TY - CHAP
T1 - Quantitative ratiometric imaging of FRET-biosensors in living cells
AU - Spiering, Désirée
AU - Bravo-Cordero, Jose Javier
AU - Moshfegh, Yasmin
AU - Miskolci, Veronika
AU - Hodgson, Louis
N1 - Funding Information:
This work was funded by GM093121 (D. S., J. J. B.-C., and L. H.), “Sinsheimer Foundation Young Investigator Award” (L. H.), and T32GM007491 (Y. M. and V. M.).
PY - 2013
Y1 - 2013
N2 - Biosensors based on FRET have been useful in deciphering the dynamics of protein activation events in living cells at subcellular resolutions and in time scales of seconds. These new systems allow observations of dynamic processes which were not possible previously using more traditional biochemical and cell biological approaches. The image data sets obtained from these sensors require careful processing in order to represent the actual protein activation events. Here, we will cover the basic approaches useful for processing the raw image data sets into relativistic ratiometric measurements, capable of depicting relative differences in the protein activation states within a single cell. We will discuss in detail the approaches for genetically encoded, single-chain biosensor systems based on FRET, as well as those that are based on intermolecular, dual-chain design. Additionally, the same analysis can be utilized for biosensor systems using solvatochromic dyes (Nalbant, Hodgson, Kraynov, Toutchkine, & Hahn, 2004), useful for detection of endogenous protein activation states.
AB - Biosensors based on FRET have been useful in deciphering the dynamics of protein activation events in living cells at subcellular resolutions and in time scales of seconds. These new systems allow observations of dynamic processes which were not possible previously using more traditional biochemical and cell biological approaches. The image data sets obtained from these sensors require careful processing in order to represent the actual protein activation events. Here, we will cover the basic approaches useful for processing the raw image data sets into relativistic ratiometric measurements, capable of depicting relative differences in the protein activation states within a single cell. We will discuss in detail the approaches for genetically encoded, single-chain biosensor systems based on FRET, as well as those that are based on intermolecular, dual-chain design. Additionally, the same analysis can be utilized for biosensor systems using solvatochromic dyes (Nalbant, Hodgson, Kraynov, Toutchkine, & Hahn, 2004), useful for detection of endogenous protein activation states.
KW - Biosensors
KW - FRET
KW - Ratio Imaging
KW - Rho GTPase
UR - http://www.scopus.com/inward/record.url?scp=84881501195&partnerID=8YFLogxK
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U2 - 10.1016/B978-0-12-407761-4.00025-7
DO - 10.1016/B978-0-12-407761-4.00025-7
M3 - Chapter
C2 - 23931524
AN - SCOPUS:84881501195
T3 - Methods in Cell Biology
SP - 593
EP - 609
BT - Methods in Cell Biology
PB - Academic Press Inc.
ER -