本文選題：非線性光學(xué)顯微成像　切入點(diǎn)：無(wú)標(biāo)記成像　出處：《哈爾濱工業(yè)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：受激拉曼散射(stimulated Raman scattering,SRS)顯微成像技術(shù)是一種新興的無(wú)標(biāo)記顯微成像技術(shù),其成像襯度來(lái)源于分子振動(dòng)特性,因而具有優(yōu)異的化學(xué)選擇性和化學(xué)特異性。除此之外,由于SRS顯微成像具有高分辨率、高靈敏度和無(wú)侵入性等特點(diǎn),在不到十年的時(shí)間里已經(jīng)發(fā)展成為生命科學(xué)領(lǐng)域研究中的一項(xiàng)重要工具�；诟吖庾VSRS和多元SRS的受激拉曼光譜顯微成像更是在每一個(gè)像素點(diǎn)都擁有一段光譜,因此其不僅可以區(qū)分擁有重疊拉曼譜帶的不同分子,還能提供更加豐富的化學(xué)信息,成為最近兩三年來(lái)研究的前沿和熱點(diǎn)。然而受發(fā)展時(shí)間限制,目前受激拉曼光譜顯微成像的研究還有很多方面需要完善,其應(yīng)用仍然需要開(kāi)拓。鑒于此,本文利用光譜聚焦以及非線性光譜壓縮和脈沖整形相結(jié)合的方法開(kāi)展了高分辨受激拉曼光譜成像技術(shù)以及其在膜電位檢測(cè)和木質(zhì)素化學(xué)成分分析中的應(yīng)用研究。理論上,本文首先利用非線性耦合波方程對(duì)SRS信號(hào)的產(chǎn)生過(guò)程進(jìn)行了推導(dǎo),給出了SRS信號(hào)的表達(dá)式。分析并比較了不同非共振背景下的SRS和CARS光譜線型的差異。研究了入射激光偏振對(duì)不同退偏比拉曼振動(dòng)模SRS信號(hào)強(qiáng)度的影響,揭示了偏振調(diào)制SRS成像的基本原理。詳細(xì)討論了SRS成像中的噪聲、信噪比以及背景等問(wèn)題,指出在理想情況下SRS顯微成像系統(tǒng)應(yīng)具有散粒噪聲極限探測(cè)能力。分析了光譜聚焦方法實(shí)現(xiàn)高光譜相干拉曼散射顯微成像的機(jī)理,并利用玻璃棒引入線性啁啾建立了一套基于光譜聚焦方法的高光譜SRS顯微成像系統(tǒng),針對(duì)該方法中存在的拉曼頻移校準(zhǔn)和信號(hào)強(qiáng)度校準(zhǔn)問(wèn)題,給出了具體的解決方案。同時(shí)在應(yīng)用研究方面,首次探討了利用SRS信號(hào)無(wú)標(biāo)記探測(cè)細(xì)胞膜電位的可行性。以紅細(xì)胞血影為模型,完成了單個(gè)自然細(xì)胞膜的振動(dòng)光譜成像,證實(shí)了高光譜SRS顯微成像探測(cè)單個(gè)細(xì)胞膜的靈敏度。通過(guò)操控細(xì)胞膜內(nèi)外離子成分改變跨膜電位,同時(shí)利用受激拉曼光譜成像觀察不同電勢(shì)下的細(xì)胞膜,發(fā)現(xiàn)SRS光譜線型隨膜電位改變發(fā)生顯著變化,結(jié)果表明SRS成像有望用于細(xì)胞膜電位的無(wú)標(biāo)記測(cè)量�？紤]光譜聚焦方法光譜分辨率較差,難以滿(mǎn)足指紋區(qū)的成像應(yīng)用,而脈沖整形技術(shù)又不能有效利用激勵(lì)激光功率,通過(guò)理論分析非線性光譜壓縮機(jī)理和總結(jié)已報(bào)導(dǎo)的實(shí)驗(yàn)方案,開(kāi)發(fā)了一套靈活、緊湊的非線性光譜壓縮裝置,該裝置可將寬帶飛秒激光線寬壓縮至幾個(gè)波數(shù),同時(shí)維持一半以上的激光功率,利用搭建的非線性光譜壓縮裝置所提供的高功率斯托克斯激勵(lì)光源,分別在指紋區(qū)和靜默區(qū)建立了兩套光譜分辨率優(yōu)于10 cm-1的高光譜SRS顯微成像系統(tǒng),同時(shí)在靜默區(qū)系統(tǒng)中改進(jìn)了光譜掃描裝置,設(shè)計(jì)了一種基于檢流計(jì)振鏡的新型脈沖內(nèi)部光譜掃描器。針對(duì)當(dāng)前高光譜SRS成像主要用于動(dòng)物細(xì)胞、組織和模型生物研究的現(xiàn)狀,利用分子指紋振動(dòng),開(kāi)展了高光譜SRS顯微成像用于分析描繪木質(zhì)素化學(xué)成分分布的研究。以擬南芥作為模型,運(yùn)用高光譜SRS顯微成像觀察對(duì)比轉(zhuǎn)基因擬南芥與野生型擬南芥,確立了高光譜SRS顯微成像定量區(qū)分木質(zhì)素中不同化學(xué)組份并實(shí)時(shí)監(jiān)測(cè)木質(zhì)素化學(xué)成分變化的能力。將研究進(jìn)一步拓展至長(zhǎng)狗尾草和玉米秸稈,利用高光譜SRS顯微成像觀察維管束內(nèi)纖維細(xì)胞,結(jié)合多元曲線分辨(multivariate curve resolution,MCR)分析,首次揭示了木質(zhì)素中醇和醛兩種不同組份在植物細(xì)胞壁內(nèi)的漸變分布。最后,針對(duì)目前多元SRS成像光譜探測(cè)范圍較窄的問(wèn)題,提出了一種利用反向啁啾脈沖實(shí)現(xiàn)多元SRS顯微成像的新方法,利用玻璃棒和光柵對(duì)分別對(duì)泵浦光和斯托克斯光引入正、負(fù)啁啾,建立了一套基于反向啁啾脈沖的多元SRS顯微成像系統(tǒng),同時(shí)通過(guò)對(duì)DMSO樣品進(jìn)行成像觀察,驗(yàn)證了該方法的可行性。本文的研究成果推動(dòng)了受激拉曼光譜顯微成像技術(shù)的進(jìn)步,豐富并拓展了其成像應(yīng)用研究,對(duì)SRS顯微成像的進(jìn)一步發(fā)展具有重要的科學(xué)意義。
[Abstract]:Stimulated Raman scattering (stimulated Raman, scattering, SRS) microscopic imaging technology is a kind of label free imaging technology emerging, the imaging contrast from the molecular vibration characteristics, so it has chemical selectivity and excellent chemical specificity. In addition, due to the SRS imaging with high resolution, high sensitivity and non-invasive etc., in less than ten years of development has become an important research tool in the field of life science. Based on the stimulated Raman spectrum imaging hyperspectral SRS and multi SRS is in every pixel has a spectrum, so it can not only distinguish between different molecules having overlapping Raman spectrum the chemical can provide more abundant information, become the research frontier of the last two or three years. However, the development of the time limit, the stimulated Raman spectrum imaging research and Many aspects need to be improved, the application still needs to develop. In view of this, this paper using spectral method for nonlinear focusing and spectrum compression and pulse shaping combined to carry out high resolution Raman spectral imaging technology and the element analysis of chemical constituents of membrane potential detection and application of wood research. In theory, this paper uses nonlinear coupling wave equation produced in the process of SRS signal was derived. The expression of the SRS signal is presented. Analyze and compare the differences between SRS and CARS spectrum of different non resonant background. The effects of laser polarization on different depolarization Raman vibration modes of SRS signal intensity, and reveals the basic principle of polarization modulated imaging SRS noise in SRS imaging was discussed in detail. The problem of SNR and background, pointed out that the SRS microscopic imaging system should have shot noise limit detection in ideal condition The mechanism analysis ability. The spectral focusing method to achieve high spectral coherent Raman scattering microscopic imaging, and the use of glass rod into linear chirp is established based on the hyperspectral imaging system SRS spectrum focusing method, aiming at the existing method of Raman frequency shift in calibration and signal intensity calibration problems, gave specific solutions at the same time. In the aspect of application research, for the first time to discuss the feasibility of detecting markers of cell membrane potential by SRS signal. The erythrocyte ghost model, completed the vibration spectrum image of single natural cell membrane, confirmed the high sensitivity spectrum of SRS imaging detection of single cell membrane. The cell membrane ion composition controlled by internal and external change across the membrane at the same time using potential stimulated Raman imaging under different cell membrane potential, found SRS spectrum with the membrane potential change changed significantly, the results show that S RS imaging is expected to be used for label free detection of cell membrane potential. Considering spectral focusing method of spectral resolution is poor, it is difficult to meet the application of fingerprint imaging area, and pulse shaping technology cannot effectively use laser through theoretical analysis, experimental scheme of nonlinear spectral compression mechanism and the total nodes have been reported, developed a set of flexible, nonlinear spectroscopy compact compression device, the device can be compressed to several broadband femtosecond laser linewidth wavenumber, while maintaining the laser power more than half of the high power Stokes using nonlinear spectral structures compression devices provide excitation light source, high spectral SRS imaging system two sets of spectral resolution better than 10 cm-1 respectively based on fingerprint and silence at the same time zone, improved spectral scanning device in a silent zone system, based on the design of a new type of vibrating mirror galvanometer in optical pulse Spectrum scanner. In view of the current high spectral SRS imaging is mainly used in animal cells, tissues and biological research status model, using molecular fingerprint vibration, carried out SRS hyperspectral microscopic imaging for analyzing and describing the distribution of wood chemical components. Using Arabidopsis as a model, using SRS hyperspectral microscopic imaging observation of transgenic and wild type of Arabidopsis Arabidopsis, established a high spectral SRS imaging quantitative ability to distinguish between different chemical components of lignin and lignin real-time monitoring of chemical composition changes. The research is extended to long bristlegrass and maize straw, the observation of fiber cells in vascular bundle SRS hyperspectral imaging, combined with multivariate curve resolution (multivariate curve, resolution, MCR) analysis reveals the first element in wood alcohol and aldehyde two different components in the plant cell wall in gradient distribution. Finally, according to the The narrow range of problems of SRS imaging spectral detection, puts forward a new method of using reverse chirped pulse multiple SRS microscopic imaging, the use of glass rod and the grating of the pump and Stokes light into the positive and negative chirp is established based on the multi SRS imaging system at the same time through reverse chirped pulse. The imaging observation of the DMSO sample, verify the feasibility of the method. This thesis promotes stimulated Raman spectrum imaging technology, enrich and expand the application of imaging research has important scientific significance of SRS imaging in further development.

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：O437.3;O439

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