FLUORESCENT SPECKLE MICROSCOPY, A METHOD TO VISUALIZE THE DYNAMICS OFPROTEIN ASSEMBLIES IN LIVING CELLS

Fluorescence microscopic visualization of fluorophore-conjugated prote ins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information ab out protein localization and dynamics El]. This approach has, however, been limited by high background fluorescence and the difficulty of de tecting movement of fluorescent structures because of uniform labeling . These problems have been partially alleviated by the use of more cum bersome methods such as three-dimensional confocal microscopy, laser p hotobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field flu orescence right microscopy and digital imaging with a low-noise, coole d charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of pr otein assemblies throughout the image field of view at diffraction-lim ited resolution. We found that FSM also significantly reduces out-of-f ocus fluorescence and greatly improves visibility of fluorescently lab eled structures and their dynamics in thick regions of living cells. O ur initial applications include the measurement of microtubule movemen ts in mitotic spindles and actin retrograde flow in migrating cells.