u-track3D: Measuring, navigating, and validating dense particle trajectories in three dimensions.

We describe u-track3D, a software package that extends the versatile u-track framework established in 2D to address the specific challenges of 3D particle tracking. First, we present the performance of the new package in quantifying a variety of intracellular dynamics imaged by multiple 3D microcopy platforms and on the standard 3D test dataset of the particle tracking challenge. These analyses indicate that u-track3D presents a tracking solution that is competitive to both conventional and deep-learning-based approaches. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability that automatically defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track3D provides a complete framework for unbiased studies of molecular processes in complex volumetric sequences.

Resolution doubling in light-sheet microscopy via oblique plane structured illumination.

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.

In vivo 3D profiling of site-specific human cancer cell morphotypes in zebrafish.

Tissue microenvironments affect the functional states of cancer cells, but determining these influences in vivo has remained a challenge. We present a quantitative high-resolution imaging assay of single cancer cells in zebrafish xenografts to probe functional adaptation to variable cell-extrinsic cues and molecular interventions. Using cell morphology as a surrogate readout of cell functional states, we examine environmental influences on the morphotype distribution of Ewing Sarcoma, a pediatric cancer associated with the oncogene EWSR1-FLI1 and whose plasticity is thought to determine disease outcome through non-genomic mechanisms. Computer vision analysis reveals systematic shifts in the distribution of 3D morphotypes as a function of cell type and seeding site, as well as tissue-specific cellular organizations that recapitulate those observed in human tumors. Reduced expression of the EWSR1-FLI1 protein product causes a shift to more protrusive cells and decreased tissue specificity of the morphotype distribution. Overall, this work establishes a framework for a statistically robust study of cancer cell plasticity in diverse tissue microenvironments.

Real-time multi-angle projection imaging of biological dynamics.

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.

Community standards for open cell migration data.

Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited owing to the diversity of experimental protocols and non-standardized output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardized data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardization efforts by the Cell Migration Standardisation Organisation (CMSO), an open community-driven organization to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration.

3D flow field estimation and assessment for live cell fluorescence microscopy.

MOTIVATION: The revolution in light sheet microscopy enables the concurrent observation of thousands of dynamic processes, from single molecules to cellular organelles, with high spatiotemporal resolution. However, challenges in the interpretation of multidimensional data requires the fully automatic measurement of those motions to link local processes to cellular functions. This includes the design and the implementation of image processing pipelines able to deal with diverse motion types, and 3D visualization tools adapted to the human visual system. RESULTS: Here, we describe a new method for 3D motion estimation that addresses the aforementioned issues. We integrate 3D matching and variational approach to handle a diverse range of motion without any prior on the shape of moving objects. We compare different similarity measures to cope with intensity ambiguities and demonstrate the effectiveness of the Census signature for both stages. Additionally, we present two intuitive visualization approaches to adapt complex 3D measures into an interpretable 2D view, and a novel way to assess the quality of flow estimates in absence of ground truth. AVAILABILITY AND IMPLEMENTATION: https://team.inria.fr/serpico/data/3d-optical-flow-data/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways.

Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0-2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.

Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1P29S Melanoma Cells.

Actin assembly supplies the structural framework for cell morphology and migration. Beyond structure, this actin framework can also be engaged to drive biochemical signaling programs. Here, we describe how the hyperactivation of Rac1 via the P29S mutation (Rac1P29S) in melanoma hijacks branched actin network assembly to coordinate proliferative cues that facilitate metastasis and drug resistance. Upon growth challenge, Rac1P29S-harboring melanoma cells massively upregulate lamellipodia formation by dendritic actin polymerization. These extended lamellipodia form a signaling microdomain that sequesters and phospho-inactivates the tumor suppressor NF2/Merlin, driving Rac1P29S cell proliferation in growth suppressive conditions. These biochemically active lamellipodia require cell-substrate attachment but not focal adhesion assembly and drive proliferation independently of the ERK/MAPK pathway. These data suggest a critical link between cell morphology and cell signaling and reconcile the dichotomy of Rac1's regulation of both proliferation and actin assembly by revealing a mutual signaling axis wherein actin assembly drives proliferation in melanoma.

Mutant p53 amplifies a dynamin-1/APPL1 endosome feedback loop that regulates recycling and migration.

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors downstream of gain-of-function (GOF) mutant p53. We report that dynamin-1 (Dyn1) is up-regulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Dyn1 is required for the recruitment and accumulation of the signaling scaffold, APPL1, to a spatially localized subpopulation of endosomes at the cell perimeter. We developed new tools to quantify peripherally localized early endosomes and measure the rapid recycling of integrins. We report that these perimeter APPL1 endosomes modulate Akt signaling and activate Dyn1 to create a positive feedback loop required for rapid recycling of EGFR and ß1 integrins, increased focal adhesion turnover, and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus that integrates signaling and receptor trafficking, which can be co-opted and amplified in mutant p53-driven cancer cells to increase migration and invasion.

Augmin accumulation on long-lived microtubules drives amplification and kinetochore-directed growth.

Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.

Universal light-sheet generation with field synthesis.

We introduce field synthesis, a theorem and method that can be used to synthesize any scanned or dithered light sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. Compared to LLSM, this user-friendly and modular approach offers a simplified optical design, higher light throughput and simultaneous multicolor illumination. Further, field synthesis achieves lower rates of photobleaching than light sheets generated by lateral beam scanning.

Imaging Subcellular Dynamics with Fast and Light-Efficient Volumetrically Parallelized Microscopy.

In fluorescence microscopy, the serial acquisition of 2D images to form a 3D volume limits the maximum imaging speed. This is particularly evident when imaging adherent cells in a light-sheet fluorescence microscopy format, as their elongated morphologies require ~200 image planes per image volume. Here, by illuminating the specimen with three light-sheets, each independently detected, we present a light-efficient, crosstalk free, and volumetrically parallelized 3D microscopy technique that is optimized for high-speed (up to 14 Hz) subcellular (300 nm lateral, 600 nm axial resolution) imaging of adherent cells. We demonstrate 3D imaging of intracellular processes, including cytoskeletal dynamics in single cell migration and collective wound healing for 1500 and 1000 time points, respectively. Further, we capture rapid biological processes, including trafficking of early endosomes with velocities exceeding 10 microns per second and calcium signaling in primary neurons.

Piecewise-Stationary Motion Modeling and Iterative Smoothing to Track Heterogeneous Particle Motions in Dense Environments.

One of the major challenges in multiple particle tracking is the capture of extremely heterogeneous movements of objects in crowded scenes. The presence of numerous assignment candidates in the expected range of particle motion makes the tracking ambiguous and induces false positives. Lowering the ambiguity by reducing the search range, on the other hand, is not an option, as this would increase the rate of false negatives. We propose here a piecewise-stationary motion model (PMM) for the particle transport along an iterative smoother that exploits recursive tracking in multiple rounds in forward and backward temporal directions. By fusing past and future information, our method, termed PMMS, can recover fast transitions from freely or confined diffusive to directed motions with linear time complexity. To avoid false positives, we complemented recursive tracking with a robust inline estimator of the search radius for assignment (a.k.a. gating), where past and future information are exploited using only two frames at each optimization step. We demonstrate the improvement of our technique on simulated data especially the impact of density, variation in frame to frame displacements, and motion switching probability. We evaluated our technique on the 2D particle tracking challenge dataset published by Chenouard et al. in 2014. Using high SNR to focus on motion modeling challenges, we show superior performance at high particle density. On biological applications, our algorithm allows us to quantify the extremely small percentage of motor-driven movements of fluorescent particles along microtubules in a dense field of unbound, diffusing particles. We also show with virus imaging that our algorithm can cope with a strong reduction in recording frame rate while keeping the same performance relative to methods relying on fast sampling.

Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells.

In subcellular light-sheet fluorescence microscopy (LSFM) of adherent cells, glass substrates are advantageously rotated relative to the excitation and emission light paths to avoid glass-induced optical aberrations. Because cells are spread across the sample volume, three-dimensional imaging requires a light-sheet with a long propagation length, or rapid sample scanning. However, the former degrades axial resolution and/or optical sectioning, while the latter mechanically perturbs sensitive biological specimens on pliant biomimetic substrates (e.g., collagen and basement membrane). Here, we use aberration-free remote focusing to diagonally sweep a narrow light-sheet along the sample surface, enabling multicolor imaging with high spatiotemporal resolution. Further, we implement a dithered Gaussian lattice to minimize sample-induced illumination heterogeneities, significantly improving signal uniformity. Compared with mechanical sample scanning, we drastically reduce sample oscillations, allowing us to achieve volumetric imaging at speeds of up to 3.5 Hz for thousands of Z-stacks. We demonstrate the optical performance with live-cell imaging of microtubule and actin cytoskeletal dynamics, phosphoinositide signaling, clathrin-mediated endocytosis, polarized blebbing, and endocytic vesicle sorting. We achieve three-dimensional particle tracking of clathrin-associated structures with velocities up to 4.5 µm/s in a dense intracellular environment, and show that such dynamics cannot be recovered reliably at lower volumetric image acquisition rates using experimental data, numerical simulations, and theoretical modeling.

Lifetime estimation of moving subcellular objects in frequency-domain fluorescence lifetime imaging microscopy.

Fluorescence lifetime is usually defined as the average nanosecond-scale delay between excitation and emission of fluorescence. It has been established that lifetime measurements yield numerous indications on cellular processes such as interprotein and intraprotein mechanisms through fluorescent tagging and Förster resonance energy transfer. In this area, frequency-domain fluorescence lifetime imaging microscopy is particularly appropriate to probe a sample noninvasively and quantify these interactions in living cells. The aim is then to measure the fluorescence lifetime in the sample at each location in space from fluorescence variations observed in a temporal sequence of images obtained by phase modulation of the detection signal. This leads to a sensitivity of lifetime determination to other sources of fluorescence variations such as intracellular motion. In this paper, we propose a robust statistical method for lifetime estimation for both background and small moving structures with a focus on intracellular vesicle trafficking.

Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy.

The use of propagation invariant Bessel beams has enabled high-resolution subcellular light sheet fluorescence microscopy. However, the energy within the concentric side lobe structure of Bessel beams increases significantly with propagation length, generating unwanted out-of-focus fluorescence that enforces practical limits on the imaging field of view size. Here, we present a light sheet fluorescence microscope that achieves 390 nm isotropic resolution and high optical sectioning strength (i.e., out-of-focus blur is strongly suppressed) over large field of views, without the need for structured illumination or deconvolution-based postprocessing. We demonstrate simultaneous dual-color, high-contrast, and high-dynamic-range time-lapse imaging of migrating cells in complex three-dimensional microenvironments, three-dimensional tracking of clathrin-coated pits, and long-term imaging spanning >10 h and encompassing >2600 time points.

Objective comparison of particle tracking methods.

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.