We examined the steady-state and time-resolved emission of NADH and NAMH 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. 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 average lifetimes of NADH fluorescence resulting from one- and two-photon excitation. Intramolecular interactions with adenine part of NADH 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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR008119-08
Application #
6315378
Study Section
Project Start
2000-04-01
Project End
2001-03-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
8
Fiscal Year
2000
Total Cost
$28,294
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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