Design and characterization of two-photon line excitation array detection (2p-LEAD) microscopy for monitoring in vivo neuronal activity

The functional meaning associated with neuronal activity in the mammalian brain and sensory systems remains to be fully understood. Exploring this area of neuroscience requires high-speed 3D imaging operating at >1 kHz volumetric scan rates with sub-cellular resolution, as neuronal signals propagate on sub-millisecond time scales. Additionally, since these studies must be performed in vivo, care must be taken to avoid invasive or damaging methods. Multi-photon imaging allows for non-invasive studies that deeply penetrate brain tissue, but has traditionally been limited to volumetric imaging between 10-100 Hz. We propose an improvement upon these systems with the novel imaging modality 2-photon Line Excitation and Array Detection (2p-LEAD) microscopy. 2p-LEAD is built on the main concept in our previous work where we developed single photon LEAD microscopy operating at 0.8 million FPS for 3D flow cytometry. In 2p-LEAD, we scan a 1035 nm excitation line of 2.4 mu m x 220 mu m (1/e(2) beam intensity diameter) at the focal plane. The resulting fluorescence is collected by a 16-channel linear PMT array. With a scanning mirror, we scan the line over a 140 mu m x 160 mu m FOV at 3,000 FPS, creating a frame of 16 x 320 pixels. Here we will present the design and imaging capabilities of our current 2p-LEAD instrument. This system lays the groundwork for higher speed imaging at 125 kHz frame rates with an acousto-optic deflector replacing the scanning mirror. When combined with vertical scanning, we will be able to volumetrically image at sub-millisecond time scales to allow for in vivo calcium imaging of the visual cortex.