Cellular signaling networks are the foundation which determines the fate and function of cells as they respond to various cues and stimuli. The discovery of fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we endeavored to assemble a comprehensive list of published engineered biosensors, and we discuss many of the molecular designs utilized in their development. Then, we review how the high temporal and spatial resolution afforded by fluorescent biosensors has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research in both biosensor design and applications that are on the forefront of biosensor development.

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with their compartmentalization. Here we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to three-fold better than the diffraction limit. Utilizing specific, binding-induced changes in protein fluorescence dynamics, these biosensors translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A Protein Kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membrane of living cells and uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture, whose structural and functional characteristics can be revealed by these new biosensors.

Distance for cAMP diffusion before degradation by PDE:Aqueous diffusion rate: sqrt(2*444*t D ) = 21-210 µm Cardiac myocyte cytosol: 12-117 µm Restricted diffusion cardiac myocyte = 1-10 µm CHO cytosol: 22-220 µm Rate constants were obtained from example models as determined from biochemical experiments. Diffusion calculations performed using the solution to the 1-D diffusion equation, = 2Dt, where is the mean-squared distance traveled, D is the diffusion coefficient, and t is time (Codling et al., 2008). Using a linear approximation of cAMP degradation by PDE, the time to degradation was calculated by t deg = [cAMP]/Vmax  PDE. The time to degradation was then used in the 1-D diffusion equation to determine the diffusion distance before degradation. Note that the range of action of cAMP is predicted to increase with increasing [cAMP] and decrease with increasing [PDE].

PEG-LA hydrogels have potential for delivering chondrocytes in vivo to replace damaged cartilage with a tissue-engineered native equivalent, overcoming many limitations associated with current clinical treatments.