以多光子螢光生命週期影像顯微術來鑑別正常與口腔癌細胞

Abstract

我們成功的以雙光子螢光生命週期影像之成份來辨別不同形態與階段的口腔癌細胞，並以單光子、雙光子螢光光譜與螢光生命週期研究了許多會造成其差異的因素。使用紅外光的鉻貴橄欖石飛秒鎖模雷射作為激發雙光子螢光的光源，可以避免在活體樣品中對細胞造成光傷害，並能有效的激發出細胞中膽綠素的紅螢光。我們以DOK、OECM-1、SCC-15、HSC-3及SG這五株人體口腔株化細胞株作為初步辨別，並以兩種生命週期(t1、t2)的成分來分析曲線。藉由細胞中紅螢光生命週期影像的表現，對應到膽綠素在細胞代謝中的差異，細胞中由於膽綠素代謝的能力不同，並會結合細胞中不同的蛋白而產生不同的生命週期，我們發現，DOK與OECM-1的t1時間較長(~500ps)，SCC-15與HSC-3相對的較短(~370ps)，而正常口腔細胞SG有明顯更短的t1(~270ps)，輔以t2的結果作分類，便可明顯的區別出五株細胞。藉由螢光生命週期影像不同的表現，我們未來可以在活體中檢測細胞的狀態，並可能早期的檢驗出類癌症而不需要在病變後才以粘膜來確認。

We have observed the red fluorescence of five oral cell lines with multi-photon fluorescence lifetime and intensity. With lifetime trace, single photon, and two photon fluorescence spectrum, we found out that biliverdin is one of red fluorescence source (exciting spectrum peak around ~660nm). Four oral cancer cells from tongue and a non-cancer oral cell lines: S-G (normal oral cell as control group), DOK (pre-cancer oral cell), OECM-1, SCC-15, and HSC-3(different stage of malignancy) were investigated by a femtosecond Cr:forsterite laser -based multi-photon fluorescence lifetime image. Due to shift of metabolic status, we used two component to fit lifetime histgrom and detected that DOK and OECM-1 has longer t1 lifetime(~500ps), SCC-15 and HSC-3 have relatively short 370ps, and normal oral, S-G cells have much shorter t1 lifetime(~270ps). In addition, S-G cell have visible shorter t2 lifetime (~1.6ns), cancer cells general longer (~1.9ns). With our study result, we believe this lifetime of biliverdin fluorescence is potential to serve as an diagnostic index on the stage and malignancy of oral cancers in the future.