發(fā)布時間：2018-06-15 16:25

  本文選題：時間相關(guān)單光子技術(shù)(TCSPC) + 熒光壽命成像��； 參考：《華中科技大學》2013年博士論文

【摘要】：熒光壽命成像技術(shù)因其具有對微環(huán)境敏感、空間分辨率高、不依賴于熒光物濃度和激發(fā)光強度等特點,使它成為生物微環(huán)境測量的一種重要工具。然而,基于TCSPC的FLIM技術(shù)存在成像信噪比低、可測量最短壽命受響應(yīng)函數(shù)寬度限制等問題,導致其測量精度較低。本文通過增強激發(fā)熒光效率、降低系統(tǒng)噪聲和優(yōu)化分析方法三個方面發(fā)展出高精度熒光壽命成像方法,并將該方法用于熒光鼠腦樣本制備方案篩選、FRET效率準確性測量和超短熒光壽命測量的研究中。本文內(nèi)容如下： (1)發(fā)展了一種簡單易行熒光壽命成像系統(tǒng)的性能評估及優(yōu)化方法。定量評估了TCSPC-FLIM的空間分辨率、成像信噪比、測量準確性等,發(fā)現(xiàn)成像信噪比對系統(tǒng)的成像精度影響最大。提出了利用降低系統(tǒng)噪聲和增強激發(fā)熒光效率的方法兩種方法來提高成像信噪比。結(jié)果表明對探測器制冷使圖像信噪比提高了4倍,單光子激發(fā)比雙光子激發(fā)顯著增強了熒光激發(fā)效率。 (2)提出將高精度熒光壽命成像方法用于熒光鼠腦樣本制備方案篩選應(yīng)用中,該方法相比于熒光強度成像方法使測量更精確、更快捷。定量比較了三種常用固定劑和包埋劑對熒光特性的影響,發(fā)現(xiàn)利用4%多聚甲醛固定和GMA(乙二醇甲基丙烯酸酯)包埋的鼠腦腦片背景熒光最弱。該方法大大縮短了實驗樣本量,使實驗周期更短。 (3)針對高精度熒光壽命成像技術(shù)在熒光共振能量轉(zhuǎn)移研究中的應(yīng)用,對我們的TCSPC-FLIM系統(tǒng)進行了優(yōu)化,使其適用于高精度研究CFP-YFP FRET效率變化。研究表明,我們的TCSPC-FLIM系統(tǒng)可以測量3%的FRET效率變化,初步證明了高精度TCSPC-FLIM方法在微小FRET效率變化測量中的有效性。 (4)研究了系統(tǒng)極限時間分辨率與熒光信號強度之間的關(guān)系,建立了一種超高精度壽命分析方法。比較了一階力矩法(M1)與傳統(tǒng)的最小二乘擬合法的熒光壽命定量分析能力,發(fā)現(xiàn)M1方法的測量精度僅受信號強度影響,而與時間通道無關(guān)。進一步發(fā)現(xiàn),M1方法在下面兩種情況下明顯優(yōu)于擬合法：壽命為70ps-3ns；信號強度小于103個光子。最后,對超短熒光壽命樣品(CdS納米線)進行定量分析,證明了M1方法在研究短壽命樣本時比擬合法更優(yōu)。該方法為精確測量短壽命熒光樣品或是微小熒光壽命變化方面的研究提供新的途徑。
[Abstract]:Because of its sensitivity to microenvironment, high spatial resolution, no dependence on fluorescence concentration and intensity of luminescence, fluorescence lifetime imaging technology makes it an important tool for the measurement of biological microenvironment. However, the FLIM technology based on TCSPC has the problems of low imaging signal to noise ratio and the limitation of the shortest lifetime of the response function, such as the width of response function. In this paper, the high precision fluorescence lifetime imaging method is developed in this paper by enhancing the excitation fluorescence efficiency, reducing the noise of the system and optimizing the analysis method in three aspects. This method is used in the screening of fluorescent rat brain sample preparation scheme, FRET efficiency accuracy measurement and ultra short fluorescence lifetime measurement. Below:
(1) the performance evaluation and optimization method of a simple and easy fluorescence lifetime imaging system is developed. The spatial resolution, image signal to noise ratio and accuracy of TCSPC-FLIM are evaluated quantitatively, and the imaging accuracy of the imaging signal noise ratio system is found to be the most influential. Two methods to reduce the noise of the system and enhance the efficiency of the excitation fluorescence are proposed. The method is used to improve the signal to noise ratio of the imaging. The results show that the signal to noise ratio of the image is increased by 4 times for the detector refrigeration, and the fluorescence excitation efficiency is significantly enhanced by the single photon excitation than the two-photon excitation.
(2) a high precision fluorescence lifetime imaging method is proposed for the screening of fluorescent rat brain samples. Compared with the fluorescence intensity imaging method, this method makes the measurement more accurate and faster. The quantitative comparison of the effects of three common fixative and embedding agents on the fluorescence characteristics, and the use of 4% polyoxymethylene and GMA (ethylene glycol methyl C) The background fluorescence of rat brain slices embedded in enolates is the weakest. This method greatly reduces the number of experimental samples and makes the experimental cycle shorter.
(3) in view of the application of high precision fluorescence lifetime imaging technology in the study of fluorescence resonance energy transfer, our TCSPC-FLIM system is optimized so that it is suitable for high precision study of CFP-YFP FRET efficiency change. The study shows that our TCSPC-FLIM system can measure the FRET efficiency of 3%, and the high precision TCSPC-FLIM method has been proved preliminarily. The effectiveness of the measurement of small FRET efficiency changes.
(4) the relationship between the limit time resolution of the system and the intensity of the fluorescence signal is studied. A super high precision life analysis method is established. The quantitative analysis ability of the first order moment method (M1) and the traditional least square method is compared. It is found that the measurement precision of the M1 method is only influenced by the intensity of the signal, but is independent of the time channel. One step is to find that the M1 method is obviously superior to the fitting method in the following two cases: the lifetime is 70ps-3ns and the signal intensity is less than 103 photons. Finally, the quantitative analysis of the ultrashort fluorescent life sample (CdS nanowires) proves that the M1 method is more effective than the short life sample. This method is a precise measurement of short life fluorescence samples or It provides a new way for the study of micro fluorescence lifetime changes.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R310

【參考文獻】

相關(guān)期刊論文 前1條

1 徐德勝;;電子冷凍與半導體[J];電子技術(shù);1983年03期

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本文編號：2022668