Imaging Of Cellular Structures Research Articles

Space-feature measures on meshes for mapping spatial transcriptomics

Advances in the development of largely automated microscopy methods such as MERFISH for imaging cellular structures in mouse brains are providing spatial detection of micron resolution gene expression. While there has been tremendous progress made in the field of Computational Anatomy (CA) to perform diffeomorphic mapping technologies at the tissue scales for advanced neuroinformatic studies in common coordinates, integration of molecular- and cellular-scale populations through statistical averaging via common coordinates remains yet unattained. This paper describes the first set of algorithms for calculating geodesics in the space of diffeomorphisms, what we term space-feature-measure LDDMM, extending the family of large deformation diffeomorphic metric mapping (LDDMM) algorithms to accommodate a space-feature action on marked particles which extends consistently to the tissue scales. It leads to the derivation of a cross-modality alignment algorithm of transcriptomic data to common coordinate systems attached to standard atlases.We represent the brain data as geometric measures, termed as space-feature measures supported by a large number of unstructured points, each point representing a small volume in space and carrying a list of densities of features elements of a high-dimensional feature space. The shape of space-feature measure brain spaces is measured by transforming them by diffeomorphisms. The metric between these measures is obtained after embedding these objects in a linear space equipped with the norm, yielding a so-called “chordal metric”.

Sample Preparation for Multicolor STED Microscopy.

Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.

Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

TdLanYFP, a Yellow, Bright, Photostable, and pH-Insensitive Fluorescent Protein for Live-Cell Imaging and Förster Resonance Energy Transfer-Based Sensing Strategies.

Yellow fluorescent proteins (YFPs) are widely used as optical reporters in Förster resonance energy transfer (FRET)-based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pH values. Here, we characterize the yellow fluorescent protein tdLanYFP, derived from the tetrameric protein from the cephalochordate Branchiostoma lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133,000 mol-1·L·cm-1, it is, to our knowledge, the brightest dimeric fluorescent protein available. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and in live cells. As a consequence, tdLanYFP allows imaging of cellular structures with subdiffraction resolution using STED nanoscopy and is compatible with the use of spectromicroscopies in single-molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pH values. Finally, we show that tdLanYFP is a valuable FRET partner either as a donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFP a very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging including FRET experiments at acidic pH.

Microscopic optical coherence tomography (mOCT) at 600 kHz for 4D volumetric imaging and dynamic contrast.

Volumetric imaging of dynamic processes with microscopic resolution holds a huge potential in biomedical research and clinical diagnosis. Using supercontinuum light sources and high numerical aperture (NA) objectives, optical coherence tomography (OCT) achieves microscopic resolution and is well suited for imaging cellular and subcellular structures of biological tissues. Currently, the imaging speed of microscopic OCT (mOCT) is limited by the line-scan rate of the spectrometer camera and ranges from 30 to 250 kHz. This is not fast enough for volumetric imaging of dynamic processes in vivo and limits endoscopic application. Using a novel CMOS camera, we demonstrate fast 3-dimensional OCT imaging with 600,000 A-scans/s at 1.8µm axial and 1.1 µm lateral resolution. The improved speed is used for imaging of ciliary motion and particle transport in ex vivo mouse trachea. Furthermore, we demonstrate dynamic contrast OCT by evaluating the recorded volumes rather than en face planes or B-scans. High-speed volumetric mOCT will enable the correction of global tissue motion and is a prerequisite for applying dynamic contrast mOCT in vivo. With further increase in imaging speed and integration in flexible endoscopes, volumetric mOCT may be used to complement or partly replace biopsies.

Deep learning enables fast and dense single-molecule localization with high accuracy.

Single-molecule localization microscopy (SMLM) has had remarkable success in imaging cellular structures with nanometer resolution, but standard analysis algorithms necessitate the activation of only single isolated emitters which limits imaging speed and labeling density. Here, we overcome this major limitation using deep learning. We developed DECODE, a computational tool that can localize single emitters at high density in 3D with highest accuracy for a large range of imaging modalities and conditions. In a public software benchmark competition, it outperformed all other fitters on 12 out of 12 data-sets when comparing both detection accuracy and localization error, often by a substantial margin. DECODE allowed us to take fast dynamic live-cell SMLM data with reduced light exposure and to image microtubules at ultra-high labeling density. Packaged for simple installation and use, DECODE will enable many labs to reduce imaging times and increase localization density in SMLM.

High Resolution Optical Coherence Tomography

Imaging cellular and extracellular structures and processes in situ and in vivo is highly desired for the understanding and diagnosis of human diseases. High resolution optical coherence tomography (HR-OCT) is particularly suitable for this task because it can provide real-time, 3-D images of a large tissue volume at subcellular resolution. Over the past two decades, tremendous technical advances have been made to tackle the fundamental and practical limitations of HR-OCT for applications in various clinical fields. Meanwhile, novel scientific and clinical applications of HR-OCT have also been proposed and validated. This review aims to provide an update on the progress of technology development, with a focus on axial focus extension, aberration correction, and fiber-optic probe development. This review also aims to summarize the newly established capabilities of HR-OCT in fundamental and clinical research, in an effort to promote multidisciplinary research using this powerful imaging tool.

Real-time brain-wide multi-planar microscopy for simultaneous cortex and hippocampus imaging at the cellular resolution in mice.

Interactions between the cerebral cortex and the deep cerebellar nuclei play important roles in cognitive processes. However, conventional microscopes fail to dynamically record cellular structures in distinct brain regions and at different depths, which requires high resolution, large field of view (FOV), and depth of field (DOF). Here we propose a single-photon excited fluorescence microscopy technique that performs simultaneous cortex and hippocampus imaging, enabled by a customized microscope and a chronic optical window. After we implant a glass microwindow above the hippocampus, the surface of the hippocampus is shifted to the superficial plane. We demonstrate that the proposed technique is able to image cellular structures and blood vessel dynamics in the cortex and the hippocampus in in vivo experiments, and is compatible with various mesoscopic systems.

Multi‐Color, Bleaching‐Resistant Super‐Resolution Optical Fluctuation Imaging with Oligonucleotide‐Based Exchangeable Fluorophores

AbstractSuper‐resolution optical fluctuation imaging (SOFI) is a super‐resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background‐free and show confocality and enhanced spatial resolution (super‐resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore‐labeled short DNA oligonucleotides, we labeled cellular structures with target‐specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two‐color imaging of cellular structures with SOFI.

Multi-Color, Bleaching-Resistant Super-Resolution Optical Fluctuation Imaging with Oligonucleotide-Based Exchangeable Fluorophores.

Super‐resolution optical fluctuation imaging (SOFI) is a super‐resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background‐free and show confocality and enhanced spatial resolution (super‐resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore‐labeled short DNA oligonucleotides, we labeled cellular structures with target‐specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two‐color imaging of cellular structures with SOFI.

Spatial filter and its application in three-dimensional single molecule localization microscopy

Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 μm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.

Quantitative analysis of super-resolved structures using ASAP.

Super resolution microscopy allows imaging of cellular structures with high throughput and detail. However, tools for the efficient and quantitative analysis of images generated are lacking. Here, we developed ASAP (Automated Structures Analysis Program) to enable rapid and automated detection, classification and quantification of super-resolved structures. We validate ASAP on ground truth data and demonstrate its broad applicability by analyzing images of nucleoporins, TORC1 complexes, endocytic vesicles and Bax pores.

Automating Localization Microscopy

Single-molecule localization microscopy (SMLM) has become a popular tool in cell biology as it allows imaging cellular structures with a precision of tens of nanometers. However, acquiring superresolved images with SMLM is inherently slow, requiring up to hours for a single image and careful user intervention in between experiments. This effectively limits the throughput of SMLM to a handful of images of the structure of interest per study. We present tools to overcome this limitation by automating a wide-field microscope to perform SMLM experiments. This microscope is capable of SMLM imaging over the course of days thanks to advanced electronics and an open-source plugin for Micro-manager. In the meantime, SMLM experiments are analysed during acquisition using a freely accessible software in Matlab. Finally, the microscope features a flat illumination system, providing homogeneous statistics across large fields of view. Altogether these developments in optics, hardware and software can be transferred to any single-molecule wide-field microscope, thus enabling automated localization microscopy.

Mapping the Mechanome-A Protocol for Simultaneous Live Imaging and Quantitative Analysis of Cell Mechanoadaptation and Ingression.

Mechanomics, the mechanics equivalent of genomics, is a burgeoning field studying mechanical modulation of stem cell behavior and lineage commitment. Analogous to mechanical testing of a living material as it adapts and evolves, mapping of the mechanome necessitates the development of new protocols to assess changes in structure and function in live stem cells as they adapt and differentiate. Previous techniques have relied on imaging of cellular structures in fixed cells and/or live cell imaging of single cells with separate studies of changes in mechanical and biological properties. Here we present two complementary protocols to study mechanobiology and mechanoadaptation of live stem cells in adherent and motile contexts. First, we developed and tested live imaging protocols for simultaneous visualization and tracking of actin and tubulin mechanoadaptation as well as shape and volume of cells and their nuclei in adherent model embryonic murine mesenchymal stem cells (C3H/10T1/2) and in a neuroblastoma cell line. Then we applied the protocol to enable quantitative study of primary human mesenchymal stem cells in a motile state, e.g., ingression in a three-dimensional, in vitro cell culture model. Together, these protocols enable study of emergent structural mechanoadaptation of the cell's own cytoskeletal machinery while tracking lineage commitment using phenotypic (quantitative morphology measures) and genotypic (e.g., reverse transcription Polymerase Chain Reaction, rtPCR) methods. These tools are expected to facilitate the mapping of the mechanome and incipient mechanistic understanding of stem cell mechanobiology, from the cellular to the tissue and organ length scales.

Visualizing and discovering cellular structures with super-resolution microscopy.

Super-resolution microscopy has overcome a long-held resolution barrier-the diffraction limit-in light microscopy and enabled visualization of previously invisible molecular details in biological systems. Since their conception, super-resolution imaging methods have continually evolved and can now be used to image cellular structures in three dimensions, multiple colors, and living systems with nanometer-scale resolution. These methods have been applied to answer questions involving the organization, interaction, stoichiometry, and dynamics of individual molecular building blocks and their integration into functional machineries in cells and tissues. In this Review, we provide an overview of super-resolution methods, their state-of-the-art capabilities, and their constantly expanding applications to biology, with a focus on the latter. We will also describe the current technical challenges and future advances anticipated in super-resolution imaging.

Real-time 3D single-molecule localization using experimental point spread functions.

We present a real-time fitter for 3D single-molecule localization microscopy using experimental point spread functions (PSFs) that achieves optimal 3D resolution on any microscope and is compatible with any PSF engineering approach. This allowed us to image cellular structures with a 3D resolution unprecedented for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D superresolution microscopy broadly accessible, even on standard microscopes without dedicated 3D optics.

Smartphone confocal microscopy for imaging cellular structures in human skin in vivo.

We report development of a low-cost smartphone confocal microscope and its first demonstration of in vivo human skin imaging. The smartphone confocal microscope uses a slit aperture and diffraction grating to conduct two-dimensional confocal imaging without using any beam scanning devices. Lateral and axial resolutions of the smartphone confocal microscope were measured as 2 and 5 µm, respectively. In vivo confocal images of human skin revealed characteristic cellular structures, including spinous and basal keratinocytes and papillary dermis. Results suggest that the smartphone confocal microscope has a potential to examine cellular details in vivo and may help disease diagnosis in resource-poor settings, where conducting standard histopathologic analysis is challenging.

Genetic Code Expansion- and Click Chemistry-Based Site-Specific Protein Labeling for Intracellular DNA-PAINT Imaging.

Super-resolution microscopy allows imaging of cellular structures at nanometer resolution. This comes with a demand for small labels which can be attached directly to the structures of interest. In the context of protein labeling, one way to achieve this is by using genetic code expansion (GCE) and click chemistry. With GCE, small labeling handles in the form of noncanonical amino acids (ncAAs) are site-specifically introduced into a target protein. In a subsequent step, these amino acids can be directly labeled with small organic dyes by click chemistry reactions. Click chemistry labeling can also be combined with other methods, such as DNA-PAINT in which a "clickable" oligonucleotide is first attached to the ncAA-bearing target protein and then labeled with complementary fluorescent oligonucleotides. This protocol will cover both aspects: I describe (1) how to encode ncAAs and perform intracellular click chemistry-based labeling with an improved GCE system for eukaryotic cells and (2) how to combine click chemistry-based labeling with DNA-PAINT super-resolution imaging. As an example, I show click-PAINT imaging of vimentin and low-abundance nuclear protein, nucleoporin 153.

Long time-lapse nanoscopy with spontaneously blinking membrane probes.

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.

Imaging Cellular Structure and Dynamics from Molecules to Organisms

Journal Article Imaging Cellular Structure and Dynamics from Molecules to Organisms Get access Eric Betzig Eric Betzig Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 23, Issue S1, 1 July 2017, Pages 2–3, https://doi.org/10.1017/S1431927617000691 Published: 04 August 2017