biostudies-literatureUnknown30(7)Inazawa K<h4>Significance</h4>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 10  μm , 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.<h4>Aim</h4>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.<h4>Approach</h4>We measure the 3D two-photon fluorescence distributions generated by the TF-CGH and TM-ML-TF-CGH systems.<h4>Results</h4>In TM-ML-TF, the crosstalk artifacts between the target spots in two proximal planes with an axial distance of 20  μm 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.<h4>Conclusions</h4>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.Journal of biomedical optics075003https://www.ebi.ac.uk/biostudies/studies/S-EPMC12302995biostudies-literatureThree-dimensional scanless patterned illumination using time-multiplexed multiline temporal focusing for multicell manipulation with single-cell resolution.PMC12302995Miyawaki AMichikawa TInazawa KMidorikawa KNamiki KYamada MImayoshi IIsobe KfalseThree-dimensional scanless patterned illumination using time-multiplexed multiline temporal focusing for multicell manipulation with single-cell resolution.<h4>Significance</h4>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 10  μm , 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.<h4>Aim</h4>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.<h4>Approach</h4>We measure the 3D two-photon fluorescence distributions generated by the TF-CGH and TM-ML-TF-CGH systems.<h4>Results</h4>In TM-ML-TF, the crosstalk artifacts between the target spots in two proximal planes with an axial distance of 20  μm 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.<h4>Conclusions</h4>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.2025-01-01T00:00:00Z2025 Jul2026-03-27T17:00:09.941Z2025-08-28T03:08:51.863ZS-EPMC123029954072659310.1117/1.JBO.30.7.075003