Nonlinear optical imaging and spectral-lifetime computational analysis of endogenous and exogenous fluorophores in breast cancer.
Multiphoton laser scanning microscopy (MPLSM) utilizing techniques such as multiphoton excitation (MPE), second harmonic generation (SHG), and multiphoton fluorescence lifetime imaging and spectral lifetime imaging (FLIM and SLIM, respectively) are greatly expanding the degree of information obtainable with optical imaging in biomedical research. The application of these nonlinear optical approaches to the study of breast cancer holds particular promise. These noninvasive, multidimensional techniques are well suited to image exogenous fluorophores that allow relevant questions regarding protein localization and signaling to be addressed both in vivo and in vitro. Furthermore, MPLSM imaging of endogenous signals from collagen and fluorophores such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD), address important questions regarding the tumor-stromal interaction and the physiologic state of the cell. We demonstrate the utility of multimodal MPE/SHG/FLIM for imaging both exogenous and/or endogenous fluorophores in mammary tumors or relevant 3-D systems. Using SLIM, we present a method for imaging and differentiating signals from multiple fluorophores that can have overlapping spectra via SLIM Plotter-a computational tool for visualizing and analyzing large spectral-lifetime data sets.
Provenzano
Paolo P
PP
University of Wisconsin, Departments of Pharmacology and Biomedical Engineering and Laboratory for Optical and Computational Instrumentation and Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin 53706, USA.
Rueden
Curtis T
CT
Trier
Steve M
SM
Yan
Long
L
Ponik
Suzanne M
SM
Inman
David R
DR
Keely
Patricia J
PJ
Eliceiri
Kevin W
KW
D000465:Algorithms; D001943:Breast Neoplasms / Q000175:diagnosis*; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D017711:Nonlinear Dynamics; D012984:Software
D049452:Green Fluorescent Proteins