Figure 1.

System diagram for the sequential VSD and calcium imaging microscope. A Prairie Technologies Ultima Multi-Photon Microscopy system mounted on an Olympus BX-61 microscope equipped with a sliding mirror (A) and a filter wheel, by which either a 565 nm LP (long-pass) filter (B) or a 660 nm LP filter (C) can be selected. For VSD imaging, the mirror (A) is removed and the LP filter (B) was inserted. Excitation light (505 nm center wavelength with 30 nm full width at half-maximum) from a collimated LED (LEDC9, Thorlab) was guided through a 535 nm/50 nm (center wavelength/width) BP (band-pass) filter (F), reflected by a LP filter (B) and focused on the specimen by an objective lens (Olympus XLUMPlanFl, 20×, NA = 0.95). Fluorescence emission was collected by the same objective lens and guided to the CCD camera (NeuroCCD-SM, Redshirt Imaging) through the LP filter (B), a 610 nm/70 nm BP filter (E), and an optical coupler. For calcium imaging, the mirror (A) and the LP filter (C) were selected and 780 nm excitation light from the Ti:Sapphire laser (Chameleon, Coherent) was guided by the mirror (A), through the LP filter (C) and focused on the specimen. Fluorescence emission was reflected by the LP filter (C) and separated into two channels by a 495 nm LP filter (D). A 460 nm/50 nm BP filter (G) and 607/45 nm BP filter (H) were placed in front of each PMT detector. PMT 2 detected the emission signals from the calcium indicator.