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

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.