Super-resolution for everybody: An image processing workflow to obtain high-resolution images with a standard confocal microscope

- We propose a workflow that allows obtaining confocal super-resolution images.
- We improve the resolution of confocal imaging 2-fold in the lateral direction.
- Axial resolution is improved 3-fold up to a depth of 60 μm.
- We evaluate two different deconvolution algorithms and denoising on the deconvolution result.
- With our approach, super-resolution may be obtained in the meiotic spindle of mouse oocytes.

In the presented work we aimed at improving confocal imaging to obtain highest possible resolution in thick biological samples, such as the mouse oocyte. We therefore developed an image processing workflow that allows improving the lateral and axial resolution of a standard confocal microscope. Our workflow comprises refractive index matching, the optimization of microscope hardware parameters and image restoration by deconvolution. We compare two different deconvolution algorithms, evaluate the necessity of denoising and establish the optimal image restoration procedure. We validate our workflow by imaging sub resolution fluorescent beads and measuring the maximum lateral and axial resolution of the confocal system. Subsequently, we apply the parameters to the imaging and data restoration of fluorescently labelled meiotic spindles of mouse oocytes. We measure a resolution increase of approximately 2-fold in the lateral and 3-fold in the axial direction throughout a depth of 60 μm. This demonstrates that with our optimized workflow we reach a resolution that is comparable to 3D-SIM-imaging, but with better depth penetration for confocal images of beads and the biological sample.

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