%0 Generic
%A Kaydanov, Nikita
%C Heidelberg
%D 2026
%F heidok:36518
%R 10.11588/heidok.00036518
%T Towards photoacoustic neuroimaging in mice: development and validation of novel calcium sensors with tailored photoacoustic instrumentation
%U https://archiv.ub.uni-heidelberg.de/volltextserver/36518/
%X Recording the activity of large neuronal populations is crucial for understanding brain function but is  constrained by current technologies. Multiphoton microscopy (MPM) with calcium-sensitive  fluorescence sensors offers sub-cellular imaging of neuronal activity but is limited to ~1 mm depth  and a narrow field of view. Functional magnetic resonance imaging (fMRI), despite whole-brain  coverage, uses blood-oxygenation as a neural activity proxy and suffers from poor spatial (~0.4 mm)  and temporal (~1 sec) resolution.    Photoacoustic imaging (PAI) uniquely combines the molecular contrast of light absorbance with  ultrasound imaging, thereby achieving ~cm penetration and bridging the scales of MPM and fMRI.  Combined with calcium probes tailored for photoacoustic emission, PAI could enable brain-wide  calcium imaging, advancing neurobiological research. However, suitable far-red (600–700 nm)  calcium reporters are currently unavailable.    This thesis presents advancements toward whole-brain neuroimaging using photoacoustic methods.  A custom Fabry-Perot-based Photoacoustic Tomography system (FP-PAT) was developed, enabling 3D  brain tissue imaging with a 15*15*15 mm³ field of view and ~90 μm spatial resolution. System  improvements in speed and sensitivity are introduced to make it suitable for calcium imaging  applications.    Novel calcium photoacoustic reporters based on synthetic dyes and HaloTag-based self-labeling  proteins were developed in collaboration with the Deo lab at EMBL. A multimodal spectroscopy  platform combining photoacoustic, absorption, and fluorescence readouts was designed to screen  and characterize these probes. Selected probes were then tested in tissue-mimicking phantoms with  FP-PAT, demonstrating superior performance in signal intensity, sensitivity, and photostability  compared to existing calcium sensors.    Next, we demonstrated successful in vivo labelling and PA imaging of mouse brain slices, thereby  showing the feasibility of using HaloTag-based probes to label neurons and detect the signal with FP-  PAT. Moreover, preliminary data on a calcium-sensitive version of the probe, HaloCaMP, shows  successfully labeled mouse brain slices with a detectable change in signal after incubation in calcium  buffer.    Finally, acute slice preparation and PAI were used to assess calcium sensors in live tissue stimulated  with high potassium. Proof-of-concept data for the fluorescent calcium indicator jGCaMP8m showed  detectable PA signal changes upon stimulation. However, the HaloCaMP probe did not yet  demonstrate calcium-mediated signal changes, likely due to low calcium affinity and brightness of  this first-generation sensor.    This work introduces a novel class of far-red photoacoustic calcium reporters with superior  performance, providing critical spectroscopic characterization and methods for labeling mouse brain  tissue. While challenges remain for realistic in vivo applications, this research establishes a  foundation for developing photoacoustic neuroimaging with tailored calcium sensors, paving the way  for future in vivo brain-wide calcium imaging.