本文選題：超分辨定位成像　切入點：弱光探測器　出處：《華中科技大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：超分辨定位成像(Super-resolution localization microscopy,或簡稱定位成像)技術(shù)實現(xiàn)了納米量級的空間分辨率,成為生命科學(xué)領(lǐng)域極具潛力的研究工具之一。定位成像本質(zhì)上是依賴單分子成像實現(xiàn)的,然而單分子的熒光信號通常在幾百至幾千個光子,并且要覆蓋多個像素,所以單分子微弱信號的探測是實現(xiàn)定位成像的關(guān)鍵技術(shù)之一。傳統(tǒng)的定位成像通常使用讀出速度較慢(10MHz)的弱光探測器--電子倍增電荷耦合器件(Electron multiplying charge coupled device, EMCCD),成像通量較小,限制了該技術(shù)在大樣本超分辨成像(如亞突觸水平神經(jīng)回路成像)等方面的應(yīng)用。因此研究適用于定位成像的新型弱光探測方法對提高成像通量是必要的。本文圍繞該問題,建立了一套研究弱光探測器的超分辨定位成像能力的方法。具體內(nèi)容如下： (1)通過表征探測器噪聲和光子傳遞曲線等特性,從弱光探測靈敏度和成像信噪比等方面定量研究不同類型弱光探測器的整體成像性能。結(jié)果表明：(i)在弱光探測靈敏度方面,以Andor iXon897為代表的EMCCD相機在電子倍增工作模式下具有最高靈敏度(最小可探測器限為2photon),以Hamamatsu Flash4.0為代表的sCMOS相機的靈敏度稍差(最小可探測限為3photon);(ii)在成像信噪比方面,EMCCD相機在極微弱信號強度下(13photon/pixel)成像信噪比最高,但是隨著信號強度的增加,sCMOS相機的成像信噪比優(yōu)于EMCCD相機；(iii)sCMOS相機具備并行高速數(shù)據(jù)讀出能力,其讀出速度(以Hamamatsu Flash4.0為例,400MHz)比EMCCD目機(iXon897為10MHz)快四十倍,但是sCMOS相機的成像均勻性較差。綜合以上發(fā)現(xiàn),sCMOS有望為提高定位成像的成像速度和通量提供新的有效途徑。 (2)基于點狀發(fā)光體的重復(fù)成像與定位,發(fā)展了一種通過定量表征探測器的單分子探測和定位能力的實驗方法。結(jié)果表明,該方法對定位精度的測量達到了～1nm的精度。進一步利用該方法,我們首次演示了在超分辨定位成像的典型信號范圍(50-2000photon/pixel)內(nèi),商品化sCMOS相機(Hamamatsu Flash4.0)可以表現(xiàn)出優(yōu)于EMCCD (Andor iXon897)的成像性能。此發(fā)現(xiàn)證實sCMOS相機有能力代替常用的EMCCD相機,將定位成像的成像通量提高～40倍,為亞突觸水平神經(jīng)回路成像提供了新的探測途徑。 (3)基于仿真方法,利用單分子成像模型及固定圖形噪聲模型,系統(tǒng)研究了sCMOS相機的成像不均勻性對單分子定位的影響。結(jié)果發(fā)現(xiàn)：(i)固定圖形噪聲幾乎對定位精度沒有影響,但是會引入一定的定位偏差；(ii)當額外增加像素固定圖形噪聲一半強度的列固定圖形噪聲時定位偏差相應(yīng)的只增加了～20%。對Hamamatsu Flash4.0sCMOS相機而言,其像素固定圖形噪聲要高于列固定圖形噪聲～3-5倍。因此,該sCMOS相機的列固定圖形噪聲對單分子定位的影響很難察覺。該結(jié)論消除了研究人員對sCMOS相機的成像不均勻性的顧慮,有利于推動sCMOS相機在定位成像領(lǐng)域中的應(yīng)用。
[Abstract]:Super-resolution localization microscopy- (or "location imaging") technology has become one of the most promising research tools in the field of life science because of its nanoscale spatial resolution, which is essentially dependent on single molecule imaging. However, the fluorescence signal of a single molecule is usually in the range of hundreds to thousands of photons, and it has to cover multiple pixels. So the detection of single molecular weak signal is one of the key techniques to realize the localization imaging. The traditional localization imaging usually uses the weak light detector (Electron multiplying charge coupled device, EMC CDD), which has a slow readout speed of 10 MHz), and the imaging flux is relatively small. The application of this technique in large sample superresolution imaging (such as subsynaptic horizontal neural loop imaging) is limited. Therefore, it is necessary to study a new weak light detection method suitable for localization imaging to improve the imaging flux. A set of methods to study the super-resolution imaging ability of weak light detectors are established. The main contents are as follows:. 1) by characterizing the characteristics of detector noise and photon transfer curve, we quantitatively study the whole imaging performance of different types of weak light detectors from the aspects of low light detection sensitivity and imaging signal-to-noise ratio. The results show that: 1) in low light detection sensitivity, The EMCCD camera, represented by Andor iXon897, has the highest sensitivity (the minimum detector limit is 2 photontons) in the mode of electron multiplication, and the sensitivity of the sCMOS camera represented by Hamamatsu Flash4.0 is slightly lower (the minimum detectable limit is 3photonine / ii) in imaging signal to noise ratio (SNR). The EMCCD camera has the highest signal-to-noise ratio (SNR) of 13photon / pixel at very weak signal intensity. However, with the increase of signal intensity, the signal-to-noise ratio of EMCCD camera is better than that of EMCCD camera. Its readout speed is 40 times faster than that of EMCCD camera iXon897 (taking Hamamatsu Flash4.0 as an example). But the imaging uniformity of sCMOS camera is poor. Based on the repeated imaging and localization of the spot luminescence, an experimental method for quantificationally characterizing the detection and localization ability of single molecule detector is developed. The results show that, By using this method, we have demonstrated for the first time that we are in the range of 50-2000photon / pixel in the typical signal range of super-resolution positioning imaging. The commercial sCMOS camera Hamamatsu Flash 4.0) can show better imaging performance than EMCCD Andor iXon897. the findings confirm that the sCMOS camera has the ability to replace the conventional EMCCD camera and increase the imaging flux by 40 times. It provides a new approach for the imaging of subsynaptic horizontal neural circuits. (3) based on the simulation method, the effects of imaging inhomogeneity of sCMOS camera on single molecule positioning are systematically studied by using monolayer imaging model and fixed figure noise model. The results show that the fixed figure noise has little effect on the positioning accuracy. But a certain positioning bias will be introduced.) when the column fixed figure noise with half the intensity of the pixel fixed figure noise is added, the positioning deviation will only increase by 20%. For the Hamamatsu Flash4.0sCMOS camera, The pixel fixed figure noise is 3-5 times higher than the column fixed figure noise. Therefore, the effect of the column fixed figure noise on the single molecular positioning of the sCMOS camera is difficult to detect. This conclusion eliminates the researchers' concern about the inhomogeneity of the sCMOS camera's imaging. It is helpful to promote the application of sCMOS camera in the field of positioning imaging.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R318

【共引文獻】

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