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Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

ABSTRACT: There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 ?m), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 ?m from the surface of a coverglass.

SUBMITTER: Lee J

PROVIDER: S-EPMC3475383 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

Lee Jinwoo J Miyanaga Yukihiro Y Ueda Masahiro M Hohng Sungchul S

Biophysical journal 20121016 8

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-ima ...[more]

PMID: 23083712

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Project description:To be able to resolve molecular-clusters it is crucial to access vital information (such as, molecule density, cluster-size, and others) that are key in understanding disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precision (LP)) to reconstruct a super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single-molecules) that undermine image resolution. Ideally, it should be possible to identify the brightest molecules (also termed as the fortunate molecules) to reconstruct ultra-superresolution map, provided sufficient statistics is available from the recorded data. Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy explores this possibility by introducing a narrow probability size-distribution of single-molecules (narrow size-spectrum about a predefined mean-size). The reconstruction begins by presetting the mean and variance of the narrow distribution function (Gaussian function). Subsequently, the dataset is processed and single-molecules are filtered by the Gaussian function to remove unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct an ultra-superresolution map. In-principle, the POSSIBLE microscopy technique is capable of infinite resolution (resolution of the order of actual single-molecule size) provided enough fortunate molecules are experimentally detected. In short, bright molecules (with large emissivity) holds the key. Here, we demonstrate the POSSIBLE microscopy technique and reconstruct single-molecule images with an average PSF sizes of σ ± Δσ = 15 ± 10 nm, 30 ± 2 nm & 50 ± 2 nm. Results show better-resolved Dendra2-HA clusters with large cluster-density in transfected NIH3T3 fibroblast cells as compared to the traditional SMLM techniques. Cluster analysis indicates densely-packed HA molecules, HA-HA interaction, and a surge in the number of HA molecules per cluster post 24 Hrs of transfection. The study using POSSIBLE microscopy introduces new insights in influenza biology. We anticipate exciting applications in the multidisciplinary field of disease biology, oncology, and biomedical imaging.

Dataset Information

Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

Publications

Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

Similar Datasets

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