Fast live simultaneous multiwavelength four-dimensional optical microscopy

Department of Biochemistry and Biophysics, University of California, San Francisco, The Keck Center for Advanced Microscopy, CA 94158-2517, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2010; 107(37):16016-22. DOI: 10.1073/pnas.1004037107
Source: PubMed


Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.

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    • "A major obstacle to filming cells at high resolution is phototoxicity (Carlton et al., 2010). We found that budding was delayed or blocked as light exposure was increased (Figure S1 available online). "
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