules or structures within a sample with fluorescent dyes and then electronically photographing the sample through a microscope. However, the resolution of conventional fluorescence microscopy is limited by diffraction within the microscope's optical path, which causes blurring of the image and reduces the ability to resolve structures in close proximity with one another. Deconvolution microscopy overcomes this limitation by means of computer-based image processing whereby mathematical algorithms are used to eliminate the blurring caused by the microscope's optics and thus obtain a higher-resolution image that reveals the fine details of the sample with greater accuracy. Deconvolution microscopy, which can be applied to a range of image acquisition modalities, including widefield, confocal, and super-resolution microscopy, has become an essential tool for studying the structure and function of biological systems at the cellular and molecular levels. In this perspective, the latest deconvolution techniques have been introduced and image-processing methods for medical purposes have been presented.

Proximity Ligation Assay (PLA) for Fillets of Drosophila Larvae.

The ability to detect protein-protein interactions is critical for understanding the mechanisms underlying protein and cell function. Current methods to assay protein-protein interactions, such as co-immunoprecipitation (Co-IP) and fluorescence resonance energy transfer (FRET), have limitations; for example, Co-IP is an in vitro technique and may not reflect the situation in vivo, and FRET typically suffers from low signal-to-noise ratio. The proximity ligation assay (PLA) is an in situ method for inferring protein-protein interaction with a high signal-to-noise ratio. The PLA technique can indicate that two different proteins are closely associated by the ability of two secondary antibody oligonucleotide probes to hybridize when they are close to each other. This interaction generates a signal with rolling-circle amplification using fluorescent nucleotides. Although a positive result does not indicate that two proteins directly interact, it implies a potential in vivo interaction that can then be verified in vitro. PLA uses primary antibodies against the two proteins (or epitopes) of interest, one raised in mouse and the other raised in rabbit. When these antibodies bind to proteins that lie within 40 nm of each other in the tissue, complementary oligonucleotides conjugated individually to mouse and rabbit secondary antibodies can anneal to form a template for rolling-circle amplification. Using fluorescently labeled nucleotides, rolling circle amplification generates a strong fluorescent signal in areas of the tissue where the two proteins are found together that is detected using conventional fluorescence microscopy. This protocol describes how to perform PLA in vivo on the central nervous system and peripheral nervous system of third-instar larvae of the fruit fly Drosophila melanogaster.

A fluorescence-based opto-mechatronic screening module (OMSM) for automated 3D cell culture workflows

Cost-effective automated solutions for hydrogel-based 3D cell culture workflows are required to increase throughput, reproducibility and user-independent results. 3D bioprinters can handle viscous biomaterials, however, throughput is low, they are limited to specific biomaterials, and lack post-processing and analysis methods. Conversely, liquid handling robots have higher throughput but miss viscous materials handling capabilities. Furthermore, commercial systems are not only expensive but mostly not ‘open-source’ and hence are not adaptable to specific experimental requirements.The aim of this study was the implementation of an opto-mechatronic screening module (OMSM) for automated 3D cell culture workflows from production to analysis into a previously developed biomanufacturing workstation. An analysis module with a fluorescence wide-field microscope and a motorised XYZ stage was engineered to transport tissue-culture plates from the storage rack of the workstation to the custom-made microscope and to assess fluorescence-based cell parameters. The microscope has two fluorescence channels and a resolution greater than 228 lp/mm. The XYZ-stage achieved an accuracy of 0.8 μm in X-direction (repeatability 2.4 μm) and 1.4 μm in Z-direction (repeatability <0.1 μm). Viability assays and drug-response studies were conducted on 2D and 3D prostate cancer gelatin-methacryloyl hydrogel models demonstrating its capabilities comparable to a conventional fluorescence microscope. Altogether, the integration of an opto-mechatronic screening module into the biomanufacturing workstation enables automated, reproducible, and user-independent production, imaging and analysis of hydrogel-based 3D cell cultures.

What do we know about blood-testis barrier? current understanding of its structure and physiology.

Blood-testis barrier (BTB) creates a particular compartment in the seminiferous epithelium. Contacting Sertoli cell-Sertoli cell plasma membranes possess specialized junction proteins which present a complex dynamic of formation and dismantling. Thus, these specialized structures facilitate germ cell movement across the BTB. Junctions are constantly rearranged during spermatogenesis while the BTB preserves its barrier function. Imaging methods are essential to studying the dynamic of this sophisticated structure in order to understand its functional morphology. Isolated Sertoli cell cultures cannot represent the multiple interactions of the seminiferous epithelium and in situ studies became a fundamental approach to analyze BTB dynamics. In this review, we discuss the contributions of high-resolution microscopy studies to enlarge the body of morphofunctional data to understand the biology of the BTB as a dynamic structure. The first morphological evidence of the BTB was based on a fine structure of the junctions, which was resolved with Transmission Electron Microscopy. The use of conventional Fluorescent Light Microscopy to examine labelled molecules emerged as a fundamental technique for elucidating the precise protein localization at the BTB. Then laser-scanning confocal microscopy allowed the study of three-dimensional structures and complexes at the seminiferous epithelium. Several junction proteins, like the transmembrane, scaffold and signaling proteins, were identified in the testis using traditional animal models. BTB morphology was analyzed in different physiological conditions as the spermatocyte movement during meiosis, testis development, and seasonal spermatogenesis, but also structural elements, proteins, and BTB permeability were studied. Under pathological, pharmacological, or pollutant/toxic conditions, there are significant studies that provide high-resolution images which help to understand the dynamic of the BTB. Notwithstanding the advances, further research using new technologies is required to gain information on the BTB. Super-resolution light microscopy is needed to provide new research with high-quality images of targeted molecules at a nanometer-scale resolution. Finally, we highlight research areas that warrant future studies, pinpointing new microscopy approaches and helping to improve our ability to understand this barrier complexity.