Objective: UPlanSApo 100x oil/1.40	Exposure: 600 ms
Microscope: Olympus DSU/IX81	Gain: 3
Camera: Hamamatsu ImagEM	Interval: 500 ms

Calcium biosensors were quickly followed by genetic indicators for pH, phosphorylation, and protease activity. Two general approaches can be used to adapt fluorescent proteins as sensors of pH. The first relies on the fluorescence sensitivity of EGFP (pKa = 5.9) and EYFP (pKa = 6.5) to acidic environments coupled to the relative insensitivity of other proteins, such as ECFP (pKa = 4.7) or DsRed (pKa = 4.5). Fusions of EGFP or EYFP with a less sensitive fluorescent protein create a ratiometric probe that can be used to measure the acidity of intracellular compartments. The second approach relies on protonation changes of native GFP that result in a shift in the bimodal spectral profiles of the native protein. A class of probes named pHluorins, derived from GFP, exhibits a shift in the excitation peak from 470 to 410 nm as the pH decreases. Dual-emission pH sensors have also been developed, which have peaks in the green and blue spectral regions. Although unable to report kinase activity in real time, phosphorylation biosensors consist of a peptide containing a phosphorylation motif from a specific kinase and a binding domain for a phosphopeptide sandwiched between two FRET-capable fluorescent proteins. When the biosensor is phosphorylated by the kinase, the phosphopeptide binding domain binds to the phosphorylated sequence, thus invoking or destroying FRET. This simple strategy has proven to generate robust and highly specific biosensors. As with many other biosensors, the major drawback is reduced dynamic range. In this digital video, a cameleon biosensor composed of cyan and yellow fluorescent proteins sandwiching a calmodulin domain and the m13 domain is being expressed transiently in human cervical carcinoma cells.