Stain‐free 3D Nanoscopy of Living Cells

Pollaro, Lisa; Equis, Sébastien; Piazza, B. Dalla; Cotte, Yann

doi:10.1002/opph.201600008

Cited by 13 publications

(9 citation statements)

References 6 publications

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“…The synthesis of the rotational series of scattering spectra is achieved using complex field deconvolution [8–10] in order to reconstruct a full 3D refractive index (RI) tomogram of even live [11] samples. Importantly, dynamically adjustable mirrors allow the optics of the HTM device to self-adjust during an acquisition experiment in order to adapt to sample changes such as medium evaporation [12]. While RI distributions have begun to be used in life sciences studies [13–16] and specific RI signatures for cell structures [17,18] have been partially analyzed, HTM systems can suffer from coherent noise created by scattered light from the rotational scanning mechanism [19].…”

Section: Introductionmentioning

confidence: 99%

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux

et al. 2019

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Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell’s refractive indices (RIs) in three dimensions at high spatial and temporal resolution. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells’ division that have so far, to the best of our knowledge, been out of reach. We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images.

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Section: Introductionmentioning

confidence: 99%

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux

et al. 2019

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“…Moreover, HTM combines this holographic approach with rotational scanning of the specimen(5) ( Fig 1A). The direct synthesis of thus gained series of scattering spectra offers a fast 3D reconstruction of even live(6) sample's refractive index (RI) distribution at a resolution below the diffractive limit of light defined by the Rayleigh criterion (7). While RI distributions start to be used in life sciences studies (8)(9)(10)(11) and specific refractive index signatures for cell structures (12,13) have been partially analyzed, current HTM systems typically suffer from coherent noise created by scattered light from the rotational scanning mechanism such as digital micro-mirror devices.…”

Section: Introductionmentioning

confidence: 99%

Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells

Sandoz

Tremblay²,

Equis³

et al. 2018

Preprint

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Holo-tomographic microscopy (HTM) is a label-free non-phototoxic microscopy method reporting the fine changes of a cell's refractive indexes (RI) in 3D. By combining HTM with epifluorescence, we demonstrate that cellular organelles such as Lipid droplets and mitochondria show a specific RI signature that distinguishes them with high resolution and contrast. We further show that HTM allows to follow in unprecedented ways the dynamics of mitochondria, lipid droplets as well as that of endocytic structures in live cells over long period of time, which led us to observe to our knowledge for the first time a global organelle spinning occurring before mitosis.

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“…Importantly, digital staining revealed details of an intricate network of fibrin fibrils with smaller and more circular structures trapped in between the fibrils. Similar to fluorescent markers in immunofluorescence, digital staining allows for specific labeling of multiple structures based on the refractive index values, but without altering the inherent features of the sample ( 43 ). Although we were able to demonstrate the structural characterization of fixed blood clot fragments in an aqueous solution, we cannot explicitly identify the non-fibrin structures observed within these clot fragments.…”

Section: Discussionmentioning

confidence: 99%

Digital holo-tomographic 3D maps of COVID-19 microclots in blood to assess disease severity

Bergaglio,

Synhaivska,

Nirmalraj

2023

Preprint

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The coronavirus disease 2019 (COVID-19) has impacted health globally. Cumulative evidence points to long-term effects of COVID-19 such as cardiovascular and cognitive disorders diagnosed in patients even after the recovery period. In particular, micrometer-sized blood clots and hyperactivated platelets have been identified as potential indicators of long COVID. Here we resolve individual microclot structures in platelet-rich plasma of donors with different subphenotypes of COVID-19 in a label-free manner, using 3D digital holo-tomographic microscopy (DHTM). Based on 3D refractive index (RI) tomograms, the size, dry mass, and prevalence of microclot composites were quantified and then parametrically differentiated from fibrin-rich microclots and platelet aggregates in the plasma of COVID-19 donors. Importantly, fewer microclots and platelet aggregates were detected in the plasma of healthy controls when compared to COVID-19 donors. Our work highlights the utility of integrating DHTM in clinical settings that may allow the detection of individuals at risk of developing microvascular thrombotic disorders and for monitoring the efficiency of prescribed treatments by screening plasma samples.

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Stain‐free 3D Nanoscopy of Living Cells

Cited by 13 publications

References 6 publications

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux

Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells

Digital holo-tomographic 3D maps of COVID-19 microclots in blood to assess disease severity

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