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Ultrafast optical clearing method for three-dimensional imaging with cellular resolution.

Xinpei Zhu1, Limeng Huang1, Yao Zheng1,2

  • 1Center for Neuroscience and Department of Neurobiology of the Second Affiliated Hospital, State Key laboratory of Modern Optical Instrumentation, Zhejiang University School of Medicine, Hangzhou 310058, China.

Proceedings of the National Academy of Sciences of the United States of America
|May 19, 2019
PubMed
Summary
This summary is machine-generated.

We developed an ultrafast optical clearing method (FOCM) for rapid, high-quality biological imaging. This method preserves tissue structure and fluorescence, overcoming limitations of conventional techniques for advanced microscopy applications.

Keywords:
deep tissue imagingoptical clearingtissue clearing

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Area of Science:

  • Biomedical Imaging
  • Microscopy Techniques
  • Neuroscience Research

Background:

  • Conventional optical clearing methods for microscopy face limitations including time consumption, tissue deformation, and fluorescence quenching.
  • These limitations hinder efficient and high-fidelity imaging of biological samples, particularly in neuroscience.
  • There is a need for advanced optical clearing techniques that are rapid, preserve tissue integrity, and maintain signal quality.

Purpose of the Study:

  • To develop and validate an ultrafast optical clearing method (FOCM) that overcomes the drawbacks of existing techniques.
  • To assess the speed, tissue preservation, and fluorescence retention capabilities of FOCM.
  • To demonstrate the utility of FOCM in detailed 3D neuroimaging and analysis of stimulated neural activity.

Main Methods:

  • Development of a novel ultrafast optical clearing method (FOCM) using simple protocols and common reagents.
  • Application of FOCM to 300-µm-thick brain slices, evaluating clarification time.
  • Quantification of tissue linear expansion and preservation of Green Fluorescent Protein (GFP) fluorescence over time.
  • Utilizing FOCM for 3D reconstruction of neural circuits and analysis of c-fos expression patterns in response to stimuli.

Main Results:

  • FOCM achieved rapid optical clearing of 300-µm brain slices in just 2 minutes.
  • Tissue linear expansion was minimal (2.12% increase), and GFP fluorescence was preserved up to 86% after 11 days.
  • Detailed 3D models of nerve cells, including neuron-astrocyte-blood vessel connections, were successfully constructed.
  • Distinct c-fos expression patterns were observed in the paraventricular nucleus of the hypothalamus (PVH) following foot shock and morphine stimulation.

Conclusions:

  • FOCM offers a significant advancement in optical clearing, providing rapid clarification with excellent tissue and fluorescence preservation.
  • The method enables high-resolution 3D neuroimaging and analysis of neural activity patterns.
  • FOCM presents a promising and versatile sample mounting medium for broad applications in biological optical imaging.