title: Improving axial resolution in confocal microscopy with new high refractive index mounting media.
abstract: Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France; CNRS, UMR8246, INSERM, U1130, Neuroscience Paris Seine, F-75005, Paris, France.
authors.forename: Coralie
authors.initials: C
authors.lastname: Fouquet
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Gilles
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France; CNRS, UMR8246, INSERM, U1130, Neuroscience Paris Seine, F-75005, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Heck
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France; CNRS, UMR8246, INSERM, U1130, Neuroscience Paris Seine, F-75005, Paris, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Dos Santos
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France.
authors.forename: Richard
authors.initials: R
authors.lastname: Schwartzmann
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France; CNRS, UMR8246, INSERM, U1130, Neuroscience Paris Seine, F-75005, Paris, France.
authors.forename: Vidjeacoumary
authors.initials: V
authors.lastname: Cannaya
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France; CNRS, UMR8246, INSERM, U1130, Neuroscience Paris Seine, F-75005, Paris, France.
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Morel
authors.affiliation: Citifluor Ltd, 18, Enfield Coisters, Fanshaw Street, London, N1 6LD, United Kingdom.
authors.forename: Robert Stephen
authors.initials: RS
authors.lastname: Davidson
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France; CNRS, UMR8246, INSERM, U1130, Neuroscience Paris Seine, F-75005, Paris, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Trembleau
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris, France; Institut de Biologie Paris-Seine, CNRS FR3631, F-75005, Paris, France.
authors.forename: Susanne
authors.initials: S
authors.lastname: Bolte
mesh_terms: D003470:Culture Media / Q000737:chemistry*; D005453:Fluorescence; D016591:Histocytological Preparation Techniques / Q000379:methods*; D007089:Image Enhancement / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods*; D012029:Refraction, Ocular; D059629:Signal-To-Noise Ratio
chemical_list: D003470:Culture Media
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