LIVE CELL IMAGING Live cell microscopic imaging has been used in this lab since 1999, with the advent of fluorescent protein technology (2008 Nobel Prize to Roger Tsien). We use the method of widefield microscopy with a highly sensitive EM-CCD camera, a robotic stage to capture X,Y,Z stacks in three dimensions, and a heated and pH controlled stage to capture these 3D images over the fourth dimension of time. This is referred to as four dimensional imaging. (see video on right) We can observe cells at video speeds in real time, or capture an image at set time points for hours to days. For HD, this means we can observe how huntingtin protein moves within a cell, and how huntingtin responds to cell signaling and cell stress. We can also observe how cell react when treated with chemical compounds that inhibit kinases, or observe heat shock stress while it is occurring. The use of fusion proteins has some advantages: very specific signal, quantifiable due to ratio of 1protein:1 fluorophore, non-invasive imaging, and various biophotonic methods are applicable. The use of fusion proteins has some disadvantages: the 26KDa fusion may affect function of the fused protein, and some over-expression may be necessary to get enough signal. We address both of these caveats by comparing localization of our fusions to endogenous proteins, expressed at endogenous levels, measuring the amount of our fusion expression relative to endogenous protein, and by the use of Z-stacking and a very high sensitivity camera that amplifies light signal so that we capture 100% of possible light, even when we express at endogenous levels, and use minimal excitation light to prevent phototoxicity.