Connecting the neurons: how PISCES connects morphology, function, and gene expression
By Octavia Santis Larrain
The ability to label and track specific cells in vivo using cell-type restricted transgenes and photoconvertible proteins has been broadly used in zebrafish researchSingle-cell RNA sequencing (scRNA-seq) has increased the molecular expression data we have per cell, and spatial transcriptomics have added spatial context to this data, however the high costs of sequencing, the need for cell sorting, and the limitations of fixed samples restrict the utility of these methods for in vivo applications. Crucially, there is still no good technique for integrating morphology, function, and molecular identity within a single, arbitrarily selected cell. Researchers in the lab of Jiu-Lin Du at the Chinese Academy of Sciences in Shanghai, China address this limitation by developing a labeling technique called ‘PISCES' (Photo-inducible Single-Cell Labeling; Tao et al., 2025.)
With calcium-signaling transgenic lines and scRNA-seq, we can monitor neuronal activity and determine the brain transcriptome with unprecedented detail (Zeng, 2022). However, integrating this functional and molecular data with specific, individual neurons within the complex networks of the brain and spinal cord remains challenging. While photoconvertible proteins have been essential for labeling single cells, the morphology of neurons, characterized by long and complex axons, often makes complete cell labeling difficult. Therefore, there is a need for non-invasive, single-neuron labeling techniques compatible with high-throughput sequencing that integrate function, morphology, and gene expression across the nervous system.
To overcome these barriers, the authors developed a Photo-inducible Single-Cell Labeling, or ‘PISCES’ (Tao et al., 2025). A system that enables the rapid visualization of a single neuron's entire morphology. This technique utilizes a fusion of a photo-cleavable protein (PhoCl) (Zhang et al., 2017) and a photoconvertible protein, mMaple (McEvoy et al., 2012), in combination with Nuclear Localization (NLS) and Nuclear Export (NES) signals, allowing the protein to be translocated into the axon. Upon a brief 10-second light pulse, the proteins undergo photoconversion, transitioning from green to red, while PhoCl cleavage triggers the rapid translocation of mMaple from the nucleus to the soma. This results in a high-speed diffusion rate of approximately 1.02 +/- 0.06 feel um/s in 6 dpf larvae that remains stable for 16 hours, allowing the fluorescent signal to fill even the most distant axonal projections in record time.
Du and coworkers demonstrated the utility of the PISCES technique by mapping different neuron types. They successfully labeled long-range axons from habenular (Hb) neurons and traced the brain-wide projections of norepinephrine (NE) neurons. Also, the technique allowed the researchers to target and resolve neurons with cell bodies deep in the ventral regions of the brain. PISCES permitted the team to identify specific neurons and trace their extensive networks across multiple brain regions.
The most interesting application of this technology is when the authors combined PISCES with calcium imaging reporters, which allowed them to complement detailed morphological reconstructions with real-time functional activation data. In addition, PISCES was used to label specific neurons for transcriptomic profiling. By sorting PISCES-labeled cells for single-cell sequencing, the authors were able to identify molecular heterogeneity in neurons across the left habenula (lHb), right habenula (rHb), and midbrain. These results were further corroborated by combining PISCES with Fluorescent In Situ Hybridization (FISH), demonstrating how this method can integrate data on a neuron's physiological role and genetic identity.
Although this is an interesting imaging technique, PISCES is in its early stages. The system has yet to be validated in other model organisms like Drosophila or mice. Currently, its compatibility with two-photon microscopy remains a challenge, potentially due to mMaple's low activation efficiency (Tao et al., 2025) and/or its susceptibility to reversible photo-switching (McEvoy et al., 2012). Additionally, labeling is unidirectional: while the signal diffuses from the nucleus to reveal the neuron's full morphology, it cannot yet perform retrograde tracing from distal projections back to the soma. Nevertheless, PISCES represents an interesting technique, offering a vital new bridge between morphological data and molecular and functional data of the brain.
About the authors:
Dr. Du’s laboratory is located at the Institute of Neuroscience, within the Center for Excellence in Brain Science and Intelligence Technology (CEBSIT) of the Chinese Academy of Sciences in Shanghai. They are interested in Neuromodulation, especially in visual processing, and visual and auditory integration can modify behavior. His lab has worked with multiple techniques, including whole cell recording, calcium imaging, optogenetics, pharmacology, and behavioral assays.
About the science writer:
Octavia Santis Larrain is a PhD student in Cell and Molecular Pathology at the University of Wisconsin-Madison. A passionate zebrafish researcher, she has spent her career using this unique model across various laboratories in Chile, Spain, and the US. Octavia is very interested in microscopy and views the zebrafish model as an incredible platform to develop innovative imaging techniques. Her current research investigates the niche interactions of Hematopoietic Stem Cells within the kidney marrow, with a specific focus on how neurotransmitters influence stem cell behavior. Outside the lab, she loves reading novels and spending time with her friends, family, and cats.
REFRENCE:
McEvoy, A. L., Hoi, H., Bates, M., Platonova, E., Cranfill, P. J., Baird, M. A., Davidson, M. W., Ewers, H., Liphardt, J., & Campbell, R. E. (2012). mMaple: a photoconvertible fluorescent protein for use in multiple imaging modalities. PLoS One, 7(12), e51314. https://doi.org/10.1371/journal.pone.0051314
Tao, R. K., Sun, L., Qian, Y., Huang, Y. M., Chen, Y. H., Guan, C. Y., Wang, M. C., Sun, Y. D., & Du, J. L. (2025). Designed optogenetic tool for bridging single-neuronal multimodal information in intact animals. Nat Commun, 16(1), 7764. https://doi.org/10.1038/s41467-025-62938-w
Zeng, H. (2022). What is a cell type and how to define it? Cell, 185(15), 2739-2755. https://doi.org/10.1016/j.cell.2022.06.031
Zhang, W., Lohman, A. W., Zhuravlova, Y., Lu, X., Wiens, M. D., Hoi, H., Yaganoglu, S., Mohr, M. A., Kitova, E. N., Klassen, J. S., Pantazis, P., Thompson, R. J., & Campbell, R. E. (2017). Optogenetic control with a photocleavable protein, PhoCl. Nat Methods, 14(4), 391-394. https://doi.org/10.1038/nmeth.4222