More than just 'added value': The perils of not establishing shared core facilities in resource-constrained communities.

The accelerating pace of technological advancements necessitates specialised expertise and cutting-edge instruments to maintain competitive research in life sciences. Core facilities - collaborative laboratories equipped with state-of-the-art tools and staffed by expert personnel - are vital resources that support diverse scientific endeavours. However, their adoption in lower-income communities has been comparatively stagnant due to both financial and cultural challenges. This paper explores the perils of not supporting core facilities on national research enterprises, underscoring the need for balanced investments in discovery science and crucial infrastructure support. We explore the implications from the perspectives of funders, university leaders and lab heads. We advocate for a paradigm shift to recognise these facilities as essential components of national research efforts. Core facilities are positioned not as optional but as strategic investments that can catalyse breakthroughs, particularly in environments with limited resources.

Believing is seeing - the deceptive influence of bias in quantitative microscopy.

The visual allure of microscopy makes it an intuitively powerful research tool. Intuition, however, can easily obscure or distort the reality of the information contained in an image. Common cognitive biases, combined with institutional pressures that reward positive research results, can quickly skew a microscopy project towards upholding, rather than rigorously challenging, a hypothesis. The impact of these biases on a variety of research topics is well known. What might be less appreciated are the many forms in which bias can permeate a microscopy experiment. Even well-intentioned researchers are susceptible to bias, which must therefore be actively recognized to be mitigated. Importantly, although image quantification has increasingly become an expectation, ostensibly to confront subtle biases, it is not a guarantee against bias and cannot alone shield an experiment from cognitive distortions. Here, we provide illustrative examples of the insidiously pervasive nature of bias in microscopy experiments - from initial experimental design to image acquisition, analysis and data interpretation. We then provide suggestions that can serve as guard rails against bias.

Imagining the future of optical microscopy: everything, everywhere, all at once.

The optical microscope has revolutionized biology since at least the 17th Century. Since then, it has progressed from a largely observational tool to a powerful bioanalytical platform. However, realizing its full potential to study live specimens is hindered by a daunting array of technical challenges. Here, we delve into the current state of live imaging to explore the barriers that must be overcome and the possibilities that lie ahead. We venture to envision a future where we can visualize and study everything, everywhere, all at once - from the intricate inner workings of a single cell to the dynamic interplay across entire organisms, and a world where scientists could access the necessary microscopy technologies anywhere.

Direct observation of the conformational states of PIEZO1.

PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals1,2 and have essential roles in diverse physiological settings3. In vitro studies have proposed that PIEZO channels transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore4-8. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.

Architecture and dynamics of a desmosome-endoplasmic reticulum complex.

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

The challenges and opportunities of open-access microscopy facilities.

Microscopy core facilities are increasingly utilised research resources, but they are generally only available to users within the host institution. Such localised access misses an opportunity to facilitate research across a broader user base. Here, we present the model of an open-access microscopy facility, using the Advanced Imaging Center (AIC) at Howard Hughes Medical Institute Janelia Research Campus as an example. The AIC has pioneered a model whereby advanced microscopy technologies and expertise are made accessible to researchers on a global scale. We detail our experiences in addressing the considerable challenges associated with this model for those who may be interested in launching an open-access imaging facility. Importantly, we focus on how this model can empower researchers, particularly those from resource-constrained settings.

Combining multiple fluorescence imaging techniques in biology: when one microscope is not enough.

While fluorescence microscopy has proven to be an exceedingly useful tool in bioscience, it is difficult to offer simultaneous high resolution, fast speed, large volume, and good biocompatibility in a single imaging technique. Thus, when determining the image data required to quantitatively test a complex biological hypothesis, it often becomes evident that multiple imaging techniques are necessary. Recent years have seen an explosion in development of novel fluorescence microscopy techniques, each of which features a unique suite of capabilities. In this Technical Perspective, we highlight recent studies to illustrate the benefits, and often the necessity, of combining multiple fluorescence microscopy modalities. We provide guidance in choosing optimal technique combinations to effectively address a biological question. Ultimately, we aim to promote a more well-rounded approach in designing fluorescence microscopy experiments, leading to more robust quantitative insight.

When light meets biology - how the specimen affects quantitative microscopy.

Fluorescence microscopy images should not be treated as perfect representations of biology. Many factors within the biospecimen itself can drastically affect quantitative microscopy data. Whereas some sample-specific considerations, such as photobleaching and autofluorescence, are more commonly discussed, a holistic discussion of sample-related issues (which includes less-routine topics such as quenching, scattering and biological anisotropy) is required to appropriately guide life scientists through the subtleties inherent to bioimaging. Here, we consider how the interplay between light and a sample can cause common experimental pitfalls and unanticipated errors when drawing biological conclusions. Although some of these discrepancies can be minimized or controlled for, others require more pragmatic considerations when interpreting image data. Ultimately, the power lies in the hands of the experimenter. The goal of this Review is therefore to survey how biological samples can skew quantification and interpretation of microscopy data. Furthermore, we offer a perspective on how to manage many of these potential pitfalls.

Visualizing the Invisible: Advanced Optical Microscopy as a Tool to Measure Biomechanical Forces.

The importance of mechanical force in biology is evident across diverse length scales, ranging from tissue morphogenesis during embryo development to mechanotransduction across single adhesion proteins at the cell surface. Consequently, many force measurement techniques rely on optical microscopy to measure forces being applied by cells on their environment, to visualize specimen deformations due to external forces, or even to directly apply a physical perturbation to the sample via photoablation or optogenetic tools. Recent developments in advanced microscopy offer improved approaches to enhance spatiotemporal resolution, imaging depth, and sample viability. These advances can be coupled with already existing force measurement methods to improve sensitivity, duration and speed, amongst other parameters. However, gaining access to advanced microscopy instrumentation and the expertise necessary to extract meaningful insights from these techniques is an unavoidable hurdle. In this Live Cell Imaging special issue Review, we survey common microscopy-based force measurement techniques and examine how they can be bolstered by emerging microscopy methods. We further explore challenges related to the accompanying data analysis in biomechanical studies and discuss the various resources available to tackle the global issue of technology dissemination, an important avenue for biologists to gain access to pre-commercial instruments that can be leveraged for biomechanical studies.

A guide to accurate reporting in digital image acquisition - can anyone replicate your microscopy data?

Recent technological advances have made microscopy indispensable in life science research. Its ubiquitous use, in turn, underscores the importance of ensuring that microscopy-based experiments are replicable and that the resulting data comparable. While there has been a wealth of review articles, practical guides and conferences devoted to the topic of maintaining standard instrument operating conditions, the paucity of attention dedicated to properly documenting microscopy experiments is undeniable. This lack of emphasis on accurate reporting extends beyond life science researchers themselves, to the review panels and editorial boards of many journals. Such oversight at the final step of communicating a scientific discovery can unfortunately negate the many valiant efforts made to ensure experimental quality control in the name of scientific reproducibility. This Review aims to enumerate the various parameters that should be reported in an imaging experiment by illustrating how their inconsistent application can lead to irreconcilable results.

Time-Variant SRC Kinase Activation Determines Endothelial Permeability Response.

In the current model of endothelial barrier regulation, the tyrosine kinase SRC is purported to induce disassembly of endothelial adherens junctions (AJs) via phosphorylation of VE cadherin, and thereby increase junctional permeability. Here, using a chemical biology approach to temporally control SRC activation, we show that SRC exerts distinct time-variant effects on the endothelial barrier. We discovered that the immediate effect of SRC activation was to transiently enhance endothelial barrier function as the result of accumulation of VE cadherin at AJs and formation of morphologically distinct reticular AJs. Endothelial barrier enhancement via SRC required phosphorylation of VE cadherin at Y731. In contrast, prolonged SRC activation induced VE cadherin phosphorylation at Y685, resulting in increased endothelial permeability. Thus, time-variant SRC activation differentially phosphorylates VE cadherin and shapes AJs to fine-tune endothelial barrier function. Our work demonstrates important advantages of synthetic biology tools in dissecting complex signaling systems.

Cellular localization of tolyporphins, unusual tetrapyrroles, in a microbial photosynthetic community determined using hyperspectral confocal fluorescence microscopy.

The cyanobacterial culture HT-58-2, composed of a filamentous cyanobacterium and accompanying community bacteria, produces chlorophyll a as well as the tetrapyrrole macrocycles known as tolyporphins. Almost all known tolyporphins (A-M except K) contain a dioxobacteriochlorin chromophore and exhibit an absorption spectrum somewhat similar to that of chlorophyll a. Here, hyperspectral confocal fluorescence microscopy was employed to noninvasively probe the locale of tolyporphins within live cells under various growth conditions (media, illumination, culture age). Cultures grown in nitrate-depleted media (BG-110 vs. nitrate-rich, BG-11) are known to increase the production of tolyporphins by orders of magnitude (rivaling that of chlorophyll a) over a period of 30-45 days. Multivariate curve resolution (MCR) was applied to an image set containing images from each condition to obtain pure component spectra of the endogenous pigments. The relative abundances of these components were then calculated for individual pixels in each image in the entire set, and 3D-volume renderings were obtained. At 30 days in media with or without nitrate, the chlorophyll a and phycobilisomes (combined phycocyanin and phycobilin components) co-localize in the filament outer cytoplasmic region. Tolyporphins localize in a distinct peripheral pattern in cells grown in BG-110 versus a diffuse pattern (mimicking the chlorophyll a localization) upon growth in BG-11. In BG-110, distinct puncta of tolyporphins were commonly found at the septa between cells and at the end of filaments. This work quantifies the relative abundance and envelope localization of tolyporphins in single cells, and illustrates the ability to identify novel tetrapyrroles in the presence of chlorophyll a in a photosynthetic microorganism within a non-axenic culture.

Image co-localization - co-occurrence versus correlation.

Fluorescence image co-localization analysis is widely utilized to suggest biomolecular interaction. However, there exists some confusion as to its correct implementation and interpretation. In reality, co-localization analysis consists of at least two distinct sets of methods, termed co-occurrence and correlation. Each approach has inherent and often contrasting strengths and weaknesses. Yet, neither one can be considered to always be preferable for any given application. Rather, each method is most appropriate for answering different types of biological question. This Review discusses the main factors affecting multicolor image co-occurrence and correlation analysis, while giving insight into the types of biological behavior that are better suited to one approach or the other. Further, the limits of pixel-based co-localization analysis are discussed in the context of increasingly popular super-resolution imaging techniques.

Characterization of differential Toll-like receptor responses below the optical diffraction limit.

Many membrane receptors are recruited to specific cell surface domains to form nanoscale clusters upon ligand activation. This step appears to be necessary to initiate cell signaling, including pathways in innate immune system activation. However, virulent pathogens such as Yersinia pestis (the causative agent of plague) are known to evade innate immune detection, in contrast to similar microbes (such as Escherichia coli) that elicit a robust response. This disparity has been partly attributed to the structure of lipopolysaccharides (LPS) on the bacterial cell wall, which are recognized by the innate immune receptor TLR4. It is hypothesized that nanoscale differences exist between the spatial clustering of TLR4 upon binding of LPS derived from Y. pestis and E. coli. Although optical imaging can provide exquisite details of the spatial organization of biomolecules, there is a mismatch between the scale at which receptor clustering occurs (<300 nm) and the optical diffraction limit (>400 nm). The last decade has seen the emergence of super-resolution imaging methods that effectively break the optical diffraction barrier to yield truly nanoscale information in intact biological samples. This study reports the first visualizations of TLR4 distributions on intact cells at image resolutions of <30 nm using a novel, dual-color stochastic optical reconstruction microscopy (STORM) technique. This methodology permits distinction between receptors containing bound LPS from those without at the nanoscale. Importantly, it is also shown that LPS derived from immunostimulatory bacteria result in significantly higher LPS-TLR4 cluster sizes and a nearly twofold greater ligand/receptor colocalization as compared to immunoevading LPS.

Advanced optical imaging reveals the dependence of particle geometry on interactions between CdSe quantum dots and immune cells.

The biocompatibility and possible toxicological consequences of engineered nanomaterials, including quantum dots (QDs) due to their unique suitability for biomedical applications, remain intense areas of interest. We utilized advanced imaging approaches to characterize the interactions of CdSe QDs of various sizes and shapes with live immune cells. Particle diffusion and partitioning within the plasma membrane, cellular uptake kinetics, and sorting of particles into lysosomes were all independantly characterized. Using high-speed total internal reflectance fluorescence (TIRF) microscopy, we show that QDs with an average aspect ratio of 2.0 (i.e., rod-shaped) diffuse nearly an order of magnitude slower in the plasma membrane than more spherical particles with aspect ratios of 1.2 and 1.6, respectively. Moreover, more rod-shaped QDs were shown to be internalized into the cell 2-3 fold more slowly. Hyperspectral confocal fluorescence microscopy demonstrates that QDs tend to partition within the cell membrane into regions containing a single particle type. Furthermore, data examining QD sorting mechanisms indicate that endocytosis and lysosomal sorting increases with particle size. Together, these observations suggest that both size and aspect ratio of a nanoparticle are important characteristics that significantly impact interactions with the plasma membrane, uptake into the cell, and localization within intracellular vesicles. Thus, rather than simply characterizing nanoparticle uptake into cells, we show that utilization of advanced imaging approaches permits a more nuanced and complete examination of the multiple aspects of cell-nanoparticle interactions that can ultimately aid understanding possible mechanisms of toxicity, resulting in safer nanomaterial designs.

Increased optical contrast in imaging of epidermal growth factor receptor using magnetically actuated hybrid gold/iron oxide nanoparticles.

We describe a new approach for optical imaging that combines the advantages of molecularly targeted plasmonic nanoparticles and magnetic actuation. This combination is achieved through hybrid nanoparticles with an iron oxide core surrounded by a gold layer. The nanoparticles are targeted in-vitro to epidermal growth factor receptor, a common cancer biomarker. The gold portion resonantly scatters visible light giving a strong optical signal and the superparamagnetic core provides a means to externally modulate the optical signal. The combination of bright plasmon resonance scattering and magnetic actuation produces a dramatic increase in contrast in optical imaging of cells labeled with hybrid gold/iron oxide nanoparticles.

A far-red fluorescent contrast agent to image epidermal growth factor receptor expression.

Recent developments in optical technologies have the potential to improve the speed and accuracy of screening and diagnosis of curable precancerous lesions and early cancer, thereby decreasing the costs of detection and management of epithelial malignancies. The development of molecular-specific contrast agents for markers of early neoplastic transformation could improve the detection and molecular characterization of premalignant lesions. In the oral cavity, epidermal growth factor receptor (EGFR) overexpression has been identified in early stages of premalignant lesions of the oral squamous cell carcinoma; therefore, real-time assessment of EGFR expression could serve as a biomarker for oral neoplasia. The purpose of our study was to develop a molecular-specific optical contrast agent targeted against EGFR for in vivo assessment of epithelial neoplasia using a monoclonal antibody and the far-red fluorescent dye, Alexa Fluor 660 streptavidin. In addition to demonstrating the specificity of the contrast agent for EGFR in cell lines, we document the ability to achieve penetration through 500 microm thick epithelial layers using multilayer tissue constructs and permeability-enhancing agents. Finally, using the fluorescence intensity of the contrast agent on fresh oral cavity tissue sections, we were able to distinguish abnormal from normal oral tissue. This contrast agent should have important clinical applications for use in conjunction with fluorescence spectroscopy or imaging (or both) to facilitate tumor detection and demarcation.