eprintid: 35912
rev_number: 16
eprint_status: archive
userid: 8703
dir: disk0/00/03/59/12
datestamp: 2025-01-21 14:45:07
lastmod: 2025-01-21 15:42:26
status_changed: 2025-01-21 14:45:07
type: doctoralThesis
metadata_visibility: show
creators_name: Cook, Alexander Stratford
title: Towards photoacoustic neuroimaging in mice: 
a chemigenetic approach for acoustogenic calcium sensors
subjects: ddc-500
subjects: ddc-540
subjects: ddc-570
divisions: i-140001
adv_faculty: af-14
cterms_swd: Photoacoustic imaging
cterms_swd: Calcium sensors
cterms_swd: Neuroimaging
abstract: Photoacoustic imaging is an attractive imaging modality for in vivo applications such as neuroimaging, able to 
image large fields of view, deep in tissue, at high resolution. However, robust molecular reporters are required 
for this modality, to label specific targets of interest and visualise dynamic biochemical events. Various 
genetically-encoded or synthetic reporters have been implemented, but suffer from poor optical properties or 
lack of labelling specificity. Here, we report the development of novel “chemigenetic” contrast agents and 
calcium sensors for photoacoustic imaging, based on synthetic dyes and the self-labelling HaloTag protein.
I designed and synthesised new photoacoustic reporters based on quenched near-infrared rhodamine-like
derivatives, tailored for use with HaloTag to exploit the open-closed equilibrium of the lactone ring. These were 
characterised in vitro for high photoacoustic signal and turn-on upon binding to HaloTag. The most promising 
ligands were used for the design of calcium indicators, with the HaloTag-based HaloCaMP protein, which show 
an absorption-modulated change in calcium-dependent photoacoustic signal. Cell permeability of these ligands 
was confirmed by efficient labelling of live cells cytosolically expressing HaloTag. Our resulting 
“acoustogenic” calcium sensors show large turn-ons in response to calcium, up to 8-fold, high photoacoustic 
signal and photostability, outperforming existing far-red sensors for this modality such as NIR-GECO1 in 
tissue-mimicking phantoms. Ex vivo brain slices with neuronal expression of HaloTag, labelled with our ligands, 
could be consistently visualised via photoacoustic tomography. In vivo labelling experiments in mice, however, 
showed mixed results with the occasional specific labelling of HaloTag-expressing neurons suggesting that the 
bioavailability of our ligands needs to be improved.
In summary, we developed a novel approach for the design of photoacoustic reporters, with the first 
chemigenetic probes for this modality. Our acoustogenic dyes and first-generation photoacoustic calcium 
sensors, show superior in vitro performance to existing sensors, and we can label mice brain tissues to produce 
a localised, strong PA signal. To reach our long-term goal of whole-brain neuroimaging in mice, we have started 
protein engineering to improve the dynamic range, calcium binding affinity, and used established methods to 
assess dye bioavailability to optimise this key parameter for future in vivo applications. In general, with this 
hybrid design, we hope to stimulate photoacoustic probe and sensor development which can enable this 
powerful modality to uncover its true potential in the field of neuroimaging and beyond.
date: 2025
id_scheme: DOI
id_number: 10.11588/heidok.00035912
ppn_swb: 1915267005
own_urn: urn:nbn:de:bsz:16-heidok-359129
date_accepted: 2024-12-04
advisor: HASH(0x5565eac8c098)
language: eng
bibsort: COOKALEXANTOWARDSPHO20241007
full_text_status: public
place_of_pub: Heidelberg
citation:   Cook, Alexander Stratford  (2025) Towards photoacoustic neuroimaging in mice: a chemigenetic approach for acoustogenic calcium sensors.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/35912/1/Alexander_Cook_PhD_thesis_final.pdf