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.

Hemocyte production in Biomphalaria glabrata snails after exposure to different Schistosoma mansoni infection protocols / Produção de hemócitos de caramujos da espécie Biomphalaria glabrata após a exposição a diferentes protocolos de infecção por Schistosoma mansoni

The objective of this work was to determine the profile of the cellular defense system during mansonic infection. Specifically, this study assessed the number of hemocytes that were produced and released into the hemolymph in response to the parasitic infection. The quantification of the Biomphalaria glabrata hemocytes was performed on groups of snails at 1, 5, 10, 15, 20 and 30 days post-infection that had been individually infected with 5, 10, 15 or 30 Schistosoma mansoni miracidia. The results revealed that B. glabrata possesses a cellular defense mechanism that is characterized by the release of hemocytes into the hemolymph. The maximum peak of cellular production occurred 24 hours after infection, and there was a significant reduction in the hemocyte concentration over the following 10 days. However, at 15 days post-infection, there was a second increase in the cellular hemocyte production, although this was not as strong as the primary peak. At 30 days post-infection, there was another moderate rise in the cellular hemocyte production. Based on this cellular response profile, the defense system of the snail appears to be effective immediately following infection, but the response does not ensure the destruction of all parasites during the course of the infection.

O objetivo deste artigo foi determinar o perfil do sistema de defesa celular durante a infecção mansônica. Especificamente, este estudo avaliou o número de hemócitos produzidos e liberados na hemolinfa em resposta à infecção pelo parasita. A quantificação dos hemócitos de Biomphalaria glabrata foi realizada em grupos de caramujos previamente infectados com 5, 10, 15 ou 30 miracídios de Schistosoma mansoni nos dias 1, 5, 10, 15, 20 e 30 pós-infecção. Os resultados revelaram que B. glabrata possui um mecanismo de defesa celular caracterizado pela liberação de hemócitos na hemolinfa. O maior registro de produção celular ocorreu 24 h após a infecção e houve uma redução significante na concentração de hemócitos durante os 10 dias seguintes. No entanto, no dia 15 pós-infecção, houve um segundo aumento na produção de hemócitos, porém não tão acentuado como o primeiro pico. No dia 30 pós-infecção, foi observado outro aumento moderado da produção de hemócitos nas células. Com base neste perfil de resposta celular, o sistema de defesa do caramujo aparenta ser eficiente nos momentos imediatamente posteriores à infecção, mas essa resposta não assegura a destruição de todos os parasitas no curso da infecção.