Subcellular Microdissection for the Identification of Organelle Proteins

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This is a new project, and consequently, proof-of-principle experiments are in progress. Initially, we are exploring expanding the limits of Expression Microdissection (xMD). Existing commercial LCM instruments fall into two broad categories: infrared laser systems which melt an absorbing thermal film in areas illuminated by the laser, capturing nearby tissue; and ultraviolet cutting systems which collect areas circumscribed by the laser. Both use microscopic imaging to define the targets for capture. In contrast, the current prototype system used for xMD requires only a fiber-coupled laser source and a computer-controlled scanning stage. A clear ethyl vinyl acetate (EVA) film is placed over an immunohistologically stained tissue section and a 120 μm laser beam rastered over the entire tissue section. The energy of the laser is absorbed only by the dark stain (DAB), which then results in local melting of the polymer film, thereby capturing only the labeled areas for downstream molecular analysis. The technique is unsupervised, automatically retrieving any antibody-stained region, and relatively rapid: a 2 cm x 3 cm tissue slice can be fully dissected in 30 minutes. In addition, the theoretical resolution is not limited by diffraction but is determined by the melt properties of the film and heat transfer from the absorber. Furthermore, the capture speed and spatial resolution are uncorrelated; dissection of a full tissue slice with 100 nm resolution could theoretically be accomplished in the same time it takes for 100 μm resolution. A significant limitation of the current xMD method for proteomics is its reliance on an immunostaining process that introduces significant chemical contamination for subsequent proteomics. Nevrtheless, we are exploring whether antibody coupled reagents, particularly metal bearing, allow capture and transfer of subcellular organelles. We are also exploring direct use of osmium or lead staining, eliminating the need for a horseradish peroxidase catalyzed reactions. Heavy metals have been widely used in electron microscopy to stain and contrast proteins and organelles. The "black reaction" first described by Camillo Golgi in 1898 results from the reduction of osmium tetroxide to a black precipitate at calcium receptors in the cisternae. Alternatively, lead citrate also stains Golgi due to enzymes ubiquitously present and active after fixation. With either osmium or lead, the spatial resolution will be defined by the extent of the metal deposit, by the contrast in absorption between the stained areas and the background, and by the ability to limit melting within the polymer film. Based on the proteomic analyses with metal staining using classical methods, we will later explore alternative strategies for specific protein metal staining, including immunogold.