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Related Experiment Video

Updated: Nov 19, 2025

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors
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In vivo NIR-II structured-illumination light-sheet microscopy.

Feifei Wang1,2, Zhuoran Ma1,2, Yeteng Zhong1,2

  • 1Department of Chemistry, Stanford University, Stanford, CA 94305.

Proceedings of the National Academy of Sciences of the United States of America
|February 2, 2021
PubMed
Summary

We developed near-infrared II structured-illumination microscopy (NIR-II SIM) for deep tissue imaging. This advanced technique allows noninvasive, high-resolution, 3D molecular mapping of immune cells in live mammals.

Keywords:
light-sheet microscopenear-infrared II imagingnoninvasive imagingstructured-illumination microscopy

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Area of Science:

  • Biomedical optics
  • In vivo imaging
  • Molecular imaging

Background:

  • Deep tissue optical imaging is crucial for studying live mammals but limited by light scattering.
  • High spatiotemporal resolution is needed for single-cell level longitudinal studies.

Purpose of the Study:

  • To develop a noninvasive optical imaging technique with deep tissue penetration and high resolution.
  • To overcome light scattering challenges in deep tissue imaging.

Main Methods:

  • Developed near-infrared II (NIR-II) structured-illumination light-sheet microscopy (SIM) using ultralong wavelengths (excitation ∼1,540 nm, emission ∼1,700 nm).
  • Integrated structured illumination into NIR-II light-sheet microscopy to reduce background and enhance spatial resolution.
  • Performed in vivo oblique NIR-II SIM for 3D volumetric multiplexed molecular imaging.

Main Results:

  • Achieved deep-axial penetration depths in tissues, suppressing light scattering.
  • Improved spatial resolution by approximately twofold compared to conventional methods.
  • Successfully mapped CD4, CD8, and OX40 immune cells in the CT26 tumor microenvironment in mice longitudinally.

Conclusions:

  • NIR-II SIM provides an effective tool for noninvasive, volumetric molecular imaging in live mammals.
  • This technique enables single-cell level longitudinal studies of the tumor microenvironment and immune responses.
  • The developed method significantly advances in vivo optical imaging capabilities for biological research.