This research focuses on the development of a non-invasive/minimally invasive optogenetic technique. The study delves into how visible (VIS) and near-infrared (NIR) light interacts with ex vivo mouse head tissues, highlighting the advantages of the NIR-II biological window for deeper tissue penetration and reduced light absorption and scattering. Our computer simulations and experimental results demonstrated that over 12% of initial light irradiation passes through 1 mm tissue (skin and skull), reaching the brain cortex, potentially enabling minimally invasive neural activation. Moreover, this work reveals the nonlinear optical properties of genetically engineered truncated monomeric and dimeric bacterial phytochromes, demonstrating their photoconversion efficiency of up to 73% in the NIR-II range and potential for optogenetics. This discovery opens new avenues in advanced neurostimulation and biomedical research by enhancing tissue penetration and minimizing invasiveness.
Current medicine might be greatly enhanced by the ability to in vivo control and monitor neurons using opsins/phytochromes expressed in neural cells. The fundamental challenge with non-invasive neural cell activity regulation is a high absorption of visible light into biological tissues. This drawback could be mitigated by the photoconversion of phytochromes in spectral ranges with higher tissue transparency. In this study, we first demonstrated two-photon Pr→Pfr conversion of monomeric phytochrome at 1.2 μm wavelength. We did a comparison of linear and nonlinear conversion of truncated DrBphP bacterial phytochromes. This work provides a structured understanding of the optical properties of the dimer and monomer of phytochrome as well as their potential for use in optogenetics.
Genetically encoded probes with absorbance and fluorescence spectra within a near-infrared tissue transparency window are preferable for deep-tissue imaging. On the basis of bacterial phytochromes we engineered several types of near-infrared absorbing probes for photoacoustic tomography and fluorescent probes for purely optical imaging. They can be used as protein and cell labels and as building blocks for biosensors. The probes enabled imaging of tumors and metastases, protein-protein interactions, RNA visualization, detection of apoptosis, cellular metabolites, signaling pathways and cell proliferation. The developed probes allow non-invasive visualization of biological processes across scales, from super-resolution microscopy to tissue and whole-body animal imaging.
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