Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.
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Training and test data for all experiments presented can be found at https://publications.mpi-cbg.de/publications-sites/7207. The code for network training and prediction (in Python/TensorFlow) is publicly available at https://github.com/CSBDeep/CSBDeep. Furthermore, to make our restoration models readily available, we developed user-friendly FIJI plugins and KNIME workflows (Supplementary Figs. 29 and 30).
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The authors thank P. Keller (Janelia) who provided Drosophila data. We thank S. Eaton (MPI-CBG), F. Gruber and R. Piscitello for sharing their expertise in fly imaging and providing fly lines. We thank A. Sönmetz for cell culture work. We thank M. Matejcic (MPI-CBG) for generating and sharing the LAP2b transgenic line Tg(bactin:eGFP-LAP2b). We thank B. Lombardot from the Scientific Computing Facility (MPI-CBG) for technical support. We thank the following Services and Facilities of the MPI-CBG for their support: Computer Department, Light Microscopy Facility and Fish Facility. This work was supported by the German Federal Ministry of Research and Education (BMBF) under the codes 031L0102 (de.NBI) and 031L0044 (Sysbio II) and the Deutsche Forschungsgemeinschaft (DFG) under the code JU 3110/1-1. M.S. was supported by the German Center for Diabetes Research (DZD e.V.). T.B. was supported by an ELBE postdoctoral fellowship and an Add-on Fellowship for Interdisciplinary Life Sciences awarded by the Joachim Herz Stiftung. R.H. and S.C. were supported by the following grants: UK BBSRC (grant nos. BB/M022374/1, BB/P027431/1, and BB/R000697/1), UK MRC (grant no. MR/K015826/1) and Wellcome Trust (grant no. 203276/Z/16/Z).
The authors declare no competing interests.
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Supplementary Figures 1–30, Supplementary Tables 1–3 and Supplementary Notes 1–5
Challenges in time-lapse imaging of flatworm Schmidtea mediterranea. Image stacks of RedDot1-labeled, anesthetized specimen were acquired every 2 min with a spinning disk confocal microscope (NA = 1.05), at high and low SNR (illumination) conditions (10% laser, 30-ms exposure per plane vs. 0.5% laser, 10 ms per plane). Whereas in the high-SNR case the specimen shows illumination-induced twitching, the image quality in the low-SNR case is insufficient for further analysis. Network restoration enabled us to recover high-SNR images from images acquired at low-SNR conditions without twitching of the specimen, thus providing a practical framework for live-cell imaging of Schmidtea mediterranea.
Restoration results of low-SNR acquisitions of Schmidtea mediterranea and comparison to ground truth. Shown are a 3D rendering of the results on a multi-tiled acquisition (8,192 × 3,072 × 100 pixels) and the comparison with ground truth.
Restoration of low-SNR volumetric time lapses of developing Tribolium castaneum embryos (EFA::nGFP-labeled nuclei).: Acquisition was done on a Zeiss LSM 710 NLO multiphoton laser-scanning microscope with a time step of 8 min, and stack size of a single time point 760 × 760 × 100 pixels. Shown are maximum-intensity projection and single slices of the raw stacks, the network prediction and the high-SNR ground truth.
Joint surface projection and denoising of developing Drosophila melanogaster wing epithelia. 3D image stacks of the developing wing of a membrane-labeled (Ecad::GFP) fly pupa were acquired with a spinning disk confocal (63×, NA = 1.3) microscope. We show the projected epithelial surface obtained by a conventional method (PreMosa, ref. 1), the restoration network, and the projected ground truth. We applied a random-forest-based cell segmentation pipeline and show segmentation/tracking results, demonstrating vastly improved accuracy for the restoration when compared to the conventionally processed raw stacks.
Isotropic restoration of anisotropic time-lapse acquisitions of hisGFP-tagged developing Drosophila melanogaster embryos. We used the original, preprocessed data set of ref. 2, acquired with a light-sheet microscope (NA = 1.1) with fivefold axially undersampled resolution (lateral/axial pixel size: 0.39 μm/1.96 μm). The video shows different axial (xz) regions of a single time point from both the original (input) stacks and the isotropic restoration.
Isotropic restoration of anisotropic dual-color acquisitions of developing Danio rerio retina. The data were acquired with a spinning disk confocal microscope (Olympus 60×, NA = 1.1) and exhibits a tenfold axial anisotropy (lateral/axial pixel size: 0.2 μm/2.0 μm); labeled structures are nuclei (DRAQ5, magenta) and nuclear envelope (GFP+LAP2b, green). The video shows a rendering of the dual-color input stack and its isotropic reconstruction.
Enhancement of diffraction-limited structures in widefield images of rat INS-1 (beta) cells. The video shows time lapses of several INS-1 cells, acquired with the widefield mode of a DeltaVision OMX microscope (63×, NA = 1.43). Labeled are secretory granules (pEG-hIns-SNAP, magenta) and microtubules (SiR-tubulin, green). Next to the time lapse of the widefield images we show the output of the reconstruction networks.
Enhancement of diffraction-limited widefield images of GFP-labeled microtubules in HeLa cells and comparison with SRRF (super-resolution radial fluctuations ). Images were acquired with a Zeiss Elyra PS.1 microscope in TIRF mode (100×, NA = 1.46). The video shows the widefield input sequence, the network restoration and the corresponding SRRF images. Note that the time resolution of the SRRF image sequence is 20 times less than the network restoration, as 20 times more images have to be processed for the same restoration quality.
Visualization of network predictions by sampling from the predicted distribution. The video shows for two examples (surface projection of fly wing tissue, and microtubule structure restoration in INS-1 cells) that drawing samples from the per-pixel predicted distribution is beneficial for identifying challenging image regions. For each example, we show successively the raw input, the per-pixel mean of the predicted distribution and random samples from the per-pixel distributions.
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Weigert, M., Schmidt, U., Boothe, T. et al. Content-aware image restoration: pushing the limits of fluorescence microscopy. Nat Methods 15, 1090–1097 (2018). https://doi.org/10.1038/s41592-018-0216-7
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