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Fluorescence microscopy has evolved from a purely observational tool to a platform for quantitative, hypothesis-driven research. As such, the demand for faster and less phototoxic imaging modalities has spurred a rapid growth in light sheet fluorescence microscopy (LSFM). By restricting the excitation to a thin plane, LSFM reduces the overall light dose to a specimen while simultaneously improving image contrast. However, the defining characteristics of light sheet microscopes subsequently warrant unique considerations in their use for quantitative experiments. In this Perspective, we outline many of the pitfalls in LSFM that can compromise analysis and confound interpretation. Moreover, we offer guidance in addressing these caveats when possible. In doing so, we hope to provide a useful resource for life scientists seeking to adopt LSFM to quantitatively address complex biological hypotheses.
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Microscopia de Fluorescência , Microscopia de Fluorescência/métodosRESUMO
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.
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Biologia , Luz , Anisotropia , Microscopia de Fluorescência/métodos , FotodegradaçãoRESUMO
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.
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Laboratórios , Pesquisa Biomédica , Laboratórios/economiaRESUMO
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.
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Since its initial development in 1976, fluorescence recovery after photobleaching (FRAP) has been one of the most popular tools for studying diffusion and protein dynamics in living cells. Its popularity is derived from the widespread availability of confocal microscopes and the relative ease of the experiment and analysis. FRAP, however, is limited in its ability to resolve spatial heterogeneity. Here, we combine selective plane illumination microscopy (SPIM) and FRAP to create SPIM-FRAP, wherein we use a sheet of light to bleach a two-dimensional (2D) plane and subsequently image the recovery of the same image plane. This provides simultaneous quantification of diffusion or protein recovery for every pixel in a given 2D slice, thus moving FRAP measurements beyond these previous limitations. We demonstrate this technique by mapping both intranuclear diffusion of NLS-GFP and recovery of 53BP1-mCherry, a marker for DNA damage, in live MDA-MB-231 cells. SPIM-FRAP proves to be an order of magnitude faster than fluorescence-correlation-spectroscopy-based techniques for such measurements. We observe large length-scale (>â¼500 nm) heterogeneity in the recovery times of NLS-GFP, which is validated against simulated data sets. 2D maps of NLS-GFP recovery times showed no pixel-by-pixel correlation with histone density, although slower diffusion was observed in nucleoli. Additionally, recovery of 53BP1-mCherry was observed to be slowed at sites of DNA damage. We finally developed a diffusion simulation for our SPIM-FRAP experiments to compare across techniques. Our measured diffusion coefficients are on the order of previously reported results, thus validating the quantitative accuracy of SPIM-FRAP relative to well-established methods. With the recent rise of accessibility of SPIM systems, SPIM-FRAP is set to provide a straightforward means of quantifying the spatial distribution of protein recovery or diffusion in living cells.
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Iluminação , Difusão , Recuperação de Fluorescência Após Fotodegradação , Microscopia Confocal , Espectrometria de FluorescênciaRESUMO
We developed VIEW-MOD (Versatile Illumination Engine with a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two fast-steering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications.
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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.
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Microscopia , Microscopia/métodosRESUMO
Forces controlling tissue morphogenesis are attributed to cellular-driven activities, and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can develop autonomous stresses during their assembly. Here, we examine the morphogenetic function of an ECM before reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with the exponential assembly of collagen IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4 mutations that affect assembly reveal that VNC morphodynamics is partially driven by a sudden increase in ECM-driven surface tension. These data suggest that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis.
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Drosophila , Matriz Extracelular , Animais , Drosophila/genética , Matriz Extracelular/fisiologia , Morfogênese/fisiologia , Sistema Nervoso CentralRESUMO
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.
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Biologia , Imagem Óptica , Microscopia de Fluorescência/métodosRESUMO
It is increasingly appreciated that the cell nucleus is not only a home for DNA but also a complex material that resists physical deformations and dynamically responds to external mechanical cues. The molecules that confer mechanical properties to nuclei certainly contribute to laminopathies and possibly contribute to cellular mechanotransduction and physical processes in cancer such as metastasis. Studying nuclear mechanics and the downstream biochemical consequences or their modulation requires a suite of complex assays for applying, measuring, and visualizing mechanical forces across diverse length, time, and force scales. Here, we review the current methods in nuclear mechanics and mechanobiology, placing specific emphasis on each of their unique advantages and limitations. Furthermore, we explore important considerations in selecting a new methodology as are demonstrated by recent examples from the literature. We conclude by providing an outlook on the development of new methods and the judicious use of the current techniques for continued exploration into the role of nuclear mechanobiology.
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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.
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Cancer metastasis, i.e., the spreading of tumor cells from the primary tumor to distant organs, is responsible for the vast majority of cancer deaths. In the process, cancer cells migrate through narrow interstitial spaces substantially smaller in cross-section than the cell. During such confined migration, cancer cells experience extensive nuclear deformation, nuclear envelope rupture, and DNA damage. The molecular mechanisms responsible for the confined migration-induced DNA damage remain incompletely understood. Although in some cell lines, DNA damage is closely associated with nuclear envelope rupture, we show that, in others, mechanical deformation of the nucleus is sufficient to cause DNA damage, even in the absence of nuclear envelope rupture. This deformation-induced DNA damage, unlike nuclear-envelope-rupture-induced DNA damage, occurs primarily in S/G2 phase of the cell cycle and is associated with replication forks. Nuclear deformation, resulting from either confined migration or external cell compression, increases replication stress, possibly by increasing replication fork stalling, providing a molecular mechanism for the deformation-induced DNA damage. Thus, we have uncovered a new mechanism for mechanically induced DNA damage, linking mechanical deformation of the nucleus to DNA replication stress. This mechanically induced DNA damage could not only increase genomic instability in metastasizing cancer cells but could also cause DNA damage in non-migrating cells and tissues that experience mechanical compression during development, thereby contributing to tumorigenesis and DNA damage response activation.
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Núcleo Celular/genética , Núcleo Celular/patologia , Dano ao DNA , Replicação do DNA , Estresse Fisiológico , Carcinogênese , Linhagem Celular , Movimento Celular , Instabilidade Genômica , Humanos , Neoplasias/genética , Neoplasias/patologia , Membrana Nuclear/patologiaRESUMO
Cell nuclei are paramount for both cellular function and mechanical stability. These two roles of nuclei are intertwined as altered mechanical properties of nuclei are associated with altered cell behavior and disease. To further understand the mechanical properties of cell nuclei and guide future experiments, many investigators have turned to mechanical modeling. Here, we provide a comprehensive review of mechanical modeling of cell nuclei with an emphasis on the role of the nuclear lamina in hopes of spurring future growth of this field. The goal of this review is to provide an introduction to mechanical modeling techniques, highlight current applications to nuclear mechanics, and give insight into future directions of mechanical modeling. There are three main classes of mechanical models-schematic, continuum mechanics, and molecular dynamics-which provide unique advantages and limitations. Current experimental understanding of the roles of the cytoskeleton, the nuclear lamina, and the chromatin in nuclear mechanics provide the basis for how each component is subsequently treated in mechanical models. Modeling allows us to interpret assay-specific experimental results for key parameters and quantitatively predict emergent behaviors. This is specifically powerful when emergent phenomena, such as lamin-based strain stiffening, can be deduced from complimentary experimental techniques. Modeling differences in force application, geometry, or composition can additionally clarify seemingly conflicting experimental results. Using these approaches, mechanical models have informed our understanding of relevant biological processes such as migration, nuclear blebbing, nuclear rupture, and cell spreading and detachment. There remain many aspects of nuclear mechanics for which additional mechanical modeling could provide immediate insight. Although mechanical modeling of cell nuclei has been employed for over a decade, there are still relatively few models for any given biological phenomenon. This implies that an influx of research into this realm of the field has the potential to dramatically shape both future experiments and our current understanding of nuclear mechanics, function, and disease.
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Núcleo Celular/metabolismo , Animais , Cromatina/metabolismo , Citoesqueleto/metabolismo , Humanos , Lamina Tipo A/metabolismo , Modelos Biológicos , Lâmina Nuclear/metabolismoRESUMO
Nuclei are often under external stress, be it during migration through tight constrictions or compressive pressure by the actin cap, and the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area, respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.