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Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans.

ABSTRACT: The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ?25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible.

SUBMITTER: Wu Y

PROVIDER: S-EPMC3203761 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans.

Wu Yicong Y Ghitani Alireza A Christensen Ryan R Santella Anthony A Du Zhuo Z Rondeau Gary G Bao Zhirong Z Colón-Ramos Daniel D Shroff Hari H

Proceedings of the National Academy of Sciences of the United States of America 20111017 43

The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating S ...[more]

PMID: 22006307

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Project description:Selective plane illumination microscopy (SPIM) is an emerging fluorescent imaging technique suitable for noninvasive volumetric imaging of C. elegans. These promising microscopy systems, however, are scarce in academic and research institutions due to their high cost and technical complexities. Simple and low-cost solutions that enable conversion of commonplace wide-field microscopes to rapid SPIM platforms promote widespread adoption of SPIM by biologist for studying neuronal expressions of C. elegans. We sought to develop a simple and low-cost optofluidic add-on device that enables rapid and immobilization-free volumetric SPIM imaging of C. elegans with conventional fluorescent microscopes. A polydimethylsiloxane (PDMS)-based device with integrated optical and fluidic elements was developed as a low-cost and miniaturized SPIM add-on for the conventional wide-field microscope. The developed optofluidic chip contained an integrated PDMS cylindrical lens for on-chip generation of the light-sheet across a microchannel. Cross-sectional SPIM images of C. elegans were continuously acquired by the native objective of microscope as worms flowed in an L-shape microchannel and through the light sheet. On-chip SPIM imaging of C. elegans strains demonstrated possibility of visualizing the entire neuronal system in few seconds at single-neuron resolution, with high contrast and without worm immobilization. Volumetric visualization of neuronal system from the acquired cross-sectional two-dimensional images is also demonstrated, enabling the standard microscope to acquire three-dimensional fluorescent images of C. elegans. The full-width at half-maximum width of the point spread function was measured as 1.1 and 2.4 μm in the lateral and axial directions, respectively. The developed low-cost optofluidic device is capable of continuous SPIM imaging of C. elegans model organism with a conventional fluorescent microscope, at high speed, and with single neuron resolution.

Dataset Information

Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans.

Publications

Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans.

Similar Datasets

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