High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing.
Live imaging in cell biology requires three-dimensional data acquisition with the best resolution and signal-to-noise ratio possible. Depth aberrations are a major source of image degradation in three-dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide-field fluorescence microscope that incorporates a large-throw deformable mirror to simultaneously focus and correct for depth aberration in three-dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2-fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.
Kner
P
P
Keck Advanced Microscopy Laboratory and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA. kner@engr.uga.edu
Sedat
J W
JW
Agard
D A
DA
Kam
Z
Z
D000818:Animals; D045744:Cell Line, Tumor; D042783:Endothelial Cells / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007814:Larva / Q000166:cytology; D008544:Melanocytes / Q000166:cytology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*
D049452:Green Fluorescent Proteins