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Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index
Open accessAbstract
SUMMARY: The effect of refractive‐index mismatch, as encountered in the observation of biological specimens, on the image acquisition process in confocal fluorescence microscopy is investigated theoretically. The analysis takes the vectorial properties of light into account and is valid for high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practical situations. When observing with a numerical aperture of 1·3 (oil immersion) and an excitation wavelength of 514 nm the centre of the focus shifts 1·7 μm per 10 μm of axial displacement in an aqueous medium, thus yielding an image that is scaled by a factor of 1·2 in the axial direction. Furthermore, it can be expected that for a fluorescent plane 20 μm deep inside an aqueous medium the peak intensity is 40% less than for a plane which is 10 μm deep. In addition, the axial resolution is decreased by a factor of 1·4. The theory was experimentally verified for test samples with different refractive indices.
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Cited by
Xu, C., & Webb, W. W. (1996) · Journal of the Optical Society of America B · doi:10.1364/josab.13.000481 · Cited by 2,198
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission
Klar, T. A., Jakobs, S., Dyba, M., Egner, A., & Hell, S. W. (2000) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.97.15.8206 · Cited by 1,869
Self-citationRichardson, D. S., & Lichtman, J. W. (2015) · Cell · doi:10.1016/j.cell.2015.06.067 · Cited by 1,095
Rajadhyaksha, M., González, S., Zavislan, J. M., Rox Anderson, R., & Webb, R. H. (1999) · Journal of Investigative Dermatology · doi:10.1046/j.1523-1747.1999.00690.x · Cited by 834
Török, P., Varga, P., Laczik, Z., & Booker, G. R. (1995) · Journal of the Optical Society of America A · doi:10.1364/josaa.12.000325 · Cited by 448
Metadata
- Authors
- HELL, S., REINER, G., CREMER, C., & STELZER, E. H. K.
- Venue
- Journal of Microscopy
- Publisher
- Wiley
- Published
- 1993-03
- Volume
- 169
- Issue
- 3
- Pages
- 391-405
- ISSN
- 0022-2720 (ISSN-L)
- 1365-2818
- OpenAlex
- W2171788130
- Cited by
- 640
- References
- 0
- FWCI
- 21.83
- Type
- journal article
- Language
- en
- Open access
- green
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- HELL, S., REINER, G., CREMER, C., & STELZER, E. H. K. (1993). Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. Journal of Microscopy. https://doi.org/10.1111/j.1365-2818.1993.tb03315.x
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