Xiaowei Zhuang

Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here, we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 to 1000 distinct RNA species in hundreds of individual cells. Correlation analysis of the ~10(4) to 10(6) pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins.

Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.

Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.

The hypothalamus controls essential social behaviors and homeostatic functions. However, the cellular architecture of hypothalamic nuclei-including the molecular identity, spatial organization, and function of distinct cell types-is poorly understood. Here, we developed an imaging-based in situ cell-type identification and mapping method and combined it with single-cell RNA-sequencing to create a molecularly annotated and spatially resolved cell atlas of the mouse hypothalamic preoptic region. We profiled ~1 million cells, identified ~70 neuronal populations characterized by distinct neuromodulatory signatures and spatial organizations, and defined specific neuronal populations activated during social behaviors in male and female mice, providing a high-resolution framework for mechanistic investigation of behavior circuits. The approach described opens a new avenue for the construction of cell atlases in diverse tissues and organisms.