Multiphoton fluorescence lifetime contrast in deep tissue imaging: prospects in redox imaging and disease diagnosis.
Turbid tissues pose serious problems of strong absorption and scattering that make steady state fluorescence imaging methods less successful in imaging tissue layers deeper than a few tens of micrometers. Complications arise as one progresses from imaging cells to tissues to whole animal--which include enormous autofluorescence background in tissues and poor signal from regions of interest. Since the steady state, intensity-based methods cannot discriminate the photons arising from the fluorophores and the autofluorescence background, it is almost impractical to isolate these two signals. We describe multiphoton fluorescence lifetime imaging methods in the time domain to demonstrate fluorescence lifetime contrast in discriminating autofluorescence background from the fluorescent signals. Since the photophysical schemes of the fluorophore and autofluorescence contributions are distinct, it is feasible to isolate these two contributions in every pixel based only on their decay constants without compromising the SNR. We present preliminary lifetime measurements to characterize autofluorescence in various cell lines and ex vivo tissues obtained from mouse models. Together, these results suggest a novel direction in obtaining quantitative information from endogenous tissue fluorescence without any exogenous staining. The prospects for this approach in metabolic redox imaging and disease diagnosis are discussed.
Ramanujan
V Krishnan
VK
University of Texas Health Science Center at San Antonio, Department of Cellular and Structural Biology, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA. krsna@uthscsa.edu
Zhang
Jian-Hua
JH
Biener
Eva
E
Herman
Brian
B
D000818:Animals; D003952:Diagnostic Imaging / Q000379:methods* / Q000639:trends; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D010084:Oxidation-Reduction