onfocal microscopy of hick specimens

bstract. Confocal microscopy is an excellent tool to ain structural information from deep within a biological ample. The depth from which information can be exracted as well as the resolution of the detection system re limited by spherical aberrations in the laser pathway. hese spherical aberrations of the visible light can be efciently canceled by optimizing the refractive index of the mmersion media. Another way of canceling spherical abrrations is by changing tube length, or alternatively, by hanging the objective from infinite correction to finite orrection, or vice versa, depending on which microscope s used. A combination of these two methods allows for onfocal imaging at continuous depths. Presently, confoal microscopes typically operate at a maximum depth of 0 m in the sample, but with the methods presented ere, we show that information can easily be gained from epths up to 100 m. Additionally, the precision of localzation of a single fluorophore in the axial direction, limted by spherical aberrations, can be significantly imroved, even if the fluorophore is located deep within the ample. In principle, this method can improve the effiiency of any kind of microscopy based on visible light. © 009 Society of Photo-Optical Instrumentation Engineers. DOI: 10.1117/1.3156813