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Key Organism Strain Cell Line Gene Symbol Protein name Protein tag Reporter Project ID Dataset ID Title Description Data Type Gene Ontology Biological Imaging Methods Method Summary Paper Information Medical Subject Headlines Person

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530-Isobe-SpheroidCellPhotoconversion

https://ssbd.riken.jp/database/530-Isobe-SpheroidCellPhotoconversion/

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ssbd-repos-000530

https://ssbd.riken.jp/repository/530/

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x-y cross-sectional images of EpH4-spheroids expressing nucleus-localized PSmOrange2 before and after photostimulation of target cells

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 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. 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 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. 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.

2026-10-01

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CC BY 4.0

Image data based on Experiment

2 ( Image datasets: 2, Quantitative data datasets: 0 )

80.0 MB ( Image datasets: 80.0 MB, Quantitative data datasets: 0 bytes )

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Mus musculus

EpH4

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cell growth

nucleus

two-photon laser scanning microscopy

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Keisuke Isobe , RIKEN RAP

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