Deblurring of 3-dimensional patterns of evoked rat cerebellar cortical activity: a study using voltage-sensitive dyes and optical sectioning.

One of the benefits of imaging neuronal activity is the capability of resolving spatial patterns in the x-y plane. With optical sectioning microscopy, the 3-dimensional (3-D) structure may also be studied without physical deformation by serially moving the focal plane of the microscope through the volume of interest along the focal axis. However, each image is blurred by contributions from neighboring planes. This degradation is most severe for low numerical aperture lenses and large amounts of defocus. In this study, an image restoration method using the optical properties of an aberration-free, defocused optical system has been developed for improving optical signals from voltage-sensitive dyes. Deblurring based on the optical transfer function (OTF) of the system was applied on two test sets of serially sectioned images: (1) fluorescent beads and (2) in vivo rat cerebellar cortex stained with the voltage-sensitive dye RH795. This method was shown to reduce significantly the out-of-focus contribution to the images, improving the spatial resolution not only in the x-y plane, but also the z axis. The algorithms were then applied to optical signals obtained by stimulation of the cerebellar surface. Optical signals having a distinct beam-like pattern were evoked and recorded over depths ranging from 0 to 300 microns prior to deblurring. Application of the deblurring algorithm reduced the depth of cerebellar cortex over which the optical signals were observed. In agreement, field potential recordings of the evoked parallel fiber volley and post-synaptic components were restricted to a narrow range of depths similar to the deblurred optical images. Removal of out-of-focus information is an essential step in the serial sectioning of central nervous system structures for neuronal imaging and 3-D reconstruction.