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

Dean, Kevin M.; Roudot, Philippe; Welf, Erik S.; Pohlkamp, Theresa; Garrelts, Gerard; Herz, Joachim; Fiolka, Reto

doi:10.1364/optica.4.000263

Cited by 43 publications

(30 citation statements)

References 34 publications

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“…In the first case, fast exposure (2.5-4.5 ms) was translated to a 5-Hz volumetric acquisition frame rate, allowing imaging of the entire animal. Along with advancing volumetric image acquisition by paralleled illumination with more than one light sheet (Dean et al, 2017), such results exemplify the potential of LSFM to monitor fast physiological events with subcellular resolution in whole organisms, organs, or tissues.…”

Section: Lsfmmentioning

confidence: 81%

Advances in Imaging Plant Cell Dynamics

et al. 2017

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Section: Lsfmmentioning

confidence: 81%

Advances in Imaging Plant Cell Dynamics

et al. 2017

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“…Light-field microscopy (LFM) simultaneously captures both the 2D spatial and 2D angular information of the incident light, allowing computational reconstruction of the full 3D volume of a specimen from a single camera frame [1][2][3][4]. Conventionally, fluorescent imaging techniques acquire 3D spatial information in a sequential or scanning fashion [5][6][7][8][9][10][11][12], inevitably compromising temporal resolution and increasing photodamage for live imaging. The 4D imaging scheme of LFM effectively liberates volume acquisition time (limited primarily by the camera's frame rate) from the spatial parameters (e.g.…”

Section: Introductionmentioning

confidence: 99%

Fast, volumetric live-cell imaging using high-resolution light-field microscopy

Guo

Kim

et al. 2018

Preprint

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Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatiotemporal levels. Fig. 13. Imaging Golgi-derived membrane vesicles in living COS-7 cells at a volume acquisition time of 5 ms using HR-LFM. (a) Reconstructed 3D images of vesicles in living COS-7 cells labeled with mEmerald-Golgi-7 acquired at a volume acquisition time of 5 ms. The left and right are reconstructed 3D images at t = 0.005s and 5.000s of a 1000-time-point series, respectively. The depth-information is color-coded according to the color scale bar. (b) Zoomedin images of the corresponding boxed region in (a) at t = 0.005s, 3.000s, 3.500s, 4.000s, 4.500s and 5.000s, respectively. White arrows indicate vesicles moving during time-points. (c) Zoomed-in image (leftmost) of the boxed region in (b) at t = 0.005s and its z-stack images from z = 0.2 µm to +1.3 µm at an axial step size of 100 nm, resolving several nearby vesicles. (d) Zoomed-in images of the corresponding boxed region in (a) at t = 0.005s, 0.500s, 1.000s, 1.500s, 2.000s, 2.500s, 3.000s and 3.500s, respectively. Scale bars: 10 µm (a), 1 µm (b-d).

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“…For live cell adhesion analyses, cells were infected with a lentivirus encoding an mRuby2-Paxillin fusion protein under the control of a crippled CMV promoter (Dean et al, 2017). This construct was generated by substituting the mNeonGreen (mNG) from pLVX-CMV100-mNG-Paxillin plasmid, gifted by Kevin Dean (Mohan et al, 2018), for mRuby2 fluorescent protein.…”

Section: O Cell Fractionationmentioning

confidence: 99%

Mutant p53 triggers a dynamin-1/APPL1 endosome feedback loop that regulates β1 integrin recycling and migration

Lakoduk

Roudot

Mettlen³

et al. 2018

Preprint

Self Cite

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Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors. We report that gain-of-function (GOF) mutant p53 expression enhances b integrin and EGF receptor recycling and increases cell migration by triggering a positive feedback loop involving the activation of dynamin-1 (Dyn1) and accumulation of a spatially-restricted subpopulation of APPL1-positive 'perimeter' endosomes. DNM1 is upregulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53.Perimeter APPL1 endosomes are required for rapid recycling of EGFR and b1 integrins and modulate Akt signaling and Dyn1 activation to create the positive feedback loop that culminates in increased focal adhesion turnover and cell migration. Thus, Dyn1-and Aktdependent perimeter APPL1 endosomes function as a nexus, integrating signaling and receptor trafficking, that can be co-opted by cancer cells for mutant p53-driven migration and invasion. 2015). Difficulties in quantitatively measuring fast recycling render this the least mechanistically understood of these trafficking pathways.

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

Cited by 43 publications

References 34 publications

Advances in Imaging Plant Cell Dynamics

Advances in Imaging Plant Cell Dynamics

Fast, volumetric live-cell imaging using high-resolution light-field microscopy

Mutant p53 triggers a dynamin-1/APPL1 endosome feedback loop that regulates β1 integrin recycling and migration

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