Fast live simultaneous multiwavelength four-dimensional optical microscopy.
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.
Carlton
Peter M
PM
Department of Biochemistry and Biophysics, University of California, San Francisco, The Keck Center for Advanced Microscopy, CA 94158-2517, USA.
Boulanger
Jérôme
J
Kervrann
Charles
C
Sibarita
Jean-Baptiste
JB
Salamero
Jean
J
Gordon-Messer
Susannah
S
Bressan
Debra
D
Haber
James E
JE
Haase
Sebastian
S
Shao
Lin
L
Winoto
Lukman
L
Matsuda
Atsushi
A
Kner
Peter
P
Uzawa
Satoru
S
Gustafsson
Mats
M
Kam
Zvi
Z
Agard
David A
DA
Sedat
John W
JW
D000465:Algorithms; D000818:Animals; D002470:Cell Survival; D004331:Drosophila melanogaster / Q000166:cytology; D008856:Microscopy, Fluorescence / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology; D012984:Software