Fluorescence-based transient state monitoring for biomolecular spectroscopy and imaging.
To increase read-out speed, sensitivity or specificity, an often applied strategy in fluorescence-based biomolecular spectroscopy and imaging is to simultaneously record two or more of the fluorescence parameters: intensity, lifetime, polarization or wavelength. This review highlights how additional, to-date largely unexploited, information can be extracted by monitoring long-lived, photo-induced transient states of organic dyes and their dynamics. Two major approaches are presented, where the transient state information is obtained either from fluorescence fluctuation analysis or by recording the time-averaged fluorescence response to a time-modulated excitation. The two approaches combine the detection sensitivity of the fluorescence signal with the environmental sensitivity of the long-lived transient states. For both techniques, proof-of-principle experiments are reviewed, and advantages, limitations and possible applications for biomolecular cellular biology studies are discussed.
Widengren
Jerker
J
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology (KTH), Albanova University Center, Stockholm 106 91, Sweden. jwideng@kth.se
D003952:Diagnostic Imaging / Q000379:methods*; D005453:Fluorescence; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods