Lattice Light-sheet Microscopy Research Articles

Cellular Bases of Olfactory Circuit Assembly Revealed by Systematic Time-Lapse Imaging

Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 28 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations, and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibit “exploring branches” consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral interactions in contralateral target selection, and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics.

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

Design and Validation of a Human Brain Endothelial Microvessel-on-a-Chip Open Microfluidic Model Enabling Advanced Optical Imaging.

We describe here the design and implementation of an in vitro microvascular open model system using human brain microvascular endothelial cells. The design has several advantages over other traditional closed microfluidic platforms: (1) it enables controlled unidirectional flow of media at physiological rates to support vascular function, (2) it allows for very small volumes which makes the device ideal for studies involving biotherapeutics, (3) it is amenable for multiple high resolution imaging modalities such as transmission electron microscopy (TEM), 3D live fluorescence imaging using traditional spinning disk confocal microscopy, and advanced lattice light sheet microscopy (LLSM). Importantly, we miniaturized the design, so it can fit within the physical constraints of LLSM, with the objective to study physiology in live cells at subcellular level. We validated barrier function of our brain microvessel-on-a-chip by measuring permeability of fluorescent dextran and a human monoclonal antibody. One potential application is to investigate mechanisms of transcytosis across the brain microvessel-like barrier of fluorescently-tagged biologics, viruses or nanoparticles.

Systematic and quantitative comparison of lattice and Gaussian light-sheets

The axial resolving power of a light-sheet microscope is determined by the thickness of the illumination beam and the numerical aperture of its detection optics. Bessel-beam based optical lattices have generated significant interest owing to their reportedly narrow beam waist and propagation-invariant characteristics. Yet, despite their significant use in lattice light-sheet microscopy and recent incorporation into commercialized systems, there are very few quantitative reports on their physical properties and how they compare to standard Gaussian illumination beams. Here, we measure the beam properties in the transmission of dithered square lattices, which is the most commonly used variant of lattice light-sheet microscopy, and Gaussian-based light-sheets. After a systematic analysis, we find that square lattices are very similar to Gaussian-based light-sheets in terms of thickness, confocal parameter, propagation length and overall imaging performance.

Digital Spindle: A New Way to Explore Mitotic Functions by Whole Cell Data Collection and a Computational Approach.

From cells to organisms, every living system is three-dimensional (3D), but the performance of fluorescence microscopy has been largely limited when attempting to obtain an overview of systems’ dynamic processes in three dimensions. Recently, advanced light-sheet illumination technologies, allowing drastic improvement in spatial discrimination, volumetric imaging times, and phototoxicity/photobleaching, have been making live imaging to collect precise and reliable 3D information increasingly feasible. In particular, lattice light-sheet microscopy (LLSM), using an ultrathin light-sheet, enables whole-cell 3D live imaging of cellular processes, including mitosis, at unprecedented spatiotemporal resolution for extended periods of time. This technology produces immense and complex data, including a significant amount of information, raising new challenges for big image data analysis and new possibilities for data utilization. Once the data are digitally archived in a computer, the data can be reused for various purposes by anyone at any time. Such an information science approach has the potential to revolutionize the use of bioimage data, and provides an alternative method for cell biology research in a data-driven manner. In this article, we introduce examples of analyzing digital mitotic spindles and discuss future perspectives in cell biology.

Isotropic three-dimensional imaging with lattice light-sheet difference microscopy

Through the interference of several beams with the same phase at the rear pupil, an optical lattice is generated and used as the excitation light field in lattice light-sheet microscopy (LLSM). Although, LLSM offers fast imaging ability through the dithered mode, it suffers from poor axial resolution. On the other hand, the structured illumination microscopy (SIM) mode in LLSM provides a high but anisotropic spatial resolution at the price of a lower imaging speed. In this Letter, we propose a new, to the best of our knowledge, type of optical lattice by defining the composed beam vectors with different phases. The lattice light-sheet difference microscopy (LLSDM) proposed in this work utilizes the new optical lattice along with the conventional one by simply changing the pattern loaded on the spatial light modulator. Through simulations with parameters obtained from the real acquisition procedures and the microscope properties, we examine the feasibility of LLSDM, and obtain nearly isotropic spatial resolution and great alleviation of the sidelobes. These characteristics enable LLSDM to broaden the accessible imaging content of light-sheet microscopes and further facilitate their applications in life sciences.

Hard-wired lattice light-sheet microscopy for imaging of expanded samples.

Light-sheet fluorescence microscopy (LSFM) helps investigate small structures in developing cells and tissue for three-dimensional localization microscopy and large-field brain imaging in neuroscience. Lattice light-sheet microscopy is a recent development with great potential to improve axial resolution and usable field sizes, thus improving imaging speed. In contrast to the commonly employed Gaussian beams for light-sheet generation in conventional LSFM, in lattice light-sheet microscopy an array of low diverging Bessel beams with a suppressed side lobe structure is used. We developed a facile elementary lattice light-sheet microscope using a micro-fabricated fixed ring mask for lattice light-sheet generation. In our setup, optical hardware elements enable a stable and simple illumination path without the need for spatial light modulators. This setup, in combination with long-working distance objectives and the possibility for simultaneous dual-color imaging, provides optimal conditions for imaging extended optically cleared tissue samples. We here present experimental data of fluorescently stained neurons and neurites from mouse hippocampus following tissue expansion and demonstrate the high homogeneous resolution throughout the entire imaged volume. Utilizing our purpose-built lattice light-sheet microscope, we reached a homogeneous excitation and an axial resolution of 1.2 µm over a field of view of (333 µm)2.

Lattice Light-Sheet Microscopy Multi-dimensional Analyses (LaMDA) of T-Cell Receptor Dynamics Predict T-Cell Signaling States.

Lattice light-sheet microscopy provides large amounts of high-dimensional, high-spatiotemporal resolution imaging data of cell surface receptors across the 3D surface of live cells, but user-friendly analysis pipelines are lacking. Here, we introduce lattice light-sheet microscopy multi-dimensional analyses (LaMDA), an end-to-end pipeline comprised of publicly available software packages that combines machine learning, dimensionality reduction, and diffusion maps to analyze surface receptor dynamics and classify cellular signaling states without the need for complex biochemical measurements or other prior information. We use LaMDA to analyze images of T-cell receptor (TCR) microclusters on the surface of live primary Tcells under resting and stimulated conditions. We observe global spatial and temporal changes of TCRs across the 3D cell surface, accurately differentiate stimulated cells from unstimulated cells, precisely predict attenuated T-cell signaling after CD4 and CD28 receptor blockades, and reliably discriminate between structurally similar TCR ligands. All instructions needed to implement LaMDA are included in this paper.

Lattice light-sheet microscopy Multi-Dimensional Analyses (LaMDA) of T-cell receptor dynamics predict T-cell signaling states

Abstract Cell surface receptors dynamically change in response to cell signaling, but a direct linkage between receptor dynamics and cell state has not been established. Here we report the development of lattice light-sheet microscopy multi-dimensional analyses (LaMDA), a pipeline that combines high spatiotemporal-resolution four-dimensional lattice light-sheet microscopy, machine learning, and diffusion maps to analyze T-cell receptor (TCR) dynamics and predict T-cell signaling states without the need for complex biochemical measurements. LaMDA images thousands of TCR microclusters on the surface of live primary cells to collect high-dimensional dynamic data for machine learning, which extracts key dynamic features to build predictive diffusion maps. LaMDA spatiotemporally reveals global changes of TCRs across the 3D cell surface, accurately differentiates stimulated cells from unstimulated cells, precisely predicts attenuated T-cell signaling after CD4 and CD28 receptor blockades, and reliably discriminates between structurally similar TCR ligands. We anticipate broad usage of this approach for other receptors and cells, as well as for guiding the design and development of future immunotherapies for cancer, infection, and autoimmunity.

Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy.

Neutrophil and macrophage (Mϕ) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.

Spatiotemporal Organization of Metabolic Networks in Living Cells

Cellular metabolic networks are dynamic. Organization between membrane‐bound organelles and cytoplasmic metabolic pathways are crucial to cell survival and function. This is the case for the glucose metabolism, where cytoplasmic enzymes have been observed to form spatially organized enzyme assemblies. We have quantified the sub‐cellular organization of these glucose metabolic assemblies with the mitochondria and studied a mitochondrial‐associated population of assemblies that are functionally linked to the mitochondrial metabolism. This research was completed with a home‐built lattice light sheet microscope and other biophysical and biochemical methods, which provided crucial spatiotemporal insight into the orchestration of metabolic functions that provides a starting point to map 4D functional metabolic networks.Support or Funding InformationNIH Research Project Grant R01 GM134086, NIH Research Project Grant R01 GM125981, Research Grant R03 CA219609, Student support provided by NIH Chemistry Biology Interface Program (T32 GM066706), Student support provided by an NIGMS Initiative for Maximizing Student Development Grant (2 R25‐GM55036)

Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM

The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.

Lattice Light-Sheet Microscopy Multi-Dimensional Analyses (LaMDA) of T-Cell Receptor Dynamics Predict T-Cell Signaling States

Cell surface receptors dynamically change in response to cell signaling, but a direct linkage between receptor dynamics and cell state has not been established. Here we report the development of lattice light-sheet microscopy multi-dimensional analyses (LaMDA), a pipeline that combines high spatiotemporal-resolution four-dimensional lattice light-sheet microscopy, machine learning, and diffusion maps to analyze T-cell receptor (TCR) dynamics and predict T-cell signaling states without the need for complex biochemical measurements. LaMDA images thousands of TCR microclusters on the surface of live primary cells to collect high-dimensional dynamic data for machine learning, which extracts key dynamic features to build predictive diffusion maps. LaMDA spatiotemporally reveals global changes of TCRs across the 3D cell surface, accurately differentiates stimulated cells from unstimulated cells, precisely predicts attenuated T-cell signaling after CD4 and CD28 receptor blockades, and reliably discriminates between structurally similar TCR ligands. We anticipate broad usage of this approach for other receptors and cells, as well as for guiding the design and development of future immunotherapies for cancer, infection, and autoimmunity.

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

Imaging intercellular interaction and extracellular vesicle exchange in a co-culture model of chronic lymphocytic leukemia and stromal cells by lattice light-sheet fluorescence microscopy.

Recent advances in live cell imaging allow investigating processes that take place over the entire cell volume with unprecedented time and spatial resolution. Here we describe a protocol to study intercellular communication, including extracellular vesicle exchange, between cancer cells and their microenvironment, using lattice light sheet fluorescence microscopy. While the described protocol is intended to study the interactions between chronic lymphocytic leukemia cells and bone marrow stromal cells, many components of it can be applied to study other cancers of hematopoietic or solid tumor origin, as well as to characterize other modalities of intercellular communication.

A simple, web-based repository for the management, access and analysis of micrographic images.

Microscopy is advancing at a rapid pace, enabling high-speed, high-resolution analyses to be conducted in a wide range of cellular contexts. For example, the capacity to quickly capture high-resolution images from multiple optical sections over multiple channels with confocal microscopy has allowed researchers to gain deeper understanding of tissue morphology via techniques such as three-dimensional rendering, as have more recent advances such as lattice light sheet microscopy and superresolution structured illumination microscopy. With this, though, comes the challenge of storing, curating, analysing and sharing data. While there are ways in which this has been attempted previously, few approaches have provided a central repository in which all of these different aspects of microscopy can be seamlessly integrated. Here, we describe a web-based storage and analysis platform called Microndata, that enables relatively straightforward storage, annotation, tracking, analysis and multi-user access to micrographs. This easy to use tool will simplify and harmonise laboratory work flows, and, importantly, will provide a central storage repository that is readily accessed, even after the researcher responsible for capturing the images has left the laboratory. Microndata is open-source software, available at http://www.microndata.net/.

The Applications of Lattice Light-sheet Microscopy for Functional Volumetric Imaging of Hippocampal Neurons in a Three-Dimensional Culture System.

The characterization of individual cells in three-dimensions (3D) with very high spatiotemporal resolution is crucial for the development of organs-on-chips, in which 3D cell cultures are integrated with microfluidic systems. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. Fully differentiated and mature hippocampal neurons were observed in our system. With LLSM, we were able to monitor the behavior of individual cells in a 3D cell culture, which was very difficult under a conventional microscope due to strong light scattering from thick samples. We demonstrated that our system could study the membrane voltage and intracellular calcium dynamics at subcellular resolution in 3D under both chemical and electrical stimulation. From the volumetric images, it was found that the voltage indicators mainly resided in the cytosol instead of the membrane, which cannot be distinguished using conventional microscopy. Neuronal volumetric images were sheet scanned along the axial direction and recorded at a laser exposure of 6 ms, which covered an area up to 4800 μm2, with an image pixel size of 0.102 μm. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions.

Software for lattice light-sheet imaging of FRET biosensors, illustrated with a new Rap1 biosensor.

Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions.

1012 - MITOCHONDRIAL DYNAMICS, DAMAGE, AND INFLAMMATORY SIGNALLING

The mitochondrial (or intrinsic) apoptosis pathway plays an essential role in the development, survival and clearance of many hematopoietic lineages. It can be activated by pathophysiological insults such as anti-cancer therapy and infection, and its suppression is regarded as one of the hallmarks of cancer. The first drug that specifically targets the pathway, Venetoclax, was approved for use in chronic lymphocytic leukemia in 2016. In recent years, we and others have uncovered a link between mitochondrial apoptosis and innate immune signalling, demonstrating that the apoptotic caspase cascade (which functions downstream of mitochondrial damage during cell death) is required to prevent mitochondrial (mt) DNA from activating the cGAS/STING pathway. In the absence of caspases, dying cells behave as if virally infected, producing type I interferon as a result of mtDNA-induced STING signalling. Using a combination of lattice light sheet, 3D-SIM and cryo-electron microscopy, we have established that mtDNA release occurs via the process of mitochondrial herniation. During apoptosis, the pro-death proteins BAX and BAK permeabilise the outer mitochondria membrane and form macropores through which the inner mitochondrial membrane balloons out. This herniated inner mitochondrial membrane acts as a conduit for mtDNA to reach the cytoplasm. Current efforts are directed at understanding the events that occur post-herniation, and whether herniation represents a more general mechanism of mtDNA escape.

Abstract 488: Direct real-time visualization and quantification of T cell receptor dynamics

Abstract The mechanism by which a T cell utilizes its T-cell receptors (TCRs) to recognize foreign peptide major histocompatibility complexes (pMHCs) has not yet been fully deciphered. Much contention still exists surrounding TCR nanostructures before, during, and after synapse formation. Accurate measurements of single TCR location, diffusion/trafficking, clustering, and signaling at the live T-cell membrane have been hindered by the microscopy techniques available. Recently developed Lattice Light-Sheet Microscopy uses a structured light sheet to excite fluorescence in successive planes of a living T cell such that it can record 4D (x, y, z directions and time) images with exceptionally high temporal resolution (10ms/frame, ~1s/volume), finally allowing for precise single molecule tracking. Here we show that TCRs pre-exist in small microclusters prior to synapse formation in both naïve and stimulated resting CD4+ T cells. These clusters are very fast-moving, explaining the previous difficulties of tracking TCR motion and revealing a fast responding mechanism for antigen recognition. Upon encountering antigens, these smaller TCR nanoclusters traffic globally and quickly to the cell interface, aggregate into larger microclusters, and form the immunological synapse. Finally, we compare and contrast the receptor dynamics of CAR-T cells with those of naturally occurring T cell receptors. These findings precisely quantify TCR and CAR dynamics three-dimensionally, suggesting new mechanistic details of the speed and accuracy of TCR signaling and providing new information upon which to base future immunotherapies. Citation Format: Jillian N. Rosenberg, Guoshuai Cao, Fernanda Borja-Prieto, Yanran He, Hans Schreiber, Jun Huang. Direct real-time visualization and quantification of T cell receptor dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 488.