Photoacoustic tomography of calcium activity in the mouse brain could potentially provide whole-brain coverage of neural activity and therefore offer new insights into brain function. Here we report the development and characterization of a novel photoacoustic calcium-sensitive probe based on the HaloTag protein which is suitable for in vivo imaging in mice. The photoacoustic brightness and signal enhancement upon calcium binding were measured using a custom-built spectroscopy setup and compared to the available far-red calcium indicator NIR-GECO1. Additionally, we conducted validation experiments on tissue-mimicking phantoms using a Fabry-Perot-based photoacoustic tomography setup to determine the depth limit and concentration detection threshold of the imaged probes. Furthermore, we tested various in vivo delivery methods, by analyzing brain slices from mice labeled with the photoacoustic probe. These experiments demonstrated that we are able to specifically label neurons targeted brain regions, confirming the suitability of these photoacoustic probes for in vivo calcium imaging applications.
In order to study dynamic biological processes in-vivo in mammalian organisms techniques are required which enable non-invasive imaging at large tissue depth with sub-cellular resolution. However, optical aberrations and scattering in biological tissue lead to signal loss and a degradation of both spatial resolution and penetration depth. Here, we combine two powerful optical techniques, multi-photon microscopy and adaptive optics, to push the depth limit further while retaining diffraction limited resolution. We apply these techniques to open questions in the field of neuron and glia biology. By utilizing three-photon excitation at the 1300 nm spectral excitation window we achieve highresolution imaging of GFP-labeled neurons up to a depth of 1.2 mm in the in-vivo mouse brain. Furthermore, we have combined our approach with indirect modal-based wavefront correction and synchronization of our microscope to the animal’s heart pulsation. This allowed us to improve the resolution up to ~6-fold and achieve synaptic resolution throughout an entire cortical column. With adaptive optics correction, small structures such a dendritic branches thus become clearly visible at over 1mm depth in the hippocampus. Furthermore, our adaptive three-photon microscope system enabled, for the first time, to investigate calcium dynamics of fibrous astrocytes which reside in the corpus callosum and whose dynamics have previously not been possible to image.
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