整合高頻調變多色發光二極體與頻率域螢光生命週期量測技術之系統架設與生醫應用

Abstract

本論文發展射頻調變多色發光二極體之光源架構，整合頻率域零差量測技術，以達到微型化之螢光生命週期量測系統。發光二極體之應用可以降低螢光生命週期量測系統之價格；並且，本論文所發展的多色光源特性，將可達成更便利以及更廣泛的生醫應用。發光二極體之特性在於發光功率提升時，其頻率反應將大幅下降，特別是在綠光波段之發光二極體。為了量測奈秒解析之螢光生命週期訊號，本論文克服此困難，達成高功率且高頻反應之發光二極體光源設計。同時，本論文將具有多色晶粒之發光二極體元件搭配光導柱，使得不同波長之光源在同一光路上，且聚焦在相同位置。本論文完成兩部分的儀器架設與驗證。第一部分是射頻調變多色發光二極體光源搭配光電倍增管，並使用零差模式進行螢光生命週期量測。由實驗結果，驗證了此系統可進行即時且具備奈秒解析度之螢光生命週期量測；並且，此系統成功觀測螢光共振能量轉移現象、以及光合作用中的非光化學反應的能量淬滅機制。本論文的第二部分整合顯微鏡系統以及射頻調變多色發光二極體光源，發展一套螢光生命週期顯微影像系統。此系統應用螢光小球以及免疫螢光染色細胞樣品進行驗證與量測。本論文使用多色發光二極體光源量測螢光標定細胞時，不必變動光路架構，即可進行細胞不同構造標定的螢光分子之螢光生命週期量測，並利用影像化之螢光生命週期訊號大小以區分細胞構造。未來將可以藉由本論文所提出之發光二極體光源，將螢光生命週期技術更廣泛的應用在生醫檢測。

In this thesis, I developed a homodyne-based fluorescence lifetime measurement system, in cooperation with ratio frequency modulated multi-color light emitting diode (LED) light source. This makes possible to achieve miniaturized fluorescence lifetime measurement systems. Advantage of LEDs include low price, high integration capability, and application towards multi-color light source design. LEDs with higher illumination power usually have lower frequency responses, which hampers applications of LED in nanosecond-resolved measurements. I used a new LED design to overcome this problem; moreover, I included a homogenizing rod in the optical design, so that multi-color LEDs produced uniform light on the same focal spot. In this thesis, two different fluorescence lifetime measurement system were built; the systems were applied to demonstrate quantitative measurements of molecular dynamics from fluorescence lifetime signals. The first part of this work was to setup a photomultiplier (PMT) based fluorescence lifetime measurement system. The system was applied for measuring Förster resonance energy transfer (FRET) and the non-photochemical quenching (NPQ) pathways of photosynthesis. The second part of this work was building a fluorescence lifetime imaging microscopy (FLIM) with our ratio frequency modulated multi-color LED light source. The system was applied for lifetime measurements of fluorescence beads and fluorescence labelled fibroblasts. Fluorescence lifetime images of actin filaments and mitochondria of the fibroblasts were measured using the multi-color and switchable LED light source. The results showed that our multi-color LED based FLIM system successfully measured the fluorescence lifetime not only accurately, but also with subcellular lifetime resolution. In this thesis, I developed a new design on the radio frequency modulated multi-color LED, which might bring about wider application in biomedical detection for nanosecond-resolved fluorescence lifetime techniques.