High-resolution single-cell imaging for functional studies in the whole brain and spinal cord and thick tissue blocks using light-emitting diode illumination.

Functional studies of neuronal networks require recordings from visually identified neurons in their natural environment preservation of which may demand experimenting with a tissue of a significant depth or the entire brain. Here we describe a new technique of single-cell imaging and visually controlled patch-clamp recordings in both brain slices of unlimited thickness and the whole brain or spinal cord preparations with a cut upper surface. It utilizes an upright microscope and ultra bright light-emitting diodes (LEDs) as a source of oblique illumination. This technique provided high quality images of superficial cells regardless of slice thickness or the presence of opaque structures, like metal plate or bone, below the tissue, when conventional differential interference contrast (DIC) optics became powerless. The technique opens broad possibilities for a single-cell imaging and visually guided recordings from intact neuronal networks in the entire brain or spinal cord.