Lattice Light-sheet Microscopy Research Articles

Four-dimensional quantitative analysis of cell plate development in Arabidopsis using lattice light sheet microscopy identifies robust transition points between growth phases.

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

Co-clustering of EphB6 and ephrinB1 in trans restrains cancer cell invasion

EphB6 is an understudied ephrin receptor tyrosine pseudokinase that is downregulated in multiple types of metastatic cancers. Unlike its kinase-active counterparts which autophosphorylate and transmit signals upon intercellular interaction, little is known about how EphB6 functions in the absence of intrinsic kinase activity. Here, we unveil a molecular mechanism of cell-cell interaction driven by EphB6. We identify ephrinB1 as a cognate ligand of EphB6 and show that in trans interaction of EphB6 with ephrinB1 on neighboring cells leads to the formation of large co-clusters at the plasma membrane. These co-clusters exhibit a decreased propensity towards endocytosis, suggesting a unique characteristic for this type of cell-cell interaction. Using lattice light-sheet microscopy, 3D structured illumination microscopy and cryo-electron tomography techniques, we show that co-clustering of EphB6 and ephrinB1 promotes the formation of double-membrane tubular structures between cells. Importantly, we also demonstrate that these intercellular structures stabilize cell–cell adhesion, leading to a reduction in the invasive behavior of cancer cells. Our findings rationalize a role for EphB6 pseudokinase as a tumor suppressor when interacting with its ligands in trans.

Lattice light-sheet DNA-PAINT microscopy with dual-labeled self-quenched imager probes

Lattice light-sheet DNA-PAINT microscopy with dual-labeled self-quenched imager probes

Chemokine receptor distribution on the surface of repolarizing T cells

T cells migrate constitutively with a polarized morphology, underpinned by signaling compartmentalization and discrete cytoskeletal organizations, giving rise to a dynamic and expansive leading edge, distinct from the stable and constricted uropod at the rear. In vivo, the motion and function of T cells at various stages of differentiation is highly directed by chemokine gradients. When cognate ligands bind chemokine receptors on their surface, T cells respond by reorientating their polarity axis and migrating toward the source of the chemokine signal. Despite the significance of such chemotactic repolarization to the accurate navigation and function of T cells, the precise signaling mechanisms that underlie it remain elusive. Notably, it remained unclear whether the distribution of chemokine receptors on the T cell surface is altered during repolarization. Here, we developed parallel cell-secreted and microfluidics-based chemokine gradient delivery methods and employed both fixed imaging and live lattice light-sheet microscopy to investigate the dynamics of chemokine receptor CCR5 on the surface of primary murine CD8+ T cells. Our findings show that, during constitutive migration, chemokine receptor distribution is largely isotropic on the T cell surface. However, upon exposure to a CCL3 gradient, surface chemokine receptor distributions exhibit a transient bias toward the uropod. The chemokine receptors then progressively redistribute from the uropod to cover the T cell surface uniformly. This study sheds new light on the dynamics of surface chemokine receptor distribution during T cell repolarization, advancing our understanding of the signaling of immune cells in the complex chemokine landscapes they navigate.

Smart lattice light-sheet microscopy for imaging rare and complex cellular events.

Light-sheet microscopes enable rapid high-resolution imaging of biological specimens; however, biological processes span spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality. Here, to overcome this limitation, we present smartLLSM, a microscope that incorporates artificial intelligence-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light-sheet microscopy (LLSM). We apply this approach to two unique processes: cell division and immune synapse formation. In each context, smartLLSM provides population-level statistics across thousands of cells and autonomously captures multicolor three-dimensional datasets or four-dimensional time-lapse movies of rare events at rates that dramatically exceed human capabilities. From this, we quantify the effects of Taxol dose on spindle structure and kinetochore dynamics in dividing cells and of antigen strength on cytotoxic T lymphocyte engagement and lytic granule polarization at the immune synapse. Overall, smartLLSM efficiently detects rare events within heterogeneous cell populations and records these processes with high spatiotemporal four-dimensional imaging over statistically significant replicates.

Lattice Light-Sheet Motor-PAINT: A Method to Map the Orientations of Microtubules in Complex Three-Dimensional Arrays.

Microtubules play an essential role in many cellular functions, in part by serving as tracks for intracellular transport by kinesin and dynein. The ability of microtubules to fulfill this role fundamentally depends on the fact that they are polar, with motors moving along them toward either their plus or minus end. Given that the microtubule cytoskeleton adopts a variety of specialized architectures in different cell types, it is important to map precisely how microtubules are oriented and organized in these cells. To this end, motor-PAINT has been developed, but in its current implementation, it relies on total internal reflection fluorescence (TIRF) microscopy and is thus restricted to imaging microtubules in a thin section of the cell immediately adjacent to the coverslip. Here, we report a variant of motor-PAINT that uses lattice light-sheet microscopy to overcome this, allowing for the mapping of microtubule organization and orientation in three-dimensional samples. We describe the necessary steps to purify, label, use, and image kinesin motors for motor-PAINT and outline the analysis pipeline used to visualize the resulting data. The method described here can be used in the future to study the microtubule cytoskeleton in (thick) polarized cells such as intestinal epithelial cells.

Live Cell Light Sheet Imaging with Low- and High-Spatial-Coherence Detection Approaches Reveals Spatiotemporal Aspects of Neuronal Signaling

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca2+ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca2+ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca2+ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches.

Lattice Light-Sheet Microscopy as a Key Method for the Analysis of the Mislocalization of TDP-43 in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) is a neurodegenerative disorder that is characterized by the progressive loss of motor neurons. Consequently, patients afflicted with this disease eventually lose the motor function associated with moving, speaking, eating, and even breathing. One neuroscience team identified a common mechanism in nearly all sporadic ALS patients in which transactive response DNA binding protein 43 kDa (TDP-43) was mislocalized from the nucleus to the cytoplasm. This suggests that the mislocalization of TDP-43 into the cytoplasm is central for the onset of ALS in patients, but the underlying factors that result in this process are not fully understood as there are no current laboratory methods to study the progression of TDP-43 mislocalization. However, the development and refinement of lattice light-sheet microscopy (LLSM) as a laboratory technique has significant potential in helping scientists elucidate the unknown processes behind ALS. This review discusses the implications of lattice light-sheet microscopy and its optimization methods as a solution to the issue and identifies LLSM as a key method that could be significant in the field of ALS research.

MitoTNT: Mitochondrial Temporal Network Tracking for 4D live-cell fluorescence microscopy data.

Mitochondria form a network in the cell that rapidly changes through fission, fusion, and motility. Dysregulation of this four-dimensional (4D: x,y,z,time) network is implicated in numerous diseases ranging from cancer to neurodegeneration. While lattice light-sheet microscopy has recently made it possible to image mitochondria in 4D, quantitative analysis methods for the resulting datasets have been lacking. Here we present MitoTNT, the first-in-class software for Mitochondrial Temporal Network Tracking in 4D live-cell fluorescence microscopy data. MitoTNT uses spatial proximity and network topology to compute an optimal tracking assignment. To validate the accuracy of tracking, we created a reaction-diffusion simulation to model mitochondrial network motion and remodeling events. We found that our tracking is >90% accurate for ground-truth simulations and agrees well with published motility results for experimental data. We used MitoTNT to quantify 4D mitochondrial networks from human induced pluripotent stem cells. First, we characterized sub-fragment motility and analyzed network branch motion patterns. We revealed that the skeleton node motion is correlated along branch nodes and is uncorrelated in time. Second, we identified fission and fusion events with high spatiotemporal resolution. We found that mitochondrial skeleton nodes near the fission/fusion sites move nearly twice as fast as random skeleton nodes and that microtubules play a role in mediating selective fission/fusion. Finally, we developed graph-based transport simulations that model how material would distribute on experimentally measured mitochondrial temporal networks. We showed that pharmacological perturbations increase network reachability but decrease network resilience through a combination of altered mitochondrial fission/fusion dynamics and motility. MitoTNT's easy-to-use tracking module, interactive 4D visualization capability, and powerful post-tracking analyses aim at making temporal network tracking accessible to the wider mitochondria research community.

Characterization, comparison, and optimization of lattice light sheets.

Lattice light sheet microscopy excels at the noninvasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here, we undertake a theoretical and experimental analysis of various forms of light sheet microscopy, which demonstrates and explains why lattice light sheets provide substantial improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. We also introduce a new type of "harmonic balanced" lattice light sheet that improves performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.

Toxicity and assimilation of cellulosic copper nanoparticles require α-arrestins in S. cerevisiae.

The increased use of antimicrobial compounds such as copper into nanoparticles changes how living cells interact with these novel materials. The increased use of antimicrobial nanomaterials combats infectious disease and food spoilage. Fungal infections are particularly difficult to treat because of the few druggable targets, and Saccharomyces cerevisiae provides an insightful model organism to test these new materials. However, because of the novel characteristics of these materials, it is unclear how these materials interact with living cells and if resistance to copper-based nanomaterials could occur. Copper nanoparticles built on carboxymethylcellulose microfibril strands with copper (CMC-Cu) are a promising nanomaterial when imported into yeast cells and induce cell death. The α-arrestins are cargo adaptors that select which molecules are imported into eukaryotic cells. We screened α-arrestins mutants and identified Aly2, Rim8, and Rog3 α-arrestins, which are necessary for the internalization of CMC-Cu nanoparticles. Internal reactive oxygen species in these mutants were lower and corresponded to the increased viability in the presence of CMC-Cu. Using lattice light-sheet microscopy on live cells, we determined that CMC-Cu were imported into yeast within 30 min of exposure. Initially, the cytoplasmic pH decreased but returned to basal level 90 min later. However, there was heterogeneity in response to CMC-Cu exposure, which could be due to the heterogeneity of the particles or differences in the metabolic states within the population. When yeast were exposed to sublethal concentrations of CMC-Cu no resistance occurred. Internalization of CMC-Cu increases the potency of these antimicrobial nanomaterials and is likely key to preventing fungi from evolving resistance.

Novel insights about ribosomal DNA double strand break repair revealed by super-resolution imaging techniques and very fast CRISPR.

Ribosomal DNA (rDNA) is repetitive gene sequence that produces the RNA components for the translation machinery in eukaryotic cells - ribosomes. The maintenance of rDNA genome stability against double-strand breaks (DSBs) is critical regarding cellular aging related diseases and tumor development due to its repetitive nature and distribution over multiple chromosomes. Previous studies have shown that DSB within rDNA region will induce rDNA transcriptional repression and nucleolar segregation. However, it remains poorly understood regarding the kinetics of associated transcriptional regulation, the architecture of the nucleolar cap, and the link between them, likely due to the resolution limit of conventional imaging tools and the absence of proper system that can rapidly induce DSBs specific to rDNA region with accurately controlled timing. To resolve these limitations and provide deeper insights on how rDNA DSB repair response is regulated, we use self-built super-resolution fluorescence imaging systems, e.g., stimulated emission depletion microscopy, which allow to visualize nucleolar structures beyond the diffraction limit, and lattice light sheet microscopy, which enables to monitor fast kinetics of the repair process with minimal photo-induced disturbance, combined with a recently developed rapidly-inducible photo-caged CRISPR-Cas9 system (very fast CRISPR, vfCRISPR) in our lab, to study the spatiotemporal coordination of the DSB repair factor assembly upon rDNA DSB damage, and dissect its important role in regulating pathway choice and outcome fidelity.

Mitochondrial temporal network tracking for 4D live-cell fluorescence microscopy data.

Mitochondria form a network in the cell that rapidly changes through fission, fusion, and motility. This four-dimensional (4D, x, y, z, time) temporal network has only recently been made accessible through advanced imaging methods such as lattice light-sheet microscopy. Quantitative analysis tools for the resulting datasets however have been lacking. Here we present MitoTNT, the first-in-class software for Mitochondrial Temporal Network Tracking in 4D live-cell fluorescence microscopy data. MitoTNT uses spatial proximity and network topology to compute an optimal tracking. Tracking is >90% accurate in dynamic spatial mitochondria simulations and are in agreement with published motility results in vitro. Using MitoTNT, we reveal correlated mitochondrial movement patterns, local fission and fusion fingerprints, asymmetric fission and fusion dynamics, cross-network transport patterns, and network-level responses to pharmacological manipulations. MitoTNT is implemented in Python with a JupyterLab interface. The extendable and user-friendly design aims at making temporal network tracking accessible to the wider mitochondria community.

Lattice light-sheet microscopy and evaluation of dendritic transport in cultured hippocampal tissue reveal high variability in mobility of the KIF1A motor domain and entry into dendritic spines

The unique morphology of neurons consists of a long axon and a highly variable arbour of dendritic processes, which assort neuronal cells into the main classes. The dendritic tree serves as the main domain for receiving synaptic input. Therefore, to maintain the structure and to be able to plastically change according to the incoming stimuli, molecules and organelles need to be readily available. This is achieved mainly via bi-directional transport of cargo along the microtubule lattices. Analysis of dendritic transport is lagging behind the investigation of axonal transport. Moreover, addressing transport mechanisms in tissue environment is very challenging and, therefore, rare. We employed high-speed volumetric lattice light-sheet microscopy and single particle tracking of truncated KIF1A motor protein lacking the cargo-binding domain. We focused our analysis on dendritic processes of CA1 pyramidal neurons in cultured hippocampal tissue. Analysis of individual trajectories revealed detailed information about stalling and high variability in movement and speed, and biased directionality of KIF1A. Furthermore, we could also observe KIF1A shortly entering into dendritic spines. We provide a workflow to analyse variations in the speed and direction of motor protein movement in dendrites that are either intrinsic properties of the motor domain or depend on the structure and modification of the microtubule trails.

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters.

B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces, and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have used lattice light-sheet microscopy in combination with tailored custom-built 4D image analysis to study the cell-surface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM-BCR. Ramos B-cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membrane-localized IgM-BCR was found in clusters, which were mainly associated with the ridges and the microvilli. The dynamic ridge-network organization and the IgM-BCR cluster mobility were linked, and both were controlled by Arp2/3 complex activity. Our results suggest that dynamic topographical features of the cell surface govern the localization and transport of IgM-BCR clusters to facilitate antigen screening by B cells.

Large-scale expanded sample imaging with tiling lattice lightsheet microscopy.

The ability to observe biological nanostructures forms a vital step in understanding their functions. Thanks to the invention of expansion microscopy (ExM) technology, super-resolution features of biological samples can now be easily visualized with conventional light microscopies. However, when the sample is physically expanded, the demand for deep and precise 3D imaging increases. Lattice lightsheet microscopy (LLSM), which utilizes a planar illumination that is confined within the imaging depth of high numerical aperture (NA=1.1) detection objective, fulfils such requirements. In addition, optical tiling could be implemented to increase the field of view (FoV) by moving the lightsheet without mechanically moving the samples or the objective for high-precision 3D imaging. In this review article, we will explain the principle of the tiling lattice lightsheet microscopy (tLLSM), which combines optical tiling and lattice lightsheet, and discuss the applications of tLLSM in ExM.

Ras-Related Protein Rab5a Regulates Complement C5a Receptor Trafficking, Chemotaxis, and Chemokine Secretion in Human Macrophages

Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits β-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with β-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or β-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-β-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.

Active image optimization for lattice light sheet microscopy in thick samples.

Lattice light-sheet microscopy (LLSM) is a very efficient technique for high resolution 3D imaging of dynamic phenomena in living biological samples. However, LLSM imaging remains limited in depth due to optical aberrations caused by sample-based refractive index mismatch. Here, we propose a simple and low-cost active image optimization (AIO) method to recover high resolution imaging inside thick biological samples. AIO is based on (1) a light-sheet autofocus step (AF) followed by (2) an adaptive optics image-based optimization. We determine the optimum AIO parameters to provide a fast, precise and robust aberration correction on biological samples. Finally, we demonstrate the performances of our approach on sub-micrometric structures in brain slices and plant roots.

Rationalized deep learning super-resolution microscopy for sustained live imaging of rapid subcellular processes.

The goal when imaging bioprocesses with optical microscopy is to acquire the most spatiotemporal information with the least invasiveness. Deep neural networks have substantially improved optical microscopy, including image super-resolution and restoration, but still have substantial potential for artifacts. In this study, we developed rationalized deep learning (rDL) for structured illumination microscopy and lattice light sheet microscopy (LLSM) by incorporating prior knowledge of illumination patterns and, thereby, rationally guiding the network to denoise raw images. Here we demonstrate that rDL structured illumination microscopy eliminates spectral bias-induced resolution degradation and reduces model uncertainty by five-fold, improving the super-resolution information by more than ten-fold over other computational approaches. Moreover, rDL applied to LLSM enables self-supervised training by using the spatial or temporal continuity of noisy data itself, yielding results similar to those of supervised methods. We demonstrate the utility of rDL by imaging the rapid kinetics of motile cilia, nucleolar protein condensation during light-sensitive mitosis and long-term interactions between membranous and membrane-less organelles.

Optogenetic manipulation of cell migration with high spatiotemporal resolution using lattice lightsheet microscopy

Lattice lightsheet microscopy (LLSM) featuring three-dimensional recording is improved to manipulate cellular behavior with subcellular resolution through optogenetic activation (optoLLSM). A position-controllable Bessel beam as a stimulation source is integrated into the LLSM to achieve spatiotemporal photoactivation by changing the spatial light modulator (SLM) patterns. Unlike the point-scanning in a confocal microscope, the lattice beams are capable of wide-field optical sectioning for optogenetic activation along the Bessel beam path.We show that the energy power required for optogenetic activations is lower than 1 nW (or 24 mWcm-2) for time-lapses of CRY2olig clustering proteins, and membrane ruffling can be induced at different locations within a cell with subcellular resolution through light-triggered recruitment of phosphoinositide 3-kinase. Moreover, with the epidermal growth factor receptor (EGFR) fused with CRY2olig, we are able to demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable cellular damages.