PT  - JOURNAL ARTICLE
AU  - Galgani, Tommaso
AU  - Fedala, Yasmina
AU  - Zapata, Romeo
AU  - Caccianini, Laura
AU  - Viasnoff, Virgile
AU  - Sibarita, Jean-Baptiste
AU  - Galland, Rémi
AU  - Dahan, Maxime
AU  - Hajj, Bassam
TI  - Selective volumetric excitation and imaging for single molecule localization microscopy in multicellular systems
AID  - 10.1101/2022.12.02.518828
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.12.02.518828
4099  - http://biorxiv.org/content/early/2022/12/03/2022.12.02.518828.short
4100  - http://biorxiv.org/content/early/2022/12/03/2022.12.02.518828.full
AB  - Light sheet fluorescence microscopy (LSFM) has become a leading standard in high-resolution imaging of living samples in 2- and 3-dimensions. Biological samples are however not restricted to a single observation plane and several molecular processes evolve rapidly in 3D. The conventional mechanical scanning required in LSFM limits the range of observable dynamics and are usually restricted in resolution. Here we introduce a new strategy for instantaneous volumetric excitation and volumetric imaging of single-molecules in cell aggregates. The technique combines, for the first time, the use of light sheet microscopy and multifocus microscopy (MFM) and enables a volumetric 4D imaging of biological samples with single-molecule resolution. We engineered the excitation beam to yield a modular and uniform excitation matching the observable detection range of MFM. The strength of the method is highlighted with examples of single-molecule 3D tracking and 3D super-resolution imaging in multicellular samples.Competing Interest StatementThe authors have declared no competing interest.