Abstract
Phase spatial light modulators (SLMs) are widely used for generating multifocal three-dimensional (3D) illumination patterns, but these are limited to a field of view constrained by the pixel count or size of the SLM. Further, with two-photon SLM-based excitation, increasing the number of focal spots penalizes the total signal linearly--requiring more laser power than is available or can be tolerated by the sample. Here we analyze and demonstrate a method of using galvanometer mirrors to time-sequentially reposition multiple 3D holograms, both extending the field of view and increasing the total time-averaged two-photon signal. We apply our approach to 3D two-photon in vivo neuronal calcium imaging.
Highlights
Many applications requiring three-dimensional (3D) patterned illumination use laser light shaped by phase-only spatial light modulators (SLMs), ranging from optical tweezing [1] to optogenetics [2]
SLMs are increasingly used with infrared femtosecond pulsed lasers to implement patterned two-photon excitation for either multisite neuronal imaging [3,4,5] or multisite photoactivation [6,7,8], to either record or manipulate neural activity, using calcium indicators or light sensitive opsins, respectively
We have addressed the field of view and two-photon signal limitations of holographic SLMbased illumination approaches by using a time-division multiplexing strategy to timesequentially tile a larger field of view
Summary
Many applications requiring three-dimensional (3D) patterned illumination use laser light shaped by phase-only spatial light modulators (SLMs), ranging from optical tweezing [1] to optogenetics [2]. SLMs are increasingly used with infrared femtosecond pulsed lasers to implement patterned two-photon excitation for either multisite neuronal imaging [3,4,5] or multisite photoactivation [6,7,8], to either record or manipulate neural activity, using calcium indicators or light sensitive opsins, respectively. These two-photon approaches afford deeper tissue penetration, crucial for in vivo applications, but are often laser power limited since the signal at each simultaneous site decreases quadratically instead of linearly with the total number of sites. We consider neuronal calcium imaging applications, where sites need only be sampled at approximately the Nyquist rate for a given calcium sensor [12], rather than simultaneously
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