Stimulated Emission Depletion (STED) Microscopy and Pacific Orange Dye Optimization For H9C2 Cox-1 Imaging Via Indirect Immunocytochemistry

The fundamental barrier of traditional microscopy has always been the Abbe limit. Diffraction has served to limit image production and microscopic investigation on the sub-cellular level, greatly hindering microbiology and other forms of study. Stimulated Emission Depletion microscopy is one of many new frontiers of microscopy that has recently broken through this diffraction barrier. By utilizing multiple competing sources of light, STED has produced high-resolution, nanoscale images of both biological and non-biological samples, greatly adding to the wealth of knowledge in multiple disciplines. Despite these contributions, several obstacles remain for STED technology. These include pricing and availability of laser usage, as well as inherit qualities of component materials, such as photobleaching of standard fluorophores. Current research aims to create smaller, cheaper STED models capable of using improved dyes that withstand photobleaching. The purpose of this study is to describe a simple method for standard biological imaging using a novel system software program. Pacific Orange dye was selected for imaging and tested against the STED system: compatibility and photobleaching tests yielded testing parameters for imaging. H9c2 rat embryonic myocardium heart cell samples were passaged and stained via indirect immunofluorescence: COX-1 proteins of the mitochondrial inner-membranes were targeted by primary mouse, anti-COX-1 IgG antibodies, which in turn were targeted by Pacific Orange conjugated secondary F(ab\u27)2 fragment goat, anti-mouse IgG antibodies. Cell samples were identified via CCD camera and then imaged on the STED through the use of a novel the LabWindows/CVI computer program used to minimize testing time and photobleaching while collecting data for dye emissions under only the excitation beam was well as both the excitation beam and the STED beam. Collected data was then processed using Matlab to generate photon emission intensity plots of the cells. While improvement in the images was recognizable through the use of the added STED beam during testing, a large step size used during sample testing movement as well as other sources of error including bandpass filter selection may have prevented full realization of sub-confocal resolution levels. Overall, the experiment described in this report is the basis for an improved technique in biological imaging over standard confocal microscopy