TY - JOUR
T1 - Fluorescence of reduced nicotinamides using one- and two-photon excitation
AU - Kierdaszuk, Borys
AU - Malak, Henryk
AU - Gryczynski, Ignacy
AU - Callis, Patrik
AU - Lakowicz, Joseph R.
N1 - Funding Information:
This work was supported by grant No. RR-081 19 from National Institutes of Health, and by grant No. MCB-8804931 from the NSF; and in part by grants UM-141 and HMI 75 195-543401 (University of Warsaw. Poland). Dr. Borys Kierdaszuk is indebted to the Fulbright Scholar Program for Advanced Research Grant.
PY - 1996/11/29
Y1 - 1996/11/29
N2 - We examined the steady-state and time-resolved emission of NADH and NAME resulting from one-photon and two-photon excitation. Similar emission spectra were observed for both modes of excitation. The fundamental anisotropy of NADH is near 0.54 for two-photon excitation from 690 to 740 nm, which is 46% higher than the value of 0.37 observed for one-photon excitation. This observation of a higher anisotropy with two-photon excitation was consistent with INDO/SDCI calculations of the one- and two-photon transitions. Minor differences in the multi-exponential decays of NADH were observed for one- and two-photon excitation, but presently available resolution does not allow us to conclude the decays are distinct. NADH-LADH-IBA complex formation led to an order of magnitude larger of the average lifetimes of NADH fluorescence resulting from one- and two-photon excitation. Fluorescence intensity and fluorescence anisotropy decays of NADH was double-exponential for both modes of excitation and show that the observed heterogeneity of the fluorescence decay kinetics of reduced nicotinamides arises from the inherent photoprocess of the dihydronicotinamide chromophore and not due to any intramolecular interactions with adenine part of NADH. Such interactions are responsible for the depolarization of NADH fluorescence observed for excitation wavelength below 300 nm for OPE and 600 nm for TPE, respectively. NADH displays a low cross-section for two-photon excitation which suggests that fluorescence from NADH will be moderately difficult to observe with two-photon fluorescence microscopy, and may not interfere with observations of TPIF of other extrinsic probes used to label cells.
AB - We examined the steady-state and time-resolved emission of NADH and NAME resulting from one-photon and two-photon excitation. Similar emission spectra were observed for both modes of excitation. The fundamental anisotropy of NADH is near 0.54 for two-photon excitation from 690 to 740 nm, which is 46% higher than the value of 0.37 observed for one-photon excitation. This observation of a higher anisotropy with two-photon excitation was consistent with INDO/SDCI calculations of the one- and two-photon transitions. Minor differences in the multi-exponential decays of NADH were observed for one- and two-photon excitation, but presently available resolution does not allow us to conclude the decays are distinct. NADH-LADH-IBA complex formation led to an order of magnitude larger of the average lifetimes of NADH fluorescence resulting from one- and two-photon excitation. Fluorescence intensity and fluorescence anisotropy decays of NADH was double-exponential for both modes of excitation and show that the observed heterogeneity of the fluorescence decay kinetics of reduced nicotinamides arises from the inherent photoprocess of the dihydronicotinamide chromophore and not due to any intramolecular interactions with adenine part of NADH. Such interactions are responsible for the depolarization of NADH fluorescence observed for excitation wavelength below 300 nm for OPE and 600 nm for TPE, respectively. NADH displays a low cross-section for two-photon excitation which suggests that fluorescence from NADH will be moderately difficult to observe with two-photon fluorescence microscopy, and may not interfere with observations of TPIF of other extrinsic probes used to label cells.
KW - Anisotropy
KW - Anisotropy spectra
KW - Fluorescence
KW - One-photon excitation
KW - Reduced β-nicotinamide adenine dinucleotide
KW - Reduced β-nicotinamide mononucleotide
KW - Time-resolved fluorescence
KW - Two-photon excitation
UR - http://www.scopus.com/inward/record.url?scp=0030606333&partnerID=8YFLogxK
U2 - 10.1016/S0301-4622(96)02182-5
DO - 10.1016/S0301-4622(96)02182-5
M3 - Article
C2 - 8962467
AN - SCOPUS:0030606333
SN - 0301-4622
VL - 62
SP - 1
EP - 13
JO - Biophysical Chemistry
JF - Biophysical Chemistry
IS - 1-3
ER -