A custom-built two-photon (2P) confocal system mounted on an upright Nikon microscope, designed for advanced imaging with epifluorescence and transmitted light capabilities. This system is uniquely equipped with a fixed stage, making it particularly suitable for electrophysiology experiments. The 2P system integrates a Thorlabs resonant scanner, controlled by Thorimage software (a customized variant of ScanImage), ensuring precise and rapid scanning. It features a single 20x objective (Nikon 0.95NA) and two Gallium Arsenide Phosphide (GaAsp) photomultiplier tubes (PMTs) with GFP/dsRED filters. While it lacks environmental controls, its primary advantage lies in two-photon microscopy's ability to image deep into thick samples (up to several millimeters) with reduced phototoxicity and photobleaching compared to conventional confocal systems. Access to this system is dependent on the availability of the Ruthazer lab’s Mai Tai pulsed 2-P laser.