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Three-dimensional scanless patterned illumination using time-multiplexed multiline temporal focusing for multicell

Kenta Inazawa1,2,3, Mayumi Yamada4, Takayuki Michikawa1,5,6

  • 1RIKEN Center for Advanced Photonics, Attosecond Science Research Team, Saitama, Japan.

Journal of Biomedical Optics
|July 29, 2025
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Summary

We developed a new method for 3D two-photon patterned illumination that achieves single-cell resolution. This technique suppresses interference, enabling precise photostimulation of cells in 3D environments.

Keywords:
patterned illuminationphotostimulationtemporal focusing

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Area of Science:

  • Optics and Photonics
  • Biomedical Engineering
  • Cellular Imaging

Background:

  • Three-dimensional (3D) two-photon patterned illumination combines computer-generated holography (CGH) and temporal focusing (TF) for effective photostimulation.
  • Current CGH with TF methods struggle to achieve single-cell resolution in 3D due to interference among multiple light spots.

Purpose of the Study:

  • To achieve 3D two-photon patterned illumination with single-cell resolution.
  • To overcome the limitations of existing TF-CGH systems in resolving individual cells within 3D structures.

Main Methods:

  • Developed a novel system combining CGH with time-multiplexed multiline temporal focusing (TM-ML-TF).
  • Implemented TM-ML-TF by incorporating an echelle grating into the TF-CGH setup.
  • Measured 3D two-photon fluorescence distributions to evaluate system performance.

Main Results:

  • The TM-ML-TF system significantly suppressed crosstalk artifacts between adjacent focal planes (axial distance of 20 μm) from 81% to 15%.
  • Successfully demonstrated photoconversion of 3D target cells within spheroids at single-cell resolution.

Conclusions:

  • Time-multiplexed multiline temporal focusing effectively suppresses interference in multispot patterns.
  • The TM-ML-TF-CGH system enables precise 3D patterned illumination with single-cell resolution for advanced photostimulation applications.