title: Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein.
abstract: Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Shaohui
authors.initials: S
authors.lastname: Huang
authors.forename: Ahmed A
authors.initials: AA
authors.lastname: Heikal
authors.forename: Watt W
authors.initials: WW
authors.lastname: Webb
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D005420:Flavoproteins / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D009206:Myocardium / Q000166:cytology; D009243:NAD / Q000032:analysis*; D009249:NADP / Q000032:analysis*; D017785:Photons; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005420:Flavoproteins; D009243:NAD; D009249:NADP