Ramanujan V Krishnan, Zhang Jian-Hua, Biener Eva, Herman Brian
University of Texas Health Science Center at San Antonio, Department of Cellular and Structural Biology, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA.
J Biomed Opt. 2005 Sep-Oct;10(5):051407. doi: 10.1117/1.2098753.
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.
浑浊组织存在严重的强吸收和散射问题，这使得稳态荧光成像方法在对几十微米以上深度的组织层进行成像时不太成功。随着从细胞成像发展到组织成像再到全动物成像，会出现各种复杂情况，包括组织中巨大的自发荧光背景以及感兴趣区域的信号较弱。由于基于稳态强度的方法无法区分来自荧光团的光子和自发荧光背景，几乎不可能分离这两种信号。我们描述了时域中的多光子荧光寿命成像方法，以展示在区分自发荧光背景和荧光信号时的荧光寿命对比度。由于荧光团和自发荧光贡献的光物理机制不同，仅基于它们的衰减常数在每个像素中分离这两种贡献是可行的，而不会降低信噪比。我们展示了初步的寿命测量结果，以表征从小鼠模型获得的各种细胞系和离体组织中的自发荧光。这些结果共同表明了一个新的方向，即无需任何外源性染色就能从内源性组织荧光中获取定量信息。讨论了这种方法在代谢氧化还原成像和疾病诊断中的前景。
