Three-dimensional scanless patterned illumination using time-multiplexed multiline temporal focusing for multicell manipulation with single-cell resolution.

Significance: Three-dimensional (3D) two-photon patterned illumination using a combination of computer-generated holography (CGH) and wide-field temporal focusing (TF) has emerged as a highly effective approach for photostimulation. However, even though the axial full-width at half-maximum of a single-spot by TF is smaller than the single-cell size of , the axial resolution of 3D multispot patterns produced by CGH with TF is lower than the single-cell resolution as a result of interference among multispots.

Aim: We aim to achieve 3D two-photon patterned illumination with single-cell resolution by combining CGH with time-multiplexed multiline temporal focusing (TM-ML-TF), which is implemented by adding an echelle grating at a position conjugate to the focal plane of the TF-CGH system.

Approach: We measure the 3D two-photon fluorescence distributions generated by the TF-CGH and TM-ML-TF-CGH systems.

Results: In TM-ML-TF, the crosstalk artifacts between the target spots in two proximal planes with an axial distance of were suppressed from 81% to 15% compared with those in TF. We successfully achieved the photoconversion of 3D target cells in spheroids with single-cell resolution.

Conclusions: TM-ML-TF successfully suppresses the interference among multispots, enabling the TM-ML-TF-CGH system to provide precise 3D patterned illumination with single-cell resolution.