Confocal and multiphoton microscopy for imaging at depth in living tissue

The desire to image with sub micron resolution at ever increasing depths into living samples is providing optical physicists with the latest in a long line of challenges presented by life science researchers. The advent of confocal, and subsequently multiphoton microscopy, has opened up exciting new possibilities but simultaneously posed new challenges. As one images ever more deeply into the sample, the optical properties of the tissue distort the image significantly lowering the resolution and, in the case of multiphoton imaging in particular, decreasing the fluorescence yield as the excitation volume rises. The recent use of active optical elements has shown a way forward in restoring high contrast high resolution images at depth. However, significant issues on the actual shape required on such an element are as yet unresolved. We report on two recent advances in this area. The first is the use of a range of optimisation algorithms to restore the optical point spread function and hence improve the image quality at depth. The second is a radically new approach incorporating two active elements, a slow spatial light modulator and a fast deformable mirror, to actively lock up the system. We report on the latest advances in active image compensation where conections at over 5OOmicrons into the sample have been made using a combination of deformable mirrors and spatial light modulators