TY - JOUR
T1 - Analysis of fluorescence decay kinetics measured in the frequency domain using distributions of decay times
AU - Lakowicz, Joseph R.
AU - Cherek, Henryk
AU - Gryczynski, Ignacy
AU - Joshi, Nanda
AU - Johnson, Michael L.
N1 - Funding Information:
The authors thank Susan Frasier for her thoughtful suggestions for improving the manuscript. Supported by grants from the National Institutes of Health (GM 35154) and from the National Science Foundation (DMB-8502835 and DMB-8511065).
PY - 1987/10
Y1 - 1987/10
N2 - We describe the theoretical and practical aspects of analyzing complex fluorescence decay kinetics using continuous distributions of decay times. Our analysis uses frequency-domain data, provides for global analysis of multiple data sets and includes the possibility of excited-state processes. Simulated data were used to estimate the types of distributions which can be reasonably recovered from the measurements. Additionally, we describe a variety of distributions recovered from experimental data. For mixtures of one, two or three exponentially decaying fluorophores we recovered narrow lifetime distributions, which are essentially identical to a multi-exponential decay. Similarly, a two-state excited-state reaction also yielded a narrow distribution with negative preexponential factors. The presence of time-dependent spectral relaxation of labeled lipids results in a wide distribution of decay times, which becomes narrower for faster relaxation rates at higher temperatures. Hence, the decay-time distributions appear to be sensitive to the dynamics of the environment surrounding the fluorophore. Additionally, distributions of decay times were observed to result from transient effects in collisional quenching, from energy transfer in the presence of a range of donor-to-acceptor distances, and for several single-tryptophan proteins.
AB - We describe the theoretical and practical aspects of analyzing complex fluorescence decay kinetics using continuous distributions of decay times. Our analysis uses frequency-domain data, provides for global analysis of multiple data sets and includes the possibility of excited-state processes. Simulated data were used to estimate the types of distributions which can be reasonably recovered from the measurements. Additionally, we describe a variety of distributions recovered from experimental data. For mixtures of one, two or three exponentially decaying fluorophores we recovered narrow lifetime distributions, which are essentially identical to a multi-exponential decay. Similarly, a two-state excited-state reaction also yielded a narrow distribution with negative preexponential factors. The presence of time-dependent spectral relaxation of labeled lipids results in a wide distribution of decay times, which becomes narrower for faster relaxation rates at higher temperatures. Hence, the decay-time distributions appear to be sensitive to the dynamics of the environment surrounding the fluorophore. Additionally, distributions of decay times were observed to result from transient effects in collisional quenching, from energy transfer in the presence of a range of donor-to-acceptor distances, and for several single-tryptophan proteins.
KW - Decay-time distribution
KW - Fluorescence decay
KW - Frequency-domain fluorometry
KW - Kinetics
UR - http://www.scopus.com/inward/record.url?scp=0023627166&partnerID=8YFLogxK
U2 - 10.1016/0301-4622(87)80073-X
DO - 10.1016/0301-4622(87)80073-X
M3 - Article
C2 - 3689869
AN - SCOPUS:0023627166
SN - 0301-4622
VL - 28
SP - 35
EP - 50
JO - Biophysical Chemistry
JF - Biophysical Chemistry
IS - 1
ER -