超分辨光學(xué)波動(dòng)顯微成像技術(shù)研究
發(fā)布時(shí)間：2018-10-21 09:38
【摘要】：超分辨熒光波動(dòng)顯微技術(shù)(Super-resolution Optical Fluctuation Imaging,SOFI)以熒光波動(dòng)統(tǒng)計(jì)分析為基礎(chǔ),獲取超分辨圖像。通過(guò)拍攝一段連續(xù)的圖像序列,使用累積算法對(duì)熒光波動(dòng)進(jìn)行統(tǒng)計(jì)分析,分析比較圖像序列在這段時(shí)間內(nèi)每個(gè)像素點(diǎn)的熒光強(qiáng)度閃爍情況,根據(jù)階數(shù)不同,得到不同程度的超分辨圖像,具有成像速度較快、信噪比較高、光毒性較小、系統(tǒng)復(fù)雜度較低等優(yōu)點(diǎn),可長(zhǎng)時(shí)間觀測(cè)活體生物樣本,同時(shí)實(shí)現(xiàn)多個(gè)熒光分子高速成像。通過(guò)此課題研究的展開,可以提高SOFI成像的實(shí)時(shí)性,擴(kuò)展其應(yīng)用范圍,也為將來(lái)儀器的研發(fā)提供理論指導(dǎo)。本文在對(duì)SOFI技術(shù)調(diào)研的基礎(chǔ)上,分別從SOFI成像原理,圖像重構(gòu)算法及實(shí)驗(yàn)裝置三個(gè)方面對(duì)超分辨光學(xué)波動(dòng)顯微成像技術(shù)進(jìn)行了深入的研究。主要研究?jī)?nèi)容為:(1)對(duì)SOFI成像理論深入研究,結(jié)合SOFI成像條件,分析SOFI圖像重構(gòu)算法原理、樣本的光學(xué)特性和有效像元尺寸對(duì)圖像的影響。(2)研究了兩種提高SOFI圖像空間分辨率的圖像重構(gòu)算法,高階SOFI算法和傅里葉SOFI算法。對(duì)仿真數(shù)據(jù)進(jìn)行驗(yàn)證,分析兩種算法的優(yōu)缺點(diǎn)及局限性。(3)為了提高SOFI算法的實(shí)時(shí)性,提出基于空間disk濾波的SOFI算法。對(duì)獲取的多幀圖像先進(jìn)行濾波處理,再根據(jù)多幀圖像中熒光粒子的時(shí)間自相關(guān)性進(jìn)行SOFI算法處理,可快速得到高信噪比的超分辨圖像。通過(guò)數(shù)據(jù)分析驗(yàn)證了基于空間高斯disk濾波的SOFI算法可以在低信噪比的圖像序列中快速得到信噪比較高的超分辨圖像。(4)基于倒置熒光顯微鏡,搭建SOFI系統(tǒng)成像光路,采用汞燈作為激發(fā)光,使用商用熒光量子點(diǎn)QDot525作為樣本,利用sCMOS相機(jī)記錄寬場(chǎng)顯微圖像。對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行分析找到最佳的圖像幀數(shù)、曝光時(shí)間、有效像元尺寸。結(jié)合本文提出的算法和SOFI圖像的影響因素,達(dá)到能夠在最短時(shí)間內(nèi)得到最優(yōu)的超分辨圖像的目的。
[Abstract]:Super-resolution fluorescence wave microscopy (Super-resolution Optical Fluctuation Imaging,SOFI) is based on statistical analysis of fluorescence fluctuations to obtain super-resolution images. By taking a continuous sequence of images and using the cumulative algorithm to analyze the fluorescence fluctuation, the fluorescence intensity flicker of each pixel in the image sequence during this period is analyzed and compared. According to the different order, the fluorescence intensity flicker of each pixel in the image sequence is analyzed and compared. The super-resolution images with different degrees have the advantages of high imaging speed, high signal-to-noise ratio, low phototoxicity and low system complexity. It can be used to observe living biological samples for a long time and to realize high speed imaging of several fluorescent molecules at the same time. Through the development of this research, the real-time of SOFI imaging can be improved, its application scope can be expanded, and the theoretical guidance for the future research and development of the instrument can also be provided. Based on the investigation of SOFI technology, the super-resolution optical wave microscopic imaging technology is studied from three aspects: the principle of SOFI imaging, the image reconstruction algorithm and the experimental device. The main contents are as follows: (1) the theory of SOFI imaging is deeply studied, and the principle of SOFI image reconstruction algorithm is analyzed according to the conditions of SOFI imaging. The influence of the optical properties of the sample and the size of the effective pixel on the image. (2) two image reconstruction algorithms, high-order SOFI algorithm and Fourier SOFI algorithm, are studied to improve the spatial resolution of SOFI images. The simulation data are verified and the advantages and disadvantages of the two algorithms are analyzed. (3) in order to improve the real-time performance of the SOFI algorithm, a SOFI algorithm based on spatial disk filtering is proposed. The acquired multi-frame images are filtered first and then processed by SOFI algorithm according to the time autocorrelation of fluorescent particles in multi-frame images. The super-resolution images with high signal-to-noise ratio can be obtained quickly. The data analysis shows that the SOFI algorithm based on spatial Gao Si disk filter can quickly obtain super-resolution images with high SNR in low SNR image sequences. (4) based on inverted fluorescence microscope, the imaging optical path of SOFI system is built. Mercury lamp was used as excitation light and commercial fluorescent quantum dot (QDot525) was used as sample. Wide field microscopic images were recorded by sCMOS camera. The experimental results are analyzed to find the best image frame number, exposure time and effective pixel size. Combined with the algorithm presented in this paper and the influencing factors of SOFI images, the optimal super-resolution image can be obtained in the shortest time.
【學(xué)位授予單位】：南京理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TP391.41
本文編號(hào)：2284716
[Abstract]:Super-resolution fluorescence wave microscopy (Super-resolution Optical Fluctuation Imaging,SOFI) is based on statistical analysis of fluorescence fluctuations to obtain super-resolution images. By taking a continuous sequence of images and using the cumulative algorithm to analyze the fluorescence fluctuation, the fluorescence intensity flicker of each pixel in the image sequence during this period is analyzed and compared. According to the different order, the fluorescence intensity flicker of each pixel in the image sequence is analyzed and compared. The super-resolution images with different degrees have the advantages of high imaging speed, high signal-to-noise ratio, low phototoxicity and low system complexity. It can be used to observe living biological samples for a long time and to realize high speed imaging of several fluorescent molecules at the same time. Through the development of this research, the real-time of SOFI imaging can be improved, its application scope can be expanded, and the theoretical guidance for the future research and development of the instrument can also be provided. Based on the investigation of SOFI technology, the super-resolution optical wave microscopic imaging technology is studied from three aspects: the principle of SOFI imaging, the image reconstruction algorithm and the experimental device. The main contents are as follows: (1) the theory of SOFI imaging is deeply studied, and the principle of SOFI image reconstruction algorithm is analyzed according to the conditions of SOFI imaging. The influence of the optical properties of the sample and the size of the effective pixel on the image. (2) two image reconstruction algorithms, high-order SOFI algorithm and Fourier SOFI algorithm, are studied to improve the spatial resolution of SOFI images. The simulation data are verified and the advantages and disadvantages of the two algorithms are analyzed. (3) in order to improve the real-time performance of the SOFI algorithm, a SOFI algorithm based on spatial disk filtering is proposed. The acquired multi-frame images are filtered first and then processed by SOFI algorithm according to the time autocorrelation of fluorescent particles in multi-frame images. The super-resolution images with high signal-to-noise ratio can be obtained quickly. The data analysis shows that the SOFI algorithm based on spatial Gao Si disk filter can quickly obtain super-resolution images with high SNR in low SNR image sequences. (4) based on inverted fluorescence microscope, the imaging optical path of SOFI system is built. Mercury lamp was used as excitation light and commercial fluorescent quantum dot (QDot525) was used as sample. Wide field microscopic images were recorded by sCMOS camera. The experimental results are analyzed to find the best image frame number, exposure time and effective pixel size. Combined with the algorithm presented in this paper and the influencing factors of SOFI images, the optimal super-resolution image can be obtained in the shortest time.
【學(xué)位授予單位】：南京理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TP391.41
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