A New Low Cost Wide-Field Illumination Method for Photooxidation of Intracellular Fluorescent Markers

Analyzing cell morphology is crucial in the fields of cell biology and neuroscience. One of the main methods for evaluating cell morphology is by using intracellular fluorescent markers, including various commercially available dyes and genetically encoded fluorescent proteins. These markers can be used as free radical sources in photooxidation reactions, which in the presence of diaminobenzidine (DAB) forms an opaque and electron-dense precipitate that remains localized within the cellular and organelle membranes. This method confers many methodological advantages for the investigator, including absence of photo-bleaching, high visual contrast and the possibility of correlating optical imaging with electron microscopy. However, current photooxidation techniques require the continuous use of fluorescent or confocal microscopes, which wastes valuable mercury lamp lifetime and limits the conversion process to a few cells at a time. We developed a low cost optical apparatus for performing photooxidation reactions and propose a new procedure that solves these methodological restrictions. Our "photooxidizer" consists of a high power light emitting diode (LED) associated with a custom aluminum and acrylic case and a microchip-controlled current source. We demonstrate the efficacy of our method by converting intracellular DiI in samples of developing rat neocortex and post-mortem human retina. DiI crystals were inserted in the tissue and allowed to diffuse for 20 days. The samples were then processed with the new photooxidation technique and analyzed under optical microscopy. The results show that our protocols can unveil the fine morphology of neurons in detail. Cellular structures such as axons, dendrites and spine-like appendages were well defined. In addition to its low cost, simplicity and reliability, our method precludes the use of microscope lamps for photooxidation and allows the processing of many labeled cells simultaneously in relatively large tissue samples with high efficacy.