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Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy


Elucidating the volumetric architecture of organelles and molecules inside cells requires microscopy methods with a sufficiently high spatial resolution in all three dimensions. Current methods are limited by insufficient resolving power along the optical axis, long recording times and photobleaching when applied to live cell imaging. Here, we present a 3D, parallelized, reversible, saturable/switchable optical fluorescence transition (3D pRESOLFT) microscope capable of delivering sub-80-nm 3D resolution in whole living cells. We achieved rapid (1–2 Hz) acquisition of large fields of view (~40 × 40 µm2) by highly parallelized image acquisition with an interference pattern that creates an array of 3D-confined and equally spaced intensity minima. This allowed us to reversibly turn switchable fluorescent proteins to dark states, leading to a targeted 3D confinement of fluorescence. We visualized the 3D organization and dynamics of organelles in living cells and volumetric structural alterations of synapses during plasticity in cultured hippocampal neurons.

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Fig. 1: Interference based OFF-switching pattern in 3D pRESOLFT.
Fig. 2: 3D pRESOLFT imaging scheme and parallelization.
Fig. 3: Resolution assessment in 3D pRESOLFT.
Fig. 4: Four-dimensional imaging and rendering in full-length human cells.
Fig. 5: Structural alteration of mitochondria following oxidative stress.
Fig. 6: Intrasynaptic neuronal imaging with isotropic 3D pRESOLFT.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The software used to acquire the super-resolved data (hardware control, image reconstruction and deconvolution pipeline) were developed by our laboratory and available upon request.


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I.T. thanks the ERC (ERC_StG 638314, MoNaLISA) and the Swedish Foundation for Strategic Research (FFL15-0031) for supporting the project.

Author information

Authors and Affiliations



I.T. designed and supervised the project. A.B. engineered and built the microscope with associated software. A.B. performed the experiments and data analysis. F.P. carried out RSFP switching experiments and data analysis. G.C. and M.D. performed the bioimaging treatments and imaging. M.R. cloned the constructs and provided biological guidance. I.T. and A.B. wrote the manuscript with assistance from all the authors.

Corresponding author

Correspondence to Ilaria Testa.

Ethics declarations

Competing interests

I.T. and A.B. have filed a provisional patent on the 3D pRESOLFT technology (no. N.1930406-2, Sweden, December 2019).

Additional information

Peer review information Nature Biotechnology thanks Reto Fiolka, Valentin Nagerl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Notes 1–9, Figs. 1–15 and Tables 1–5.

Reporting Summary

Supplementary Video 1

Mitochondrial 3D dynamics.

Supplementary Video 2

Entire mitochondrial network of a U2OS cell.

Supplementary Video 3

Entire actin cytoskeleton of a U2OS cell.

Supplementary Video 4

Actin architecture in hippocampal neuron.

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Bodén, A., Pennacchietti, F., Coceano, G. et al. Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy. Nat Biotechnol 39, 609–618 (2021).

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