Impact of immersion oils and mounting media on the confocal imaging of dendritic spines

• Mounting medium and immersion oil affect resolution in confocal microscopy.
• Optimizing confocal resolution can impact biological measurements.
• Select immersion oil to match refractive index of glass.
• Mounting media may be selected based on tissue requirements.

BackgroundStructural plasticity, such as changes in dendritic spine morphology and density, reflect changes in synaptic connectivity and circuitry. Procedural variables used in different methods for labeling dendritic spines have been quantitatively evaluated for their impact on the ability to resolve individual spines in confocal microscopic analyses. In contrast, there have been discussions, though no quantitative analyses, of the potential effects of choosing specific mounting media and immersion oils on dendritic spine resolution.New methodHere we provide quantitative data measuring the impact of these variables on resolving dendritic spines in 3D confocal analyses. Medium spiny neurons from the rat striatum and nucleus accumbens are used as examples.ResultsBoth choice of mounting media and immersion oil affected the visualization of dendritic spines, with choosing the appropriate immersion oil as being more imperative. These biologic data are supported by quantitative measures of the 3D diffraction pattern (i.e. point spread function) of a point source of light under the same mounting medium and immersion oil combinations.Comparison with existing methodAlthough not a new method, this manuscript provides quantitative data demonstrating that different mounting media and immersion oils can impact the ability to resolve dendritic spines. These findings highlight the importance of reporting which mounting medium and immersion oil are used in preparations for confocal analyses, especially when comparing published results from different laboratories.ConclusionCollectively, these data suggest that choosing the appropriate immersion oil and mounting media is critical for obtaining the best resolution, and consequently more accurate measures of dendritic spine densities.