Light microscopy of thick biological samples, such as tissues, is often limited by aberrations caused by refractive index variations within the sample itself. This problem is particularly severe for live imaging, a field of great current excitement due to the development of inherently fluorescent proteins. We describe a method of removing such aberrations computationally by mapping the refractive index of the sample using differential interference contrast microscopy, modeling the aberrations by ray tracing through this index map, and using space-variant deconvolution to remove aberrations. This approach will open possibilities to study weakly labeled molecules in difficult-to-image live specimens.T he present renaissance in microscopy has been made possible in large part by progress in imaging and analysis of threedimensional (3D) information in whole cells and tissue (1, 2). The development of intrinsically fluorescent proteins (3-5) opened avenues for a wide range of dynamic studies in live sample microscopy. Much like kinetic analysis contributed to understanding mechanisms of chemical reactions, in vivo microscopy is providing new insight into complex, time-dependent cellular mechanisms. Because these mechanisms are often mediated by a small number of molecules, the ability to detect faint signals in vivo is critical.Under their design conditions, modern microscope optics produce nearly ideal aberration-free imaging. However, these conditions are in general true only if the object of interest is immediately adjacent to the coverslip. When focusing into thick samples, the 3D optical characteristics of the sample itself must be considered as part of the optical system. For homogeneous specimens, such as fixed and embedded tissue, the major problem is sample-depth-dependent spherical aberration (6-8); this can be largely corrected for by a choice of appropriate refractive index immersion oil for a particular depth (9, 10), by adjustment of correction collars on special objectives (11), by adjustable optics beyond the objective lens (12), or by computational corrections (13,14).When imaging is attempted in live cells, one has in addition to address further problems that are less severe in fixed preparations: live cells contain a number of organelles, ranging from submicron vesicles to the many micron-sized nuclei, each with its own refractive index (15, 16). The refractive index heterogeneity is equivalent to adding optical elements that locally modify the properties of the 3D microscopic imaging, and thereby cause distortions, degrade image resolution, and reduce the signal-tonoise ratio. As the index variations are local, different regions show different distortions that cannot be corrected by global approaches. A demonstration of such distortions is shown in Fig. 1. These distortions spread out the intensity of a specific fluorescent label, causing what would have been a detectable signal to disappear below the background noise level. Although single fluorophores have been imaged on slides (17-22), even mult...