Oxygen-enriched photoconversion of fluorescent dyes by means of a closed conversion chamber

The goal of the present study was an improvement of the widely used photoconversion technique, which still represents the major approach to the ultrastructural analysis of tissue labelled with fluorescent dyes. Since free access of oxygen to the tissue is essential for the dye-dependent photooxidation of diaminobenzidine (DAB), we attempted to facilitate the process using a closed conversion chamber (CCC), which allows photoconversion in an atmosphere of pure oxygen. Fixed rat tissue samples, containing 4Di-10ASP labelled retinal ganglion cells and Cy3 stained cortical perineuronal nets, were choosen to test the applicability and efficiency of the proposed system. The results are compared to corresponding structures photoconverted without pure oxygen. As a result, the employment of the CCC helps saving up to 50% of time required to achieve a comparable degree of photoconversion. Electron microscopical inspection showed no differences between both approaches regarding the distribution of DAB reaction product. However, probably due to the reduced time of irradiation, the ultrastructural integrity of tissue sometimes appeared considerably less affected after photoconversion in the CCC. Additionally, the chamber allowed for safety measures in handling DAB, as the unintentional emission of the presumable carcinogenic substance was completely avoided.