Limitations on optical sectioning in live-cell confocal microscopy.

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.

[1]  D. Agard,et al.  Computational adaptive optics for live three-dimensional biological imaging , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[2]  D. Agard,et al.  Determination of three-dimensional imaging properties of a light microscope system. Partial confocal behavior in epifluorescence microscopy. , 1990, Biophysical journal.

[3]  S. Myrdal,et al.  Time-resolved confocal analysis of antibody penetration into living, solid tumor spheroids. , 1994, Scanning.

[4]  Joseph Jackson Lister.  XIII. On some properties in achromatic object-glasses applicable to the improvement of the microscope , 1830, Philosophical Transactions of the Royal Society of London.

[5]  S. Hell,et al.  Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index , 1993 .

[6]  Stefan W. Hell,et al.  Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses , 1995 .

[7]  D A Agard,et al.  Dispersion, aberration and deconvolution in multi‐wavelength fluorescence images , 1996, Journal of microscopy.

[8]  David A. Agard,et al.  Three‐dimensional microscopy in thick biological samples: A fresh approach for adjusting focus and correcting spherical aberration , 1997 .

[9]  M Rajadhyaksha,et al.  Analysis of spherical aberration of a water immersion objective: application to specimens with refractive indices 1.33–1.40 , 2000, Journal of microscopy.

[10]  R D Allen,et al.  Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris. , 1981, Cell motility.

[11]  S. Hell,et al.  Lens Aberrations in Confocal Fluorescence Microscopy , 1995 .

[12]  G. J. Brakenhoff,et al.  Refractive index and axial distance measurements in 3-D microscopy , 1992 .

[13]  C. J. R. Sheppard,et al.  Effects of aberrating layers and tube length on con focal imaging properties , 1991 .

[14]  Kjell Carlsson,et al.  The influence of specimen refractive index, detector signal integration, and non‐uniform scan speed on the imaging properties in confocal microscopy , 1991 .