Deng, C. M. | Search bioRxiv

bioRxiv2026

Hydrogel Fiber Endomicroscopy

Multimode fibers enable minimally invasive, high-resolution imaging through ultrathin probes, thereby enhancing diagnostic precision and facilitating real-time monitoring in delicate anatomical regions. In this work, we introduce HYFEN, a hydrogel-based endomicroscopic imaging platform for flexible, biocompatible, and subcellular-scale fluorescence microscopy. HYFEN leverages the unique properties of hydrogel materials, adaptive optics, and pixel-wise image enhancement to address challenges associated with silica-based fibers, including mode scrambling, limited field of view, and mechanical rigidity. The technique achieves precise mode threading, rapid diffraction-limited focusing at kilohertz speeds, and high-fidelity fluorescence signal acquisition with subcellular resolution. Notably, the approach extends fluorescence imaging under enhanced fiber dimensions and bending conditions that are unachievable with conventional modalities. Together, these advances establish HYFEN as a versatile platform for next-generation biointerfacing and minimally invasive imaging across biomedical and clinical settings.

bioengineering↗

bioRxiv2026

An optogenetics-compatible red fluorescent calcium indicator with negligible blue light photoactivation

Red genetically encoded calcium indicators (GECIs) are important tools for live cell and in vivo imaging. However, their application in optogenetic experiments has been limited by their complex photophysics, which can yield blue-light-induced artifacts. These photophysical drawbacks arise from the fluorescent protein (FP) used to construct the GECI. To address these limitations, we engineered novel red GECIs based on photostable red FPs, including mScarlet variants. After testing multiple design topologies and screening for calcium responses, we identified a lead variant, named ScaRCaMP-1.0. ScaRCaMP-1.0 exhibits moderate Ca2+ responses ({Delta}F/F0 = -13%) relative to other red GECIs, a tradeoff that appears to have enabled remarkable blue-light photostability at power densities exceeding 200 mW/mm2. We validated the performance of ScaRCaMP-1.0 in an optogenetic regime, and in vivo via fiber photometry. Finally, guided by structural predictions, we investigated the mechanism underlying ScaRCaMP responses. A pair of lysine residues on the surface of the FP appear to be important for controlling Ca2+ responses, and mutation of one residue (K132Y) notably increased the response size ({Delta}F/F0 = -22%) without compromising blue-light photostability. We call the improved variant ScaRCaMP-2.0. Taken together, these results establish ScaRCaMP as an optogenetics-compatible red GECI and demonstrate the potential of mScarlet-based fluorophores as a basis for generating photostable red biosensors.

neuroscience↗