Super-resolution for everybody: An image processing workflow to obtain high-resolution images with a standard confocal microscope.
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 60μm. 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.
Sorbonne Universités, UPMC Univ Paris 06, CNRS, Core Facilities - Institut de Biologie Paris Seine (IBPS), 75005 Paris, France.
France
F
Lam
Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratory of Developmental Biology - Institut de Biologie Paris Seine (IBPS), 75005 Paris, France.
Damien
D
Cladière
Sorbonne Universités, UPMC Univ Paris 06, CNRS, Core Facilities - Institut de Biologie Paris Seine (IBPS), 75005 Paris, France.
Cyndélia
C
Guillaume
Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratory of Developmental Biology - Institut de Biologie Paris Seine (IBPS), 75005 Paris, France.
Katja
K
Wassmann
Sorbonne Universités, UPMC Univ Paris 06, CNRS, Core Facilities - Institut de Biologie Paris Seine (IBPS), 75005 Paris, France. Electronic address: susanne.bolte@upmc.fr.
Susanne
S
Bolte
D000465:Algorithms; D000818:Animals; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D007091:Image Processing, Computer-Assisted / Q000379:methods* / Q000706:statistics & numerical data; D008540:Meiosis; D051379:Mice; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008863:Microspheres; D009865:Oocytes / Q000648:ultrastructure*; D061251:Primary Cell Culture; D008941:Spindle Apparatus / Q000648:ultrastructure*; D057188:Workflow
D005456:Fluorescent Dyes
3D-SIM-like resolution improvement; Confocal microscopy; Deconvolution; Meiotic spindle; Refractive index matching