Fu Dan, Ye Tong, Matthews Thomas E, Yurtsever Gunay, Warren Warren S
Princeton University, Department of Chemistry, Princeton, New Jersey 08544, USA.
J Biomed Opt. 2007 Sep-Oct;12(5):054004. doi: 10.1117/1.2780173.
We develop a new approach in imaging nonfluorescent species with two-color two-photon and excited state absorption microscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes places in the medium. We can easily measure 10(-6) absorption changes caused by either two-photon absorption or excited-state absorption with a RF lock-in amplifier. Sepia melanin is studied in detail as a model system. Spectroscopy studies on the instantaneous two-photon absorption (TPA) and the relatively long-lived excited-state absorption (ESA) of melanin are carried out in solution, and imaging capability is demonstrated in B16 cells. It is found that sepia melanin exhibits two distinct excited states with different lifetimes (one at 3 ps, one lasting hundreds of nanoseconds) when pumped at 775 nm. Its characteristic TPA/ESA enables us to image its distribution in cell samples with high resolution comparable to two-photon fluorescence microscopy (TPFM). This new technique could potentially provide valuable information in diagnosing melanoma.
我们开发了一种利用双色双光子和激发态吸收显微镜对非荧光物质进行成像的新方法。如果两个不同颜色的同步锁模脉冲序列中的一个进行强度调制，当介质中发生非线性吸收时，这种调制会转移到另一个脉冲序列。我们可以使用射频锁相放大器轻松测量由双光子吸收或激发态吸收引起的10^(-6)吸收变化。以乌贼墨黑色素作为模型系统进行了详细研究。在溶液中对黑色素的瞬时双光子吸收（TPA）和相对长寿命的激发态吸收（ESA）进行了光谱研究，并在B16细胞中展示了成像能力。研究发现，当在775nm波长泵浦时，乌贼墨黑色素呈现出具有不同寿命的两种不同激发态（一种寿命为3ps，另一种持续数百纳秒）。其独特的TPA/ESA特性使我们能够以与双光子荧光显微镜（TPFM）相当的高分辨率对其在细胞样品中的分布进行成像。这项新技术有可能为黑色素瘤的诊断提供有价值的信息。
