Object detection and classification are key tasks in computer vision that can facilitate high-throughput image analysis of microscopy data. We present a set of local image descriptors for three-dimensional (3D) microscopy datasets inspired by the well-known Haar wavelet framework. We add orientation, illumination and scale information by assuming that the neighborhood surrounding points of interests in the image can be described with ellipsoids, and we increase discriminative power by incorporating edge and shape information into the features. The calculation of the local image descriptors is implemented in a Graphics Processing Unit (GPU) in order to reduce computation time to 1 millisecond per object of interest. We present results for cell division detection in 3D time-lapse fluorescence microscopy with 97.6% accuracy.

Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

3D live imaging is important for a better understanding of biological processes, but it is challenging with current techniques such as spinning-disk confocal microscopy. Bessel beam plane illumination microscopy allows high-speed 3D live fluorescence imaging of living cellular and multicellular specimens with nearly isotropic spatial resolution, low photobleaching and low photodamage. Unlike conventional fluorescence imaging techniques that usually have a unique operation mode, Bessel plane illumination has several modes that offer different performance with different imaging metrics. To achieve optimal results from this technique, the appropriate operation mode needs to be selected and the experimental setting must be optimized for the specific application and associated sample properties. Here we explain the fundamental working principles of this technique, discuss the pros and cons of each operational mode and show through examples how to optimize experimental parameters. We also describe the procedures needed to construct, align and operate a Bessel beam plane illumination microscope by using our previously reported system as an example, and we list the necessary equipment to build such a microscope. Assuming all components are readily available, it would take a person skilled in optical instrumentation \~{}1 month to assemble and operate a microscope according to this protocol.

A major frontier in single cell biology is decoding how transcriptional states result in cellular-level architectural changes, ultimately driving function. A remarkable example of this cellular remodelling program is the differentiation of airway stem cells into the human respiratory multiciliated epithelium, a tissue barrier protecting against bacteria, viruses and particulate matter. Here, we present the first isotropic three-dimensional map of the airway epithelium at the nanometre scale unveiling the coordinated changes in cellular organisation, organelle topology and contacts, occurring during multiciliogenesis. This analysis led us to discover a cellular mechanism of communication whereby motile cilia relay mechanical information to mitochondria through striated cytoskeletal fibres, the rootlets, to promote effective ciliary motility and ATP generation. Altogether, this study integrates nanometre-scale structural, functional and dynamic insights to elucidate fundamental mechanisms responsible for airway defence.

New methods in stem cell 3D organoid tissue culture, advanced imaging and big data image analytics now allow tissue scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice Light-Sheet Imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70µm x 60µm x 40µm xyz) at 5.7s/frame. We developed an open source data analysis package termed pyLattice to process the resulting large (∼60Gb) movie datasets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. Based on their localization in the organoid, we classified CME tracks into apical, lateral and basal events and found that CME dynamics are similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative, high temporal and spatial resolution analysis of subcellular events within tissues. Movie S1 Movie S1 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid showing clathrin (red) and dynamin2 (green) puncta in surface depiction. The movie zooms out from a single clathrin mediated endocytosis event that shows both clathrin and dynamin2 at the same location to eventually show the whole AO-LLSM field of view. Nuclear envelopes and the outer membranes of the tissue are depicted in transparent white. Movie S2 Movie S2 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid showing clathrin (red) and dynamin2 (green) puncta in surface depiction. The movie rotates the AO-LLSM field of view. Nuclear envelopes and the outer membranes of the tissue are depicted in transparent white. Movie S3 Movie S3 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid. The curved surface is of the spherical organoid is visible as the movie rotates. Clathrin puncta are visible throughout the tissue (white). Movie S4 Movie S4 The detection step in the data processing pipeline retrieves all clathrin puncta in the volume. Detected puncta are marked with a cube (blue). Movie S5 Movie S5 Zoom on one clathrin puncta in the thresholded 3D dataset. The punctum of interest is marked with a blue cube. Other puncta are also visible. Movie S6 Movie S6 Zoom on the same clathrin puncta as in M3 in non-thresholded 3D data. The surrounding fluorescence is visible as a transparent cloud.