A better resolution in confocal microscopy

Recently, and through a collaboration between the Cellular Imaging Facility1 and the team « Development and Plasticity of Neural Networks »2 an outstanding axial resolution in confocal microscopy in thick sections of the mouse brain has been made possible. This is due to the use of new high refractive index mounting media and systematic deconvolution of confocal data.

Resolution, high signal intensity and elevated signal to noise ratio are key parameters for biologists who use confocal microscopy to localize biological structures at the cellular and subcellular levels. When optimally used, confocal microscopy may reach lateral and axial resolutions up to 150 nm and 500 nm respectively. Axial resolution is however often biased by spherical aberrations due to the difference in refractive index between the sample and the objective. Spherical aberrations lead to decreased axial resolution, diminished signal intensity, and distorted image depth, thus leading to scaling errors.

The study published in Plos One (3) shows how the use of a new high refractive index mounting medium eliminates spherical aberration effects in thick mouse brain sections and that it increases the axial resolution and the imaging depth in immune labelled or fluorescent protein labelled tissue. Moreover, this mounting medium renders tissue transparent.

The mounting medium is currently being evaluated for the use in other types of biological samples such as zebrafish tissues, whole mouse embryos or mouse retina.
This work was funded by and IBPS grant and by Citifluor Ltd who provided custom made mounting media to the Cellular Imaging Facility.

1.Cellular imaging facility – IBPS

2.Development and plasticity of neural network - IBPS

3.Improving axial resolution in confocal microscopy with new high refractive index mounting media. Fouquet C et al. PLoS ONE (2015)