Abstract
Simultaneous, high-resolution imaging across a large number of synaptic and dendritic sites is critical for understanding how neurons receive and integrate signals. Yet, functional imaging that targets a large number of submicrometer-sized synaptic and dendritic locations poses significant technical challenges. We demonstrate a new parallelized approach to address such questions, increasing the signal-to-noise ratio by an order of magnitude compared to previous approaches. This selective access multifocal multiphoton microscopy uses a spatial light modulator to generate multifocal excitation in three dimensions (3D) and a Gaussian-Laguerre phase plate to simultaneously detect fluorescence from these spots throughout the volume. We test the performance of this system by simultaneously recording Ca2+ dynamics from cultured neurons at 98-118 locations distributed throughout a 3D volume. This is the first demonstration of 3D imaging in a "single shot" and permits synchronized monitoring of signal propagation across multiple different dendrites.
Highlights
Synaptic transmission between neurons is the most basic unit of information flow in a neural circuit
The P-polarized beam is phase-modulated by an spatial light modulator (SLM) (PLUTO-NIR-HR phase-only reflective SLM, HOLOEYE Photonics AG, Germany) that is placed in a conjugate position to the back aperture of the objective (XLUMPlanFL, 20 ×, 0.95 NA, Olympus), projecting holographic patterns that generate excitation foci defined by the region of interest (ROI)
This enables out-of-focus fluorescence to be more efficiently captured by the camera, since the Gaussian point spread function (PSF) without GL phase encoding has a much shorter depth of field (DoF) [Figs. 2(b)–2(d)]
Summary
Synaptic transmission between neurons is the most basic unit of information flow in a neural circuit. Pyramidal neuron dendritic arbor can occupy a volume in the range of 1–5 × 106 μm, depending on depth [4], with synaptic sites on the scale of a few micrometers distributed across the arbor [5]. Tackling this class of neurobiological questions requires high-speed methods for detecting activity at synaptic resolution across many distributed sites. The image signal-to-noise ratio (SNR) typically suffers due to the short signal integration time at each voxel
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
