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Related Concept Videos

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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
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Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking

Jian-Zong Lai1, Chun-Yu Lin2, Shean-Jen Chen2

  • 1Department of Optics and Photonics, National Central University, No. 300, Zhongda Rd., Zhongli Dist., Taoyuan City, 32001, Taiwan.

Angewandte Chemie (International Ed. in English)
|April 20, 2024
PubMed
Summary
This summary is machine-generated.

This study introduces a new single-molecule localization microscopy technique using temporal-focusing multiphoton excitation (TFMPE) for high-resolution 3D imaging. The method enhances photon efficiency and visualizes subcellular structures and Alzheimer

Keywords:
Amyloid-beta depositsSingle-molecule localization microscopySpontaneously blinking fluorophoreTemporal-focusing multiphoton excitationThree-dimensional imaging

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Area of Science:

  • Biophysics
  • Microscopy
  • Cell Biology

Background:

  • Single-molecule localization microscopy (SMLM) enables nanoscale imaging but faces challenges with photobleaching and photon budget in thick specimens.
  • Multiphoton excitation (MPE) offers advantages for deep tissue imaging but requires optimization for SMLM.

Purpose of the Study:

  • To develop and validate a single-wavelength temporal-focusing multiphoton excitation (TFMPE) SMLM technique for improved 3D imaging.
  • To optimize TPE wavelength for enhanced photon detection from blinking fluorophores.
  • To visualize subcellular structures and pathological features in thick biological samples.

Main Methods:

  • Implemented single-wavelength TFMPE for wide-field and axially confined two-photon excitation (TPE) of blinking fluorophores.
  • Performed TPE spectral measurements to determine optimal excitation wavelengths.
  • Combined TFMPE-SMLM with astigmatic imaging for 3D reconstructions.

Main Results:

  • Achieved nanoscale spatial resolution (approx. 51 nm) and high localization precision (18±6 nm) in 2D SMLM of cancer cell microtubules.
  • Demonstrated 3D TFMPE-SMLM imaging of amyloid-beta deposits in Alzheimer's disease mouse brain tissue.
  • Successfully reduced photobleaching effects in out-of-focus regions, improving photon budget utilization.

Conclusions:

  • TFMPE-SMLM is a powerful tool for high-resolution 3D imaging of subcellular structures and disease pathology in thick specimens.
  • The optimized TPE wavelength significantly enhances photon detection efficiency for SMLM.
  • This technique offers a promising approach for studying complex biological systems at the nanoscale.