TY - JOUR
T1 - Measurements of diffusion and chemical kinetics by fluorescence photobleaching recovery and fluorescence correlation spectroscopy
AU - Petersen, N. O.
AU - Elson, E. L.
N1 - Funding Information:
The development of FCS and FPR techniques has involved a large number of researchers in many laboratories across the world. This work was supported by NIH Grants GM 21661, GM 30299, and GM 27160 (to ELE), and NSERC, Canada Grants U0109 and E5837 as well as ADF (UWO) Grant 81-10 (to NOP).
PY - 1986/1/1
Y1 - 1986/1/1
N2 - Chemical kinetics and rates of lateral transport (diffusion or flow) can be measured by observing spontaneous fluctuations in reactant concentrations which occur even when the system rests in equilibrium. This can be accomplished by using fluorescence to monitor the numbers of molecules of specified types in a defined open region of the reaction system. The feasibility of this approach has been demonstrated, and the method, called fluorescence correlation spectroscopy (FCS) has found practical application. Because it is based on microscopic fluctuations FCS is an intrinsically statistical method, however, and so requires that the reaction system be stable during prolonged measuring intervals. For studies of less stable systems (e.g., living cells in culture) a method has been developed which is based on observation of the rate of relaxation of a local macroscopic concentration gradient. The gradient is produced by rapidly photolyzing a portion of the reactant molecules in a small open region of the system. This method, which we call fluorescence photobleaching recovery (FPR), yields results which are approximately equivalent to those obtained by FCS. Theoretical advantages of FCS are balanced by practical experimental advantages of FPR. The major applications of this combined technology up to now have been in studies of lateral diffusion in model membranes (FCS and FPR) and on animal cell surfaces (FPR).
AB - Chemical kinetics and rates of lateral transport (diffusion or flow) can be measured by observing spontaneous fluctuations in reactant concentrations which occur even when the system rests in equilibrium. This can be accomplished by using fluorescence to monitor the numbers of molecules of specified types in a defined open region of the reaction system. The feasibility of this approach has been demonstrated, and the method, called fluorescence correlation spectroscopy (FCS) has found practical application. Because it is based on microscopic fluctuations FCS is an intrinsically statistical method, however, and so requires that the reaction system be stable during prolonged measuring intervals. For studies of less stable systems (e.g., living cells in culture) a method has been developed which is based on observation of the rate of relaxation of a local macroscopic concentration gradient. The gradient is produced by rapidly photolyzing a portion of the reactant molecules in a small open region of the system. This method, which we call fluorescence photobleaching recovery (FPR), yields results which are approximately equivalent to those obtained by FCS. Theoretical advantages of FCS are balanced by practical experimental advantages of FPR. The major applications of this combined technology up to now have been in studies of lateral diffusion in model membranes (FCS and FPR) and on animal cell surfaces (FPR).
UR - http://www.scopus.com/inward/record.url?scp=0022487732&partnerID=8YFLogxK
U2 - 10.1016/0076-6879(86)30021-1
DO - 10.1016/0076-6879(86)30021-1
M3 - Article
C2 - 3773744
AN - SCOPUS:0022487732
SN - 0076-6879
VL - 130
SP - 454
EP - 484
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -