Cross-modality synthesis of EM time series and live fluorescence imaging

Abstract

Analyses across imaging modalities allow the integration of complementary spatiotemporal information about brain development, structure and function. However, systematic atlasing across modalities is limited by challenges to effective image alignment. We combine highly spatially resolved electron microscopy (EM) and highly temporally resolved time-lapse fluorescence microscopy (FM) to examine the emergence of a complex nervous system in C. elegans embryogenesis. We generate an EM time series at four classic developmental stages and create a landmark-based co-optimization algorithm for cross-modality image alignment, which handles developmental heterochrony among datasets to achieve accurate single-cell level alignment. Synthesis based on the EM series and time-lapse FM series carrying different cell-specific markers reveals critical dynamic behaviors across scales of identifiable individual cells in the emergence of the primary neuropil, the nerve ring, as well as a major sensory organ, the amphid. Our study paves the way for systematic cross-modality data synthesis in C. elegans and demonstrates a powerful approach that may be applied broadly.

A sex-specific switch in a single glial cell patterns the apical extracellular matrix

“…Approximately 100 to 300 sections were obtained per animal and transferred to a 2x4 cm silicon wafer (Burel et al, 2018; Franke and Kolotuev, 2021). Wafers were dried at ambient temperature by evaporation and subsequently incubated in a 60°C oven for further fixation as previously described (Burel et al, 2018; Franke and Kolotuev, 2021; Santella et al, 2022). Wafers were analyzed with a Helios SEM microscope (Thermo Fisher Scientific) at 2keV landing energy and 0.8 nA beam current at 2 mm distance using a Mirror Detector (MD-BSA) (Burel et al, 2018; Franke and Kolotuev, 2021).…”

“…Wafers were analyzed with a Helios SEM microscope (Thermo Fisher Scientific) at 2keV landing energy and 0.8 nA beam current at 2 mm distance using a Mirror Detector (MD-BSA) (Burel et al, 2018; Franke and Kolotuev, 2021). Images were collected manually or automatically with 4-6 µs dwell time using Maps 3.11 software (Thermo Fisher Scientific) (Burel et al, 2018; Franke and Kolotuev, 2021; Santella et al, 2022). Images were collected at 3 mm/s (1024x886) or 5 mm/s (6084x2044) to generate 20 nm and 5 nm resolution images, respectively.…”

Analysis of developmental gene expression using smFISH andin silicostaging ofC. elegansembryos

“…Given that C. elegans follows a stereotypical developmental pattern, the number of nuclei directly correlates with its developmental stage [36,37]. While it is theoretically feasible to discern each embryonic stage ranging from a single nucleus to 558 nuclei at the conclusion of embryogenesis, such an undertaking would be exceedingly challenging for a high-throughput approach [38][39][40] or require staining of additional markers [41]. Therefore, we employ an autoencoder-based image classifier to learn representations from fixed, DAPI-stained C. elegans embryos to automatically stage them in an approach similar to [42].…”

Work smart, not hard: How array tomography can help increase the ultrastructure data output

“…In this manuscript, I draw on over a decade of firsthand experience to discuss the benefits and possibilities of AT. 19,[21][22][23][24][25][26][27][28][29] Through a series of examples used to highlight the specific strengths of AT-SEM, I hope to initiate a more extensive discussion about how this true SEM revolution can expand access to electron microscopy beyond the committed EM specialists to allow researchers as well as clinicians to gain insight into the ultrastructural changes in cells, tissues and even organs. The hope is that it can help to 'democratise' electron microscopy for all.…”