Biology subjects

Murakami, T. C.

Publications and source records attributed to Murakami, T. C..

3 recordsLinked to original sources

bioRxiv2025

Open-source Photobleacher for Fluorescent Imaging of Large Pigment-Rich Tissues

Fluorescent imaging enables visualization of the specific molecules of interest with high contrast, and the use of multiple fluorophores in a single tissue sample allows visualization of complex relationships between biological molecules, cell types, and anatomy. The utility of fluorescent imaging in human tissue has been limited by endogenous pigments that can block the light path or emit an autofluorescence, thereby interfering with the specific imaging of target molecules. Although photobleachers have been developed to quench endogenous pigments, the lack of customizability limits their utility for a broad range of applications. Here, we present a high luminous-intensity photobleacher that is based on rigorous simulations of illumination patterns using the laws of radiation, along with the framework to maximize bleaching efficiency. This open-source project is designed to help researchers customize and scale according to the tissue types and the research goals. The photobleacher is applicable to both thin tissue slices and large-volume cleared tissue samples to enable serial three-dimensional imaging of postmortem human brain using multiplexed antibody or oligonucleotide probes. SIGNIFICANCE STATEMENTPhotobleaching is an effective technique for quenching endogenous pigments, enabling multiplexed fluorescent imaging of pigment-rich tissues, such as postmortem human samples. While many photobleaching strategies have been proposed, there is no standard guidance on how to design and use a photobleacher. This study introduces a general strategy for designing an effective, scalable, and customizable photobleacher, and proposes a workflow for properly treating tissues with the photobleacher. The technique enables high-contrast molecular visualization in tissues of various sizes, including large volumetric cleared tissues. Our framework will accelerate the quantitative understanding of human molecular anatomy and is applicable to diverse biological fields, including medical diagnostics.

neuroscience↗

bioRxiv2024

Spontaneously regenerative corticospinal neurons in mice

The spinal cord receives inputs from the cortex via corticospinal neurons (CSNs). While predominantly a contralateral projection, a less-investigated minority of its axons terminate in the ipsilateral spinal cord. We analyzed the spatial and molecular properties of these ipsilateral axons and their post-synaptic targets in mice and found they project primarily to the ventral horn, including directly to motor neurons. Barcode-based reconstruction of the ipsilateral axons revealed a class of primarily bilaterally-projecting CSNs with a distinct cortical distribution. The molecular properties of these ipsilaterally-projecting CSNs (IP-CSNs) are strikingly similar to the previously described molecular signature of embryonic-like regenerating CSNs. Finally, we show that IP-CSNs are spontaneously regenerative after spinal cord injury. The discovery of a class of spontaneously regenerative CSNs may prove valuable to the study of spinal cord injury. Additionally, this work suggests that the retention of juvenile-like characteristics may be a widespread phenomenon in adult nervous systems.

neuroscience↗

bioRxiv2022

Multiplexed and scalable cellular phenotyping toward the standardized three-dimensional human neuroanatomy

The advent of three-dimensional histological methods has advanced studies of cellular-resolution anatomy of the brain. The use of whole-mount staining and tissue clearing has advanced systems-level identification of cells underlying brain functions in mouse models. However, application of these methods to studies of human brains has been difficult due to their structural variability and the lack of standardized quantitative metrics. Here we report a rapid and scalable staining/imaging technique, termed mFISH3D, that enables single-cell-resolution imaging of mRNAs of more than ten genes in a large mammalian brain. To apply mFISH3D to postmortem human cerebral cortex, we have reconstructed morphogenic tracks of cortical growth, and used the tracks to provide a framework for quantitative assessment of cytoarchitecture. The workflow enabled the objective quantification of biological heterogeneity among cortical regions. We propose these techniques for standardization of 3D histology of the human cortex to provide reproducible measurements of cell-type-specific neuroanatomy.

neuroscience↗