Summary
In conclusion, we have explored methods that exploit the idea of using diffraction to optimize two-photon microscopy. We have used static DOE as well as active, computer-controlled SLM to dynamically control the distribution of excitation light on the sample. While these multibeam methods are currently limited in applications with large power requirements, we have proven their practicality and functionality. Not only are they currently useful in non-power-limited settings but stand to be equally useful in power-limited applications when more powerful lasers become available. We have devised several novel experimental schemes for improving either speed and/or sensitivity of imaging, specifically applicable to non-linear (two-photon) modalities. The use of computer-controlled phase-only SLM also allows for photostimulation of multiple regions of the sample while largely preserving excitation power. In addition, a newly developed imaging technique relies on SLM to continuously illuminate preselected regions of the sample acquire time lapse signals from those regions faster than video rate. The proposed and demonstrated techniques entail insertion of simple optical elements into a two-photon imaging rig and thus can be easily and inexpensively implemented and added to existing experimental equipment, including commercial and custom two-photon microscopes.