Figure 2. Quantitative live cell imaging techniques.
A. Time lapse microscopy. Fluorescence intensity in distinct cellular compartments of the same cell can be monitored in real time. B. Fluorescence recovery after photobleaching. A region of interest, shown by the red box, is photobleached with a strong laser pulse and the recovery of intensity within the region is monitored in time. Slower recovery arises if the GFP fusion protein is bound longer to an immobile structure and/or diffusing at a slower rate. C. Fluorescence loss in photobleaching. Repetitive bleaching within a region of interest in one cellular compartment (cytoplasm in this example) is alternated with measurement of intensity in another cellular compartment (nucleus in this example). Fluorescence decays in the second compartment if there is a steady-state exchange between the two compartments. Faster loss in fluorescence generally corresponds to faster exchange. D. Fluorescence correlation spectroscopy. Photon counting in a diffraction-limited spot enables estimates of molecular residence times due to diffusion and/or binding. E. Fluorescence resonance energy transfer. Formation of a complex can be detected when donor (green) and acceptor (red) tags are brought in close proximity (second panel). Improper positioning of the tags can lead to no FRET even when a complex forms (third panel). F. Biosensors based on FRET for detection of small molecules. Binding of the small molecule(s) to the FRET sensor leads to a change in conformation and a corresponding change in FRET. In this example, phosphorylation due to the activity of a kinase would lead to a loss of FRET.