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Current Protocols in Cytometry Mar 2020In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does...
In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does not have sufficient depth of focus, light from sample planes above and below the focal plane will also be detected. The out-of-focus light will add blur to the image, reducing the resolution. In fluorescence microscopy, any dye molecules in the field of view will be stimulated, including those in out-of-focus planes. Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high-resolution imaging in thick tissues. In a confocal microscope, the illumination and detection optics are focused on the same diffraction-limited spot in the sample, which is the only spot imaged by the detector during a confocal scan. To generate a complete image, the spot must be moved over the sample and data collected point by point. A significant advantage of the confocal microscope is the optical sectioning provided, which allows for 3D reconstruction of a sample from high-resolution stacks of images. Several types of confocal microscopes have been developed for this purpose, and each has different advantages and disadvantages. This article provides a concise introduction to confocal microscopy. © 2019 by John Wiley & Sons, Inc.
Topics: Animals; Drosophila; HeLa Cells; Humans; Larva; Microscopy, Confocal; Microtubules; Sample Size; Time Factors
PubMed: 31876974
DOI: 10.1002/cpcy.68 -
Journal of the American Chemical Society Oct 2022Strategies to visualize cellular membranes with light microscopy are restricted by the diffraction limit of light, which far exceeds the dimensions of lipid bilayers....
Strategies to visualize cellular membranes with light microscopy are restricted by the diffraction limit of light, which far exceeds the dimensions of lipid bilayers. Here, we describe a method for super-resolution imaging of metabolically labeled phospholipids within cellular membranes. Guided by the principles of expansion microscopy, we develop an all-small molecule approach that enables direct chemical anchoring of bioorthogonally labeled phospholipids into a hydrogel network and is capable of super-resolution imaging of cellular membranes. We apply this method, termed lipid expansion microscopy (LExM), to visualize organelle membranes with precision, including a unique class of membrane-bound structures known as nuclear invaginations. Compatible with standard confocal microscopes, LExM will be widely applicable for super-resolution imaging of phospholipids and cellular membranes in numerous physiological contexts.
Topics: Cell Membrane; Hydrogels; Lipid Bilayers; Microscopy, Confocal; Microscopy, Fluorescence; Phospholipids
PubMed: 36190998
DOI: 10.1021/jacs.2c03743 -
Proceedings of the National Academy of... Nov 2015Imaging reveals complex structures and dynamic interactive processes, located deep inside the body, that are otherwise difficult to decipher. Numerous imaging modalities... (Review)
Review
Imaging reveals complex structures and dynamic interactive processes, located deep inside the body, that are otherwise difficult to decipher. Numerous imaging modalities harness every last inch of the energy spectrum. Clinical modalities include magnetic resonance imaging (MRI), X-ray computed tomography (CT), ultrasound, and light-based methods [endoscopy and optical coherence tomography (OCT)]. Research modalities include various light microscopy techniques (confocal, multiphoton, total internal reflection, superresolution fluorescence microscopy), electron microscopy, mass spectrometry imaging, fluorescence tomography, bioluminescence, variations of OCT, and optoacoustic imaging, among a few others. Although clinical imaging and research microscopy are often isolated from one another, we argue that their combination and integration is not only informative but also essential to discovering new biology and interpreting clinical datasets in which signals invariably originate from hundreds to thousands of cells per voxel.
Topics: Animals; Biomarkers; Diagnostic Imaging; Engineering; Humans; Intravital Microscopy; Neoplasms; Tomography, Optical Coherence
PubMed: 26598657
DOI: 10.1073/pnas.1508524112 -
The International Journal of... 2019In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring... (Review)
Review
In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring during oogenesis and early embryonic development in mammals. In short, two modes of cellular imaging are now available: those involving fluorescent labels and those which are fluorophore-free. Fluorescence microscopy, in its various forms, is used predominantly in research, as it provides detailed information about cellular processes; however, it can involove an increased risk of photodamage. Fluorophore-free techniques provide, on the other hand, a smaller amount of biological data but they are safer for cells and therefore can be potentially used in a clinical setting. Here, we review various fluorescence and fluorophore-free visualisation approaches and discuss their applicability in developmental biology and reproductive medicine.
Topics: Blastocyst; Embryonic Development; Microscopy; Microscopy, Fluorescence; Microscopy, Polarization; Oocytes
PubMed: 31058300
DOI: 10.1387/ijdb.180300aa -
Annual Review of Biophysics May 2020Mitochondria are essential for eukaryotic life. These double-membrane organelles often form highly dynamic tubular networks interacting with many cellular structures.... (Review)
Review
Mitochondria are essential for eukaryotic life. These double-membrane organelles often form highly dynamic tubular networks interacting with many cellular structures. Their highly convoluted contiguous inner membrane compartmentalizes the organelle, which is crucial for mitochondrial function. Since the diameter of the mitochondrial tubules is generally close to the diffraction limit of light microscopy, it is often challenging, if not impossible, to visualize submitochondrial structures or protein distributions using conventional light microscopy. This renders super-resolution microscopy particularly valuable, and attractive, for studying mitochondria. Super-resolution microscopy encompasses a diverse set of approaches that extend resolution, as well as nanoscopy techniques that can even overcome the diffraction limit. In this review, we provide an overview of recent studies using super-resolution microscopy to investigate mitochondria, discuss the strengths and opportunities of the various methods in addressing specific questions in mitochondrial biology, and highlight potential future developments.
Topics: Humans; Microscopy; Mitochondria; Nanotechnology
PubMed: 32092283
DOI: 10.1146/annurev-biophys-121219-081550 -
Developmental Cell Apr 20233D cell cultures, in particular organoids, are emerging models in the investigation of healthy or diseased tissues. Understanding the complex cellular sociology in...
3D cell cultures, in particular organoids, are emerging models in the investigation of healthy or diseased tissues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter-scale live-cell light microscopy to nanometer-scale volume electron microscopy by performing 3D cell cultures in a single carrier that is amenable to all imaging steps. This allows for following organoids' growth, probing their morphology with fluorescent markers, identifying areas of interest, and analyzing their 3D ultrastructure. We demonstrate this workflow on mouse and human 3D cultures and use automated image segmentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses identify local organization of diffraction-limited cell junctions in compact and polarized epithelia. The continuum-resolution imaging pipeline is thus suited to fostering basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy.
Topics: Animals; Humans; Mice; Cell Culture Techniques, Three Dimensional; Microscopy; Microscopy, Electron; Organoids; Colorectal Neoplasms
PubMed: 36990090
DOI: 10.1016/j.devcel.2023.03.001 -
Microscopy (Oxford, England) Jun 2023Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely...
Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques.
Topics: Humans; Virus Diseases; Viruses; Microscopy, Atomic Force; Microscopy, Electron; Microscopy, Fluorescence; Cryoelectron Microscopy
PubMed: 37079744
DOI: 10.1093/jmicro/dfad024 -
Viruses Apr 2018Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging... (Review)
Review
Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.
Topics: Animals; Host-Pathogen Interactions; Humans; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Optical Imaging; Plants; Viruses
PubMed: 29670029
DOI: 10.3390/v10040202 -
Current Opinion in Chemical Biology Jun 2014Mitochondria, the powerhouses of the cell, are essential organelles in eukaryotic cells. With their complex inner architecture featuring a smooth outer and a highly... (Review)
Review
Mitochondria, the powerhouses of the cell, are essential organelles in eukaryotic cells. With their complex inner architecture featuring a smooth outer and a highly convoluted inner membrane, they are challenging objects for microscopy. The diameter of mitochondria is generally close to the resolution limit of conventional light microscopy, rendering diffraction-unlimited super-resolution light microscopy (nanoscopy) for imaging submitochondrial protein distributions often mandatory. In this review, we discuss what can be expected when imaging mitochondria with conventional diffraction-limited and diffraction-unlimited microscopy. We provide an overview on recent studies using super-resolution microscopy to investigate mitochondria and discuss further developments and challenges in mitochondrial biology that might by addressed with these technologies in the future.
Topics: Animals; Cell Nucleus; DNA; Humans; Membrane Proteins; Microscopy; Mitochondria
PubMed: 24769752
DOI: 10.1016/j.cbpa.2014.03.019 -
Philosophical Transactions of the Royal... May 2017The hostile environment of the microscope stage poses numerous challenges to successful imaging of morphogenesis in live tissues. This review aims to highlight some of... (Review)
Review
The hostile environment of the microscope stage poses numerous challenges to successful imaging of morphogenesis in live tissues. This review aims to highlight some of the main practical considerations to take into account when embarking on a project to image cell behaviour in the context of cells' normal surroundings. Scrutiny of these activities is likely to be the most informative approach to understanding mechanical morphogenesis but is often confounded by the substantial technical difficulties involved in imaging samples over extended periods of time. Repeated observation of cells in live tissue requires that strategies be adopted to prioritize the stability of the sample, ensuring that it remains viable and develops normally while being held in a manner accessible to microscopic examination. Key considerations when creating reliable protocols for time-lapse imaging may be broken down into three main criteria; labelling, mounting and image acquisition. Choices and compromises made here, however, will directly influence image quality, and even small refinements can substantially improve what information may be extracted from images. Live imaging of tissue is difficult but paying close attention to the basics along with a little innovation is likely to be well rewarded.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.
Topics: Microscopy; Morphogenesis; Time-Lapse Imaging
PubMed: 28348248
DOI: 10.1098/rstb.2015.0511