Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy

Dean, Kevin M.; Roudot, Philippe; Welf, Erik S.; Danuser, Gaudenz; Fiolka, Reto

doi:10.1016/j.bpj.2015.05.013

Cited by 216 publications

(193 citation statements)

References 39 publications

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“…2b; Baumgart and Kubitscheck, 2012). Inspired by the integration of this rolling shutter with other applications of LSFM (Baumgart and Kubitscheck, 2012; Chhetri et al ., 2015; de Medeiros et al ., 2015; Dean et al ., 2015), we added this functionality to our control code (Fig. 2c, Materials and Methods), thereby introducing partial confocality into diSPIM.…”

Section: Resultsmentioning

confidence: 99%

Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM)

Kumar

Christensen

Guo

et al. 2016

The Biological Bulletin

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Dual-view inverted selective plane illumination microscopy (diSPIM) enables high-speed, long-term, fourdimensional (4D) imaging with isotropic spatial resolution. It is also compatible with conventional sample mounting on glass coverslips. However, broadening of the light sheet at distances far from the beam waist and sample-induced scattering degrades diSPIM contrast and optical sectioning. We describe two simple improvements that address both issues and entail no additional hardware modifications to the base diSPIM. First, we demonstrate improved diSPIM sectioning by keeping the light sheet and detection optics stationary, and scanning the sample through the stationary light sheet (rather than scanning the broadening light sheet and detection plane through the stationary sample, as in conventional diSPIM). This stage-scanning approach allows a thinner sheet to be used when imaging laterally extended samples, such as fixed microtubules or motile mitochondria in cell monolayers, and produces finer contrast than does conventional diSPIM. We also used stage-scanning diSPIM to obtain high-quality, 4D nuclear datasets derived from an uncompressed nematode embryo, and performed lineaging analysis to track 97% of cells until twitching. Second, we describe the improvement of contrast in thick, scattering specimens by synchronizing light-sheet synthesis with the rolling, electronic shutter of our scientific complementary metal-oxide-semiconductor (sCMOS) detector. This maneuver forms a virtual confocal slit in the detection path, partially removing out-of-focus light. We demonstrate the applicability of our combined stage- and slit-scanning-methods by imaging pollen grains and nuclear and neuronal structures in live nematode embryos. All acquisition and analysis code is freely available online.

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Section: Resultsmentioning

confidence: 99%

Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM)

Kumar

Christensen

Guo

et al. 2016

The Biological Bulletin

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“…Optical sectioning in the axial direction for descanned and normal imaging modes was measured as described previously (Dean et al, 2015). Images were deconvolved as follows unless otherwise noted (Figure S6).…”

Section: Methodsmentioning

confidence: 99%

Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments

et al. 2016

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The microenvironment determines cell behavior, but the underlying molecular mechanisms are poorly understood because quantitative studies of cell signaling and behavior have been challenging due to insufficient spatial and/or temporal resolution and limitations on microenvironmental control. Here we introduce microenvironmental selective plane illumination microscopy (meSPIM) for imaging and quantification of intracellular signaling and submicrometer cellular structures as well as large-scale cell morphological and environmental features. We demonstrate the utility of this approach by showing that the mechanical properties of the microenvironment regulate the transition of melanoma cells from actin-driven protrusion to blebbing, and we present tools to quantify how cells manipulate individual collagen fibers. We leverage the nearly isotropic resolution of meSPIM to quantify the local concentration of actin and phosphatidylinositol 3-kinase signaling on the surfaces of cells deep within 3D collagen matrices and track the many small membrane protrusions that appear in these more physiologically relevant environments.

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“…pLSFM could also be adapted to image larger 3D environments, including synthetic hydrogels and reconstituted extracellular matrices, by scanning staggered light-sheets in their propagation direction with selective fluorescence detection [31]. In such a scheme, some out of focus cross talk from adjacent image planes will occur, which could be suppressed with two-photon excitation [32, 33].…”

Section: Discussionmentioning

confidence: 99%

“…Rab5a-associated vesicles were automatically detected and tracked with a 3D version of uTrack, as previously reported [6, 31]. Brownian and directed motion are modeled with multiple Kalman filters.…”

Section: Methodsmentioning

confidence: 99%

Imaging subcellular dynamics with fast and light-efficient volumetrically parallelized microscopy

Dean

Roudot

Welf

et al. 2017

Optica

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In fluorescence microscopy, the serial acquisition of 2D images to form a 3D volume limits the maximum imaging speed. This is particularly evident when imaging adherent cells in a light-sheet fluorescence microscopy format, as their elongated morphologies require ~200 image planes per image volume. Here, by illuminating the specimen with three light-sheets, each independently detected, we present a light-efficient, crosstalk free, and volumetrically parallelized 3D microscopy technique that is optimized for high-speed (up to 14 Hz) subcellular (300 nm lateral, 600 nm axial resolution) imaging of adherent cells. We demonstrate 3D imaging of intracellular processes, including cytoskeletal dynamics in single cell migration and collective wound healing for 1500 and 1000 time points, respectively. Further, we capture rapid biological processes, including trafficking of early endosomes with velocities exceeding 10 microns per second and calcium signaling in primary neurons.

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Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy

Cited by 216 publications

References 39 publications

Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM)

Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM)

Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments

Imaging subcellular dynamics with fast and light-efficient volumetrically parallelized microscopy

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