Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method.
Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z-stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal-to-background ratio and image contrast. Here, we present comparative studies on a variety of biological samples.
UMR 144 CNRS Institut Curie, Cell and Tissue Imaging Platform (PICT-IBiSA), Nikon Imaging Centre, Paris, France.
V
V
Fraisier
Imagine Optic, Orsay, France.
G
G
Clouvel
Imagine Optic, Orsay, France.
A
A
Jasaitis
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
A
A
Dimitrov
Institut Curie, Cell and Tissue Imaging platform (PICT-IBiSA), Paris, France.
T
T
Piolot
UMR 144 CNRS Institut Curie, Cell and Tissue Imaging Platform (PICT-IBiSA), Nikon Imaging Centre, Paris, France.|UMR 144 CNRS Institut Curie, Space Time Imaging of Endomembranes and Organelles Dynamics, Paris, France.
J
J
Salamero
D000818:Animals; D018385:Centrosome / Q000648:ultrastructure; D004330:Drosophila / Q000033:anatomy & histology / Q000648:ultrastructure; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods*; D008962:Models, Theoretical; D055095:Optics and Photonics / Q000295:instrumentation*; D017785:Photons; D014404:Tubulin / Q000648:ultrastructure
D014404:Tubulin
Aberrations; adaptive optics; deformable mirror; fluorescence; live imaging; spinning disk microscopy