PN-ImTLSM facilitates high-throughput low background single-molecule localization microscopy deep in the cell.

When imaging the nucleus structure of a cell, the out-of-focus fluorescence acts as background and hinders the detection of weak signals. Light-sheet fluorescence microscopy (LSFM) is a wide-field imaging approach which has the best of both background removal and imaging speed. However, the commonly adopted orthogonal excitation/detection scheme is hard to be applied to single-cell imaging due to steric hindrance. For LSFMs capable of high spatiotemporal single-cell imaging, the complex instrument design and operation largely limit their throughput of data collection. Here, we propose an approach for high-throughput background-free fluorescence imaging of single cells facilitated by the Immersion Tilted Light Sheet Microscopy (ImTLSM). ImTLSM is based on a light-sheet projected off the optical axis of a water immersion objective. With the illumination objective and the detection objective placed opposingly, ImTLSM can rapidly patrol and optically section multiple individual cells while maintaining single-molecule detection sensitivity and resolution. Further, the simplicity and robustness of ImTLSM in operation and maintenance enables high-throughput image collection to establish background removal datasets for deep learning. Using a deep learning model to train the mapping from epi-illumination images to ImTLSM illumination images, namely PN-ImTLSM, we demonstrated cross-modality fluorescence imaging, transforming the epi-illumination image to approach the background removal performance obtained with ImTLSM. We demonstrated that PN-ImTLSM can be generalized to large-field homogeneous illumination imaging, thereby further improving the imaging throughput. In addition, compared to commonly used background removal methods, PN-ImTLSM showed much better performance for areas where the background intensity changes sharply in space, facilitating high-density single-molecule localization microscopy. In summary, PN-ImTLSM paves the way for background-free fluorescence imaging on ordinary inverted microscopes.

MRGPRX4 is a bile acid receptor for human cholestatic itch.

Patients with liver diseases often suffer from chronic itch, yet the pruritogen(s) and receptor(s) remain largely elusive. Here, we identify bile acids as natural ligands for MRGPRX4. MRGPRX4 is expressed in human dorsal root ganglion (hDRG) neurons and co-expresses with itch receptor HRH1. Bile acids elicited Ca2+ responses in cultured hDRG neurons, and bile acids or a MRGPRX4 specific agonist induced itch in human subjects. However, a specific agonist for another bile acid receptor TGR5 failed to induce itch in human subjects and we find that human TGR5 is not expressed in hDRG neurons. Finally, we show positive correlation between cholestatic itch and plasma bile acids level in itchy patients and the elevated bile acids is sufficient to activate MRGPRX4. Taken together, our data strongly suggest that MRGPRX4 is a novel bile acid receptor that likely underlies cholestatic itch in human, providing a promising new drug target for anti-itch therapies.