High-speed, large field-of-view and deep imaging with an adaptive excitation source

Conventional multiphoton microscopy uses periodically pulsed sources as excitation and the sample is illuminated uniformly by the laser. While necessary for structural imaging, monitoring dynamic biological functions such as neuronal activity in the brain typically only requires imaging of the region of interest (ROI), e.g., the neurons. The adaptive excitation source enables imaging of the region of interest only. It reduces the requirement for the output power of the excitation source (by at least an order of magnitude) and simultaneously reduces the excitation power to the sample for obtaining the necessary information (e.g., neuronal activity). We demonstrate three-photon imaging of brain activity in awake transgenic mice (jRGECO1a), with highest speed (30 frames/s), large field-of-view (620x620 μm/512x512 pixels) and deep penetration (750 μm beneath the dura).

[1]  Bo Li,et al.  An adaptive excitation source for high-speed multiphoton microscopy , 2019, Nature Methods.

[2]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[3]  Andreas S Tolias,et al.  In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain , 2017, Nature Methods.

[4]  Timothy D. Weber,et al.  Neuronal imaging with ultrahigh dynamic range multiphoton microscopy , 2017, Scientific Reports.

[5]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[6]  Michael Häusser,et al.  Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo , 2014, Nature Methods.

[7]  David Pfau,et al.  Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data , 2016, Neuron.

[8]  K. Charan,et al.  Investigation of the long wavelength limit of soliton self-frequency shift in a silica fiber. , 2018, Optics express.

[9]  Kaspar Podgorski,et al.  Brain heating induced by near infrared lasers during multi-photon microscopy , 2016, bioRxiv.

[10]  Stefan R. Pulver,et al.  Ultra-sensitive fluorescent proteins for imaging neuronal activity , 2013, Nature.

[11]  F. Wise,et al.  In vivo three-photon microscopy of subcortical structures within an intact mouse brain , 2012, Nature Photonics.

[12]  A. Gordus,et al.  Sensitive red protein calcium indicators for imaging neural activity , 2016, bioRxiv.

[13]  E. Isacoff,et al.  Scanless two-photon excitation of channelrhodopsin-2 , 2010, Nature Methods.