Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy

Abstract

Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo. SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.

Quantitative contrast-enhanced optical coherence tomography

“…[2][3][4] However, for such contrast-enhanced OCT studies to reach their full potential, quantitative imaging capabilities during in vivo experiments are needed in order to assess biomarker expression levels, 5 disease response to therapy, 6 and developmental processes. 7 Thus, the ability to easily extract quantitative information about scattering contrast agents will be useful for realizing OCT as a molecular modality. However, accurate quantitative measurements are not readily obtained from OCT images since detected signals comprise convoluted information from single and multiple scattering events as well as interference among photons within a given imaging volume.…”

Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintű idegsejtvizsgálatokhoz

“…Számos technológia létezik már, amely képes gyors populációs és dendritikus aktivitás 3D-s rögzítésére [6,7,9,10,11,12,13,14,15,16,17,18], ám csak az akusztooptikai (AO) megoldásokon alapuló kétfoton-pásztázás volt képes a szükséges technikai paramétereket biztosítani, úgymint 20-nál több ROI pásztázása 30 μm-nél nagyobb z kiterjedésből, 100 Hz-nél nagyobb ismét-lési sebességgel [5,19,20,21]. A következőkben célunk az első olyan kétfoton-mikroszkóp megalkotása volt, amellyel lehetséges ROI-k véletlen elérésű pásztázása há-rom dimenzióban, leküzdve a korábbi megoldások korlátait.…”

A computational model of nuclear self-organisation in syncytial embryos

“…An additional challenge, is to obtain data in three dimensions at high temporal resolution rather than using the two-dimensional slices usually considered. There have been studies looking at the entire embryo in three dimensions on fixed embryos (Keränen et al (2006)), but an exciting development is the recent interest in using imaging and data analysis techniques to track live nuclei in real-time in three dimensions (Krzic et al (2012);Tomer et al (2012)). …”