Open AccessLimitations on optical sectioning in live‐cell confocal microscopy
Author(s) -
Pawley James B.
Publication year - 2002
Publication title -
scanning
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.359
H-Index - 47
eISSN - 1932-8745
pISSN - 0161-0457
DOI - 10.1002/sca.4950240504
Subject(s) - confocal , differential interference contrast microscopy , optics , confocal microscopy , optical sectioning , microscopy , interference microscopy , materials science , deconvolution , optical microscope , microscope , refractive index , living cell , bright field microscopy , fluorescence microscope , fluorescence , physics , scanning electron microscope , biology , biological system
In three‐dimensional (3‐D) live‐cell microscopy, it has been common to treat cells as having a constant refractive index (RI). Although the variations in RI associated with the nucleus and other organelles were recognized from phase‐ and differential interference contrast (DIC) images, it was assumed that they were small and would not affect 3‐D fluorescence images obtained using widefield/deconvolution, confocal of multiphoton imaging. This paper makes clear that this confidence was misplaced. Confocal images made using backscattered light (BSL) to image the flat, glass/water interfaces above and below living microscope specimens should reveal these structures as flat and featureless. That the image of the interface on the far side of the cells is neither flat nor featureless indicates that the “optical section” surface can be profoundly distorted by the RI irregularities associated with the presence of nuclei and other subcellar structures. This observation calls into question the reliability of images made using any of the current methods for performing 3‐D light microscopy of living cells.