以多光子螢光顯微術早期診斷急性腸繫膜缺血

Abstract

我們成功地以雙光子顯微術發展早期偵測急性腸繫膜缺血的技術，以單光子、雙光子螢光光譜、螢光激發光譜與螢光生命週期研究了不同時間程度的缺血及再灌流之差異。使用紅外光的鈦藍寶石飛秒鎖模雷射作為激發雙光子螢光的光源，可以避免對於血清樣品造成光傷害，並能有效的激發出血清樣品中黃素衍伸物的黃螢光。我們發現，隨著缺血時間變長，黃螢光的強度也隨之增強，再灌流後，隨著再灌流時間變長，黃螢光強度則反之下降。在雙光子螢光激發光譜中，缺血前與再灌流之後的光譜呈現類似的趨勢，相對於前兩者，缺血階段的螢光激發光譜有著一個特殊的趨勢，它在940奈米附近有個較高的峰值。藉由螢光生命週期的表現，比較缺血前、缺血階段、再灌流階段之標準化後的螢光衰減曲線，結果顯示幾乎重疊，代表三個不同階段並沒有新的螢光物質產生。由以上結果得知，缺血階段的黃螢光強度增強是由於原先即存在於血清中螢光物質濃度的增加。更進一步，我們嘗試以高效液相層析質譜儀標定造成黃螢光強度改變的螢光物質，初步結果判斷為核黃素。在未來的臨床應用上，即可以雙光子螢光激發光譜的表現檢測血液，以一種非侵入的方式，早期診斷鑑別急性腸繫膜缺血。

We have observed the yellow fluorescence of mesenteric ischemia, and ischemia-reperfusion rats with multi-photon fluorescence lifetime and intensity, which based on a femtosecond Ti:sapphire laser. With lifetime trace, single photon, two-photon fluorescence spectrum, and two-photon fluorescence excitation spectrum, we found out that intensity of yellow fluorescence increases during ischemia state, and recovered gradually after reperfusion. The characteristic change of the autofluorescence intensity ratio in two-photon excitation spectrum, which observed only in the ischemia state instead of normal state or reperfusion state. We compared fluorescence decay traces of preishemia, ischemia state, and ischemia-reperfusion state, the results showed these traces are almost overlapping, which indicated that there is no any new fluorescent substance producing during the whole process. From above results, the elevating intensity of ischemia state is due to the concentration of original fluorescent substances increase. We tried to identify that fluorescent substance by High-performance Liquid Chromatography-Mass Spectrophotometer (HPLC-MS), and the preliminary result is riboflavin. With our study result, we believe two-photon fluorescence excitation spectrum, a non-invasive way, is potential to serve as a tool for early diagnosis of acute mesenteric ischemia.