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J Microsc. 2015 Sep;259(3):219-27. doi: 10.1111/jmi.12256. Epub 2015 May 4.

Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method.

Author information

1
UMR 144 CNRS Institut Curie, Cell and Tissue Imaging Platform (PICT-IBiSA), Nikon Imaging Centre, Paris, France.
2
Imagine Optic, Orsay, France.
3
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
4
Institut Curie, Cell and Tissue Imaging platform (PICT-IBiSA), Paris, France.
5
UMR 144 CNRS Institut Curie, Space Time Imaging of Endomembranes and Organelles Dynamics, Paris, France.

Abstract

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.

KEYWORDS:

Aberrations; adaptive optics; deformable mirror; fluorescence; live imaging; spinning disk microscopy

PMID:
25940062
DOI:
10.1111/jmi.12256
[Indexed for MEDLINE]
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