Extracting calcium traces from the neurons of C. elegans is an important problem, enabling the study of individual neuronal activity and the large-scale dynamics that govern behavior. Traditionally, non-negative matrix factorization (NMF) methods have been successful in demixing and denoising cellular calcium activity in relatively motionless or pre-registered videos. However, in the case of C. elegans or other animal models where motion compensation methods fail to stabilize the effect of even mild motion in the imaging data, standard NMF methods fail to capture cellular footprints since these footprints are variable in time. In this work, we introduce deformable non-negative matrix factorization (dNMF), which models the motion trajectory of the underlying image space using a polynomial basis function. Spatial footprints and neural activity are optimized jointly with motion trajectories in a matrix tri-factorization setting. On simulated data, dNMF is demonstrated to outperform currently available demixing methods as well as methods that account for motion and demixing separately. Furthermore, we display the practical utility of our approach in extracting calcium traces from C. elegans microscopy videos. The extracted traces elucidate spontaneous neural activity as well as responses to stimuli. Open source code implementing this pipeline is available at https://github.com/amin-nejat/dNMF

Single-beam scanning electron microscopes (SEM) are widely used to acquire massive datasets for biomedical study, material analysis, and fabrication inspection. Datasets are typically acquired with uniform acquisition: applying the electron beam with the same power and duration to all image pixels, even if there is great variety in the pixels' importance for eventual use. Many SEMs are now able to move the beam to any pixel in the field of view without delay, enabling them, in principle, to invest their time budget more effectively with non-uniform imaging.

Comprehensively resolving single neurons and their cellular identities from whole-brain fluorescent images is a major challenge. We achieve this in C. elegans through the engineering and use of a multicolor transgene called NeuroPAL (a Neuronal Polychromatic Atlas of Landmarks). NeuroPAL worms share a stereotypical multicolor fluorescence map for the entire hermaphrodite nervous system that allows comprehensive determination of neuronal identities. Neurons labeled with NeuroPAL do not exhibit fluorescence in the green, cyan, or yellow emission channels, allowing the transgene to be used with numerous reporters of gene expression or neuronal dynamics. Here we showcase three studies that leverage NeuroPAL for nervous-system-wide neuronal identification. First, we determine the brainwide expression patterns of all metabotropic receptors for acetylcholine, GABA, and glutamate, completing a map of this communication network. Second, we uncover novel changes in cell fate caused by transcription factor mutations. Third, we record brainwide activity in response to attractive and repulsive chemosensory cues, characterizing multimodal coding and novel neuronal asymmetries for these stimuli. We present a software package that enables semi-automated determination of all neuronal identities based on color and positional information. The NeuroPAL framework and software provide a means to design landmark atlases for other tissues and organisms. In conclusion, we expect NeuroPAL to serve as an invaluable tool for gene expression analysis, neuronal fate studies, and for mapping whole-brain activity patterns.

Temperature is a key control parameter of biological processes, but measuring and controlling temperatures on a cellular-length scale in living organisms remains an outstanding challenge. Applying nanoscale-thermometry techniques to early embryos, we study cell divisions in a highly controlled manner using local laser heating and real-time in vivo temperature readout. Nitrogen-vacancy centers in nanodiamonds, incorporated into the cells, allow us to map out the temperature distribution of a locally heated embryo with submicrometer spatial resolution and high sensitivity. The simultaneous cell-division imaging under controlled laser heating is used to achieve cell-cycle timing control and inversion, providing insights into timing-regulation mechanisms during early embryogenesis. Understanding the coordination of cell-division timing is one of the outstanding questions in the field of developmental biology. One active control parameter of the cell-cycle duration is temperature, as it can accelerate or decelerate the rate of biochemical reactions. However, controlled experiments at the cellular scale are challenging, due to the limited availability of biocompatible temperature sensors, as well as the lack of practical methods to systematically control local temperatures and cellular dynamics. Here, we demonstrate a method to probe and control the cell-division timing in Caenorhabditis elegans embryos using a combination of local laser heating and nanoscale thermometry. Local infrared laser illumination produces a temperature gradient across the embryo, which is precisely measured by in vivo nanoscale thermometry using quantum defects in nanodiamonds. These techniques enable selective, controlled acceleration of the cell divisions, even enabling an inversion of division order at the two-cell stage. Our data suggest that the cell-cycle timing asynchrony of the early embryonic development in C. elegans is determined independently by individual cells rather than via cell-to-cell communication. Our method can be used to control the development of multicellular organisms and to provide insights into the regulation of cell-division timings as a consequence of local perturbations.