Confocal microscopy of thick specimens

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 cancelling 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 microm in the sample, but with the methods presented here, we show that information can easily be gained from depths up to 100 microm. 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.