A Sensitive Soma-localized Red Fluorescent Calcium Indicator for Multi-Modality Imaging of Neuronal Populations In Vivo.

Recent advancements in genetically encoded calcium indicators, particularly those based on green fluorescent proteins, have optimized their performance for monitoring neuronal activities in a variety of model organisms. However, progress in developing red-shifted GECIs, despite their advantages over green indicators, has been slower, resulting in fewer options for end-users. In this study, we explored topological inversion and soma-targeting strategies, which are complementary to conventional mutagenesis, to re-engineer a red genetically encoded calcium indicator, FRCaMP, for enhanced performance.

Nanoscale Resolution Imaging of Whole Mouse Embryos Using Expansion Microscopy.

Nanoscale imaging of whole vertebrates is essential for the systematic understanding of human diseases, yet this goal has not yet been achieved. Expansion microscopy (ExM) is an attractive option for accomplishing this aim; however, the expansion of even mouse embryos at mid- and late-developmental stages, which have fewer calcified body parts than adult mice, is yet to be demonstrated due to the challenges of expanding calcified tissues. Here, we introduce a state-of-the-art ExM technique, termed whole-body ExM, that utilizes cyclic digestion.

In Vivo Optical Clearing of Mammalian Brain.

Established methods for imaging the living mammalian brain have, to date, taken optical properties of the tissue as fixed; we here demonstrate that it is possible to modify the optical properties of the brain itself to significantly enhance at-depth imaging while preserving native physiology. Using a small amount of any of several biocompatible materials to raise the refractive index of solutions superfusing the brain prior to imaging, we could increase several-fold the signals from the deepest cells normally visible and, under both one-photon and two-photon imaging, visualize cells previously too dim to see. The enhancement was observed for both anatomical and functional fluorescent reporters across a broad range of emission wavelengths.

In vivo whole-brain imaging of zebrafish larvae using three-dimensional fluorescence microscopy.

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy.