Multiplexed fluorescence imaging enables the visualization of signaling dynamics within a cell by leveraging genetically encoded fluorescent reporters. Previous techniques are limited by the number of signals they can detect within a single cell, or they may require specific hardware that might not be available to all researchers. A recent technique, spatial multiplex imaging, enables the simultaneous imaging of five different reporters within a cell. However, it cannot be used to visualize the organization of proteins in living cells or organelles, or to visualize protein movement in response to a stimulus. In a recent study published in Cell, Qian and colleagues describe temporally multiplexed imaging (TMI), which makes it possible to image multiple reporters using a conventional microscope.
TMI uses reversibly photoswitchable fluorescent proteins (rsFPs) that are expressed in the same cell, can be switched ‘on’ and ‘off’ with different colored light pulses, and act as fluorescent tags to different protein sequences. These rsFPs can be the same color and — as each fluorophore will decay differently over time — their signals can be differentiated. The authors showcase the ability of TMI to track up to seven molecules in a cell, with very low crosstalk between fluorophores. Tracking TMI fluorescent proteins requires a single color channel and frees up the spectrum for imaging additional signals in other channels. The authors use TMI to image the activities of four kinase translocation reporters simultaneously following stimulation, and also look at the activity of cyclin-dependent kinases in a cell-cycle model, allowing the testing of hypotheses for how these proteins may interact in networks.
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