1 May 2009 Confocal microscopy of thick specimens
Author Affiliations +
J. of Biomedical Optics, 14(3), 030513 (2009). doi:10.1117/1.3156813
Abstract
Confocal microscopy is an excellent tool to gain structural information from deep within a biological sample. The depth from which information can be extracted as well as the resolution of the detection system are limited by spherical aberrations in the laser pathway. These spherical aberrations of the visible light can be efficiently canceled by optimizing the refractive index of the immersion media. Another way of canceling spherical aberrations is by changing tube length, or alternatively, by changing the objective from infinite correction to finite correction, or vice versa, depending on which microscope is used. A combination of these two methods allows for confocal imaging at continuous depths. Presently, confocal microscopes typically operate at a maximum depth of 40 μm in the sample, but with the methods presented here, we show that information can easily be gained from depths up to 100 μm. Additionally, the precision of localization of a single fluorophore in the axial direction, limited by spherical aberrations, can be significantly improved, even if the fluorophore is located deep within the sample. In principle, this method can improve the efficiency of any kind of microscopy based on visible light.
Nader Reihani, Lene B. Oddershede, "Confocal microscopy of thick specimens," Journal of Biomedical Optics 14(3), 030513 (1 May 2009). http://dx.doi.org/10.1117/1.3156813
Submission: Received ; Accepted
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KEYWORDS
Confocal microscopy

Visualization

Refractive index

Microscopes

Gold

Monochromatic aberrations

Nanoparticles

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