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Abstract
This chapter shows that, through suitable chimeras, green fluorescent protein (GFP) can be targeted to various intracellular locations (the cytosol, the nucleus, the mitochondria, the endoplasmic reticulum, the Golgi, the subplasmalemmal space), thus providing powerful in vivo markers of these compartments. The GFP chimeras described in this chapter are currently utilized in lab for monitoring the dynamic changes of organelle structure that occur in living cells. Two types of instruments are currently used for detecting GFP fluorescence: (a) a wide-field fluorescence imaging system, based on a Zeiss Axiovert inverted microscope and a Princeton Instruments back-illuminated camera, and (b) a Nikon RCM 8000 confocal microscope. In both cases, the microscope is equipped with motorized control of the Z stage, thus allowing the acquisition of three-dimensional images. It is obvious that by confocal imaging, the dense endoplasmic reticulum structure of HeLa cells can be finely resolved, and the continuous rearrangement of organelle structure can be clearly appreciated.
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