Spinning-disk confocal microscopy

Oreopoulos, John; Berman, R.; Browne, Mark A. Oakley

doi:10.1016/b978-0-12-420138-5.00009-4

Cited by 71 publications

(19 citation statements)

References 35 publications

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“…While the working principle of confocal microscopes (i.e., the rejection of out of focus light through a pinhole) has remained largely the same, the technical implementation has become very diverse 56 , 57 , 58 , 60 . Confocal imaging can be implemented via multipoint scanning, as in a spinning disc system 61 , point scanning of a single laser beam, as in line scanning 62 , or imaging the point spread function with subsequent pixel reassignments via a multi-element detector 57 . Recent advances that expand the utility of confocal imaging include laser scanning capability, high speed resonance scanning, and high sensitivity detectors 63 , 64 , 65 .…”

Section: Introductionmentioning

confidence: 99%

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Sandin

Aryal

Wilkop

et al. 2020

JoVE

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Techniques available for micro-and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe "pokes" the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

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Section: Introductionmentioning

confidence: 99%

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Sandin

Aryal

Wilkop

et al. 2020

JoVE

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“…Spinning‐disk confocal microscopy (SDCM) [37, 38] enables rapid 3D imaging of fluorescently labeled biological specimens. Using this modality, thickness maps of specific fluorescent‐labeled cellular and subcellular structures, such as the cell nucleus, can be reconstructed [26].…”

Section: Introductionmentioning

confidence: 99%

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

Cohen‐Maslaton

Barnea

Taieb

et al. 2020

Journal of Biophotonics

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We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.

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“…To address this, we developed a quantitative technique to rapidly detect cell sensitivity to fluorescent excitation light during short illumination durations (≤1 minute), before morphological changes occur. Recent studies have described microscopy techniques that mitigate effects of excitation light on cells, such as multi-photon, spinning-disk confocal, light sheet, and controlled light exposure microscopy 8 17 18 19 20 21 . These techniques are important for minimizing photo-induced changes in cells.…”

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confidence: 99%

Contractile dynamics change before morphological cues during fluorescence illumination

Knoll

Ahmed

Saif

2015

Sci Rep

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Illumination can have adverse effects on live cells. However, many experiments, e.g. traction force microscopy, rely on fluorescence microscopy. Current methods to assess undesired photo-induced cell changes rely on qualitative observation of changes in cell morphology. Here we utilize a quantitative technique to identify the effect of light on cell contractility prior to morphological changes. Fibroblasts were cultured on soft elastic hydrogels embedded with fluorescent beads. The adherent cells generated contractile forces that deform the substrate. Beads were used as fiducial markers to quantify the substrate deformation over time, which serves as a measure of cell force dynamics. We find that cells exposed to moderate fluorescence illumination (λ = 540–585 nm, I = 12.5 W/m2, duration = 60 s) exhibit rapid force relaxation. Strikingly, cells exhibit force relaxation after only 2 s of exposure, suggesting that photo-induced relaxation occurs nearly immediately. Evidence of photo-induced morphological changes were not observed for 15–30 min after illumination. Force relaxation and morphological changes were found to depend on wavelength and intensity of excitation light. This study demonstrates that changes in cell contractility reveal evidence of a photo-induced cell response long before any morphological cues.

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Spinning-disk confocal microscopy

Cited by 71 publications

References 35 publications

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

Contractile dynamics change before morphological cues during fluorescence illumination

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