Introduction
One approach that neuroscientists use to investigate complex learning is to assess the effects of damage to specific brain regions in rodents. Chemogenetics [1-3] can be used to temporarily, rather than permanently inactive a region of interest. This two-step approach involves viral-mediated delivery of a piece of modified, foreign DNA (a receptor) and a fluorescent tag to cells within a specific brain region. Next, administration of a pharmacological agent activates the receptor and induces temporary inactivation of cells. Thus, investigators can combine chemogenetics with learning paradigms to assess behavior with and without neural activity in the region of interest. Assessment and verification of correct placement of the viral construct is an essential component of the research and requires the use of sophisticated imaging.
Challenges
An imaging solution that can quickly screen thick 3D cortical tissue specimens and achieve sharp, high-contrast imaging, where important details deep inside the specimen are clearly resolved, is important for cortical brain regions. These cortical regions are comprised of six distinct layers of neuron; having the ability to determine which layers contain labeled neurons provides important information about the neural circuitry underlying complex learning [2,3]. Conventional widefield microscopy offers users speed and detection sensitivity, but unfortunately there is often an out-of-focus blur or haze, due to signals from out-of-focus planes of thick specimens, reducing significantly image contrast [4].
Methods
Fluorescence images of rat cortical tissue with neurons expressing enhanced green fluorescent protein (eGFP) were acquired using a THUNDER Imager Tissue with a HC PL