Two simple criteria to estimate an objective’s performance when imaging in non design tissue clearing solutions

• Published in: Journal of neuroscience methods Vol. 332; p. 108564
• Main Authors: Asteriti, Sabrina, Ricci, Valeria, Cangiano, Lorenzo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.02.2020
• Subjects: Deconvolution; Fluorescence; Microscopy; Neuron; Serial optical sectioning; Spherical aberration; Tissue clearing; PSF; OPD; Fluorescence; Serial optical sectioning; G&L; WD; Deconvolution; NA; Neuron; Microscopy; RI; Tissue clearing; Spherical aberration
• ISSN: 0165-0270; 1872-678X; 1872-678X
• DOI: 10.1016/j.jneumeth.2019.108564

[Display omitted] •Tissue clearing enables deep non invasive imaging of fluorescent neurons.•Imaging in clearing solutions may be affected by severe optical aberrations.•We developed approximate formulae that predict image quality degradation.•Easy to use numerical criteria evalute any clearing solution/objective combination. Tissue clearing techniques are undergoing a renaissance motivated by the need to image fluorescent neurons, and other cells, deep in the sample without physical sectioning. Optical transparency is achieved by equilibrating tissues with high refractive index (RI) solutions. When the microscope objective is not perfectly matched to the RI of the cleared sample, aberrations are introduced. We present two simple-to-calculate numerical criteria predicting: (i) the degradation in image quality (brightness and resolution) from optimal conditions of any clearing solution/objective combination; (ii) which objective, among several available, achieves the highest resolution in a given medium. We derived closed form approximations for image quality degradation versus RI mismatch and other parameters available to the microscopist, validated them with computed and measured aberrated point spread functions and by imaging fluorescent neurons in high RI solution. These approximations apply to the widefield fluorescent microscope but are also relevant to more advanced microscopes. Currently, to accurately predict the impact of RI mismatch-induced aberrations on imaging, the life scientist must examine theoretical or experimental point spread functions (PSFs) obtained under the optical configuration of interest. These criteria can be used to select a suitable objective for the chosen clearing method (particularly when subject to budget constraints) or to tweak a clearing solution RI to the available objectives. Even with a nominally optimal objective, one may wish to assess the impact of any small unavoidable mismatches.