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A Confocal Reflection Super‐Resolution Technique to Image Golgi‐Cox Stained Neurons

Metal‐based Golgi‐Cox (GC) staining is an established method used to visualise neurons with great morphological detail. Although GC stained samples are imaged routinely under transmitted light microscopy, this method is unable to yield information on the three‐dimensional structure of dendrites and neurons and thus help reveal the connective properties of the central nervous system. Although a few studies have attempted simultaneous visualisation of GC staining and antigen‐specific fluorescent labelling under a confocal reflection technique, the resolution of both confocal reflection and fluorescence modalities used to acquire GC reflection and fluorescently stained antibody signals are still limited by the diffraction limit of light at about 220 nm. Here, we report a confocal reflection super‐resolution technique (CRSR) to break this diffraction barrier, which is achieved by minimising the pinhole size from 1 airy unit (AU) to 0.1 AU. This is achieved by minimising or closing the confocal pinhole size and is possible in this reflection modality, unlike fluorescence, because it is not a photon limited technique. Utilising the lowest wavelength of light available in the system (405 nm), the CRSR technique results in ∼30% lateral and axial resolution improvement. We also show that the CRSR technique can be used in conjunction to visualise both GC and immunofluorescence targets to create precise and improved three‐dimensional visualisation and analysis. In addition, using these superresolution confocal reflection data sets from GC in CRSR mode significantly reduced the data overestimation, improving the accuracy of statistical analysis of dendritic spine density and average spine dimensions. Combining the 0.1 AU setting with deconvolution routines, the signal‐to‐noise ratio and resolution could further be improved an additional ∼20–25%, yielding CRSR images with resolutions up to 2‐fold over the diffraction limit both laterally and axially. The improved precision of both visualisation and quantification of subdiffraction limited dendritic spines using the CRSR technique may prove to be critical in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease.