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Miniature two-photon microscopes, such as MINI2P, are powerful tools to monitor the activity of brain cells in freely moving animals. To image deep in the brain, the MINI2P can be coupled to gradient index (GRIN) lenses, which are implanted in the tissue and relay the imaging beam to the target region of interest. However, GRIN lenses have intrinsic optical aberrations, which severely degrade imaging spatial resolution especially in lateral portions of the field-of-view (FOV) leading to restricted imaging FOV. Here, we tested the hypothesis that coupling MINI2P with aberration corrected GRIN lenses results in improved spatial resolution and extended FOV during two-photon fluorescence imaging. We coupled the MINI2P with a cylindrical GRIN lens (GRIN length, 4.07 mm; GRIN cross section, 0.5 mm), in which we corrected aberrations using a micro-fabricated polymer lens. We found that the axial dimension of both the on-axis and off-axis point-spread-function was significantly improved in MINI2P coupled with corrected GRIN lenses compared to MINI2P coupled with uncorrected GRIN lenses. Moreover, using corrected GRIN lenses enabled MINI2P imaging with > 3-fold larger FOV radius compared to using uncorrected GRIN lenses. We confirmed extended FOV imaging when MINI2P was coupled to corrected GRIN lenses by performing two-photon imaging of thalamic neurons expressing a fluorescent indicator in fixed brain tissue.
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Emerging Trends in Monitoring and Stimulating Brain Function
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.
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Real-time monitoring of tissue oxygenation within the nervous system is imperative for advancements in neuroscience research and the improvement of clinical diagnostics. Unlike blood oxygenation levels, the partial pressure of oxygen in brain tissue (PbtO2) offers a more direct insight into the localized neural activities and metabolic states. Here, we present a microscale optoelectronic probe for the wireless, real-time monitoring of in vivo partial brain tissue oxygen (PbtO2) levels. This probe measures local PbtO2 concentrations via the luminescent quenching mechanism of phosphorescent dyes. An integrated light-emitting diode (LED) and photodetector are used to generate and capture the optical signals. To facilitate capturing and wirelessly transmitting PbtO2 signals, the device includes miniaturized electronic circuits that can be powered by a battery or an inductive coil. In vitro and in vivo experiments demonstrate the ability to dynamically record oxygen partial pressure (pO2), offering novel exploration opportunities in neuroscience research and clinical applications.
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Plasmonic enhancement of fluorescence has been challenging in in vivo imaging applications. We present a study demonstrating the plasmonic enhancement of fluorescent membrane proteins within their native physiological environment using tailored metallic nanoparticles. This work highlights two schemes to influence the distance between the emitting dipoles and the enhancing nanoparticles, namely the addition of nanoparticles in the buffer solution and the incorporation in the polymer matrix at the bottom of the cells. Incorporating biological structures native to the cellular environment offers opportunities for the optimization of in vivo fluorescence imaging methods and the detection of membrane proteins.
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The emerging mid-infrared photoacoustic microscopy (MIR-PAM) is a potential imaging modality in revealing special biomolecules compositions in thick samples by utilizing the light-excited ultrasound signals. The development of a nanosecond and high-energy MIR fiber laser source is still at an early age, facing the challenges of either low peak power or large footprint. This work aims to develop a new Raman laser source for MIR-PAM based on the gas-filled anti-resonant hollow core fiber (ARHCF) technology. As a proof of concept, a MIR laser source at 3.4 μm is developed and combined with PAM for the first time targeting at the lipid-rich mouse brain sample due to main absorption band of myelin sheaths. This laser source is based on the cascading of two ARHCFs, where a high-energy (~26.5 μJ) Raman Stokes line at 1409 nm is generated through the 1st-stage nitrogen-filled ARHCF with a pump fiber laser at 1060 nm. The output Raman laser from the 1st stage ARHCF is used as a pump for the 2nd-stage hydrogen-filled ARHCF, to generate the Raman laser at 3.4 μm with ~2.7 μJ pulse energy. Our label-free ex-vivo imaging depicted the lipid-rich myelin region in the mouse brain, showing the feasibility of extending the novel gas-filled laser platform into PAM imaging modalities.
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