Widefield multiphoton imaging at depth with temporal focusing

Optical imaging has the potential to reveal high-resolution information with minimal photodamage. The recent renaissance of super-resolution, widefield, ultrafast, and computational imaging methods has broadened its horizons even further. However, a remaining grand challenge is imaging at depth over a widefield and with a high spatiotemporal resolution. This achievement would enable the observation of fast collective biological processes, particularly those underpinning neuroscience and developmental biology. Multiphoton imaging at depth, combining temporal focusing and single-pixel detection, is an emerging avenue to address this challenge. The novel physics and computational methods driving this approach offer great potential for future advances. This chapter articulates the theories of temporal focusing and single-pixel detection and details the specific approach of TempoRAl Focusing microscopy with single-pIXel detection (TRAFIX), with a particular focus on its current practical implementation and future prospects.