受激拉曼散射顯微鏡的髓鞘成像應(yīng)用及連續(xù)波激光器搭建受激拉曼散射顯微鏡
發(fā)布時(shí)間：2019-05-17 02:30
【摘要】：基于相干反斯托克斯拉曼散射(coherent ani-Stokes Raman scattering, CARS)和受激拉曼散射(stimulated Raman scattering, SRS)現(xiàn)象的相干拉曼散射成像技術(shù),不需要額外染料分子或熒光蛋白標(biāo)記,針對(duì)特定的化學(xué)鍵振動(dòng)進(jìn)行成像,具有非侵入性的優(yōu)點(diǎn),在生物學(xué)和醫(yī)學(xué)成像領(lǐng)域有著非常重要的作用。經(jīng)過二十年的發(fā)展,相干拉曼散射顯微鏡被廣泛應(yīng)用于生命科學(xué)和生物醫(yī)學(xué)成像領(lǐng)域。分子振動(dòng)成像賦予相干拉曼散射顯微鏡探測(cè)分子信息的優(yōu)勢(shì),更便于檢測(cè)疾病組織中分子組成成分的變化。病變組織分子組成成分為診斷疾病發(fā)生提供了重要信息,而相干拉曼散射顯微鏡正是探測(cè)這一過程最用力的工具。 髓鞘是神經(jīng)系統(tǒng)最重要的組成成分之一,它保證動(dòng)作電位沿著神經(jīng)軸突以跳躍式傳導(dǎo)方式傳播,確保神經(jīng)信號(hào)在長(zhǎng)距離下快速高效的傳導(dǎo)能力。髓鞘的發(fā)育和成熟,是神經(jīng)系統(tǒng)正常工作的基礎(chǔ),因而髓鞘發(fā)育和成熟的研究對(duì)我們理解大腦的工作機(jī)制具有重要意義。在髓鞘相關(guān)疾病中,損傷的髓鞘妨礙或阻止了神經(jīng)信號(hào)的傳導(dǎo),導(dǎo)致感覺、運(yùn)動(dòng)、認(rèn)知等功能的障礙和缺陷,嚴(yán)重影響人類的正常生理健康和生活質(zhì)量。因此脫髓鞘和髓鞘再生被廣泛研究,以尋求能夠治療脫髓鞘疾病的方案和策略。目前研究髓鞘的成像手段有電子顯微鏡、經(jīng)典組織形態(tài)學(xué)、熒光顯微鏡和核磁共振成像。電子顯微鏡的超高分辨率揭示了髓鞘的超微結(jié)構(gòu),為我們理解髓鞘提供了結(jié)構(gòu)基礎(chǔ)；組織形態(tài)學(xué)在形態(tài)學(xué)水平上為診斷正常和病變髓鞘提供了重要標(biāo)準(zhǔn)；熒光顯微鏡常常被用于研究髓鞘相關(guān)蛋白的功能；核磁共振成像是目前在體診斷髓鞘相關(guān)疾病最實(shí)用的工具。這幾種髓鞘成像方法為理解髓鞘的生理機(jī)能,診斷髓鞘相關(guān)疾病提供了重要的手段,然而它們都具有各自的局限性：電子顯微鏡和組織形態(tài)學(xué)只能觀察固定的樣本,不能進(jìn)行活體在位觀察；熒光顯微鏡需要對(duì)髓鞘蛋白進(jìn)行熒光探針標(biāo)記,而人們很難保證外源性的熒光探針整合后不會(huì)影響體內(nèi)內(nèi)源性髓鞘蛋白的正常功能；核磁共振成像是目前可以進(jìn)行活體在位檢查的工具,然而其成像分辨率十分有限,不能進(jìn)行超微層面的觀察。探測(cè)分子振動(dòng)的能力保證了SRS顯微鏡可以對(duì)髓鞘進(jìn)行無標(biāo)記觀察,避免了分子探針對(duì)髓鞘正常功能的干擾,非線性效應(yīng)使得SRS顯微鏡可以進(jìn)行三維層析成像,并且具有很好的成像分辨率。我們應(yīng)用模式生物非洲爪蟾蝌蚪,利用其發(fā)育期身體透明的特征,避免了暴露神經(jīng)系統(tǒng)手術(shù)操作的潛在損傷和干擾,使用SRS顯微鏡,我們分別觀察了髓鞘的形成過程、郎飛氏節(jié)的成熟過程,以及脫髓鞘過程。我們的工作闡述了新的動(dòng)物模型和成像工具用于研究髓鞘發(fā)育和髓鞘相關(guān)疾病。 目前常用的SRS顯微鏡需要兩束空問上共線的超快激光光源作為激發(fā)光源,而超快激光器的昂貴價(jià)格大大地限制了SRS顯微鏡在普通生物學(xué)實(shí)驗(yàn)室和醫(yī)學(xué)實(shí)驗(yàn)室的應(yīng)用。為了降低SRS顯微鏡的成本,我們使用成本低廉的連續(xù)波激光器作為激發(fā)光源,搭建了連續(xù)波激光受激拉曼散射(cw-SRS)顯微鏡。在搭建cw-SRS顯微鏡前,我們首先搭建了cw-SRS光譜檢測(cè)裝置。兩個(gè)連續(xù)波半導(dǎo)體激光器被用作激發(fā)光源,其中一束波長(zhǎng)可調(diào)的激光器作為泵浦(pump)光束,其波長(zhǎng)范圍為765-781nm,另一束中心波長(zhǎng)為982nm激光器作為斯托克斯(Stokes)光束。斯托克斯光束以5.4MHz的頻率進(jìn)行電壓調(diào)制,泵浦光束發(fā)生受激拉曼損耗(stimulated Raman loss, SRL)過程,其信號(hào)頻率和斯托克斯光束的調(diào)制頻率一致。泵浦光和斯托克斯光在空間上完全共線,由10mm的透鏡聚焦在樣本上,從樣本上透射出的泵浦光被光電二極管檢測(cè),斯托克斯光被濾光鏡濾除。光電二極管的輸出信號(hào)包含了泵浦光自身的光強(qiáng)和SRL信號(hào),泵浦光光強(qiáng)為直流信號(hào),SRL為交流信號(hào),其頻率為5.4MHz。用鎖相放大器將SRL信號(hào)拾取出來,經(jīng)放大后送至數(shù)據(jù)采集系統(tǒng)。進(jìn)行光譜采集時(shí),使用Lab View程序控制泵浦激光器的波長(zhǎng)范圍,波長(zhǎng)從766nm掃描至776nm,掃描步進(jìn)為0.6nm,對(duì)應(yīng)的拉曼頻譜為2700～2870cm-1,不同波長(zhǎng)對(duì)應(yīng)的SRL信號(hào)由數(shù)據(jù)采集系統(tǒng)采集,并繪制成SRS光譜輸出。我們用橄欖油、甲醇和環(huán)乙烷作為樣本,分別取得了SRL光譜,和自發(fā)拉曼光譜完全一致。由于斯托克斯激光器直接被電壓進(jìn)行調(diào)制,獲取的SRL信號(hào)具有很高的背景,為了消除背景,我們改用雙調(diào)制模式檢測(cè)和頻信號(hào),避開斯托克斯激光器電壓調(diào)制引起的背景。我們將泵浦光束以0.8MHz進(jìn)行調(diào)制,斯托克斯光束以4.6MHz進(jìn)行調(diào)制,光電二極管檢測(cè)5.4MHz的和頻信號(hào)。由于和頻信號(hào)的頻率和斯托克斯光束的調(diào)制頻率不同,雙調(diào)制模式完全去除了背景噪聲。之后我們將兩束激光送入激光掃描顯微鏡中,對(duì)橄欖油和脂肪肝切片進(jìn)行顯微成像,并取得了cw-SRS圖像。和皮秒脈沖激光器相比,連續(xù)波激光器激發(fā)的SRL信號(hào)弱103左右,這是因?yàn)檫B續(xù)波激光的能量比脈沖激光器的峰值能量弱103。連續(xù)波激光對(duì)生物組織的光損傷很小,理論上可以通過提高激發(fā)光的能量來提高SRS信號(hào)的強(qiáng)度。由于連續(xù)波激光器的低廉價(jià)格,cw-SRS顯微鏡大大降低了傳統(tǒng)SRS顯微鏡的成本,這將開拓SRS顯微鏡在生物學(xué)和醫(yī)學(xué)研究領(lǐng)域的更多應(yīng)用。
[Abstract]:A coherent Raman scattering imaging technique based on coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) phenomenon does not require an additional dye molecule or a fluorescent protein mark, Has the advantages of non-invasive property, and plays a very important role in the field of biology and medical imaging. With the development of twenty years, the coherent Raman scattering microscope is widely used in the field of life science and biomedical imaging. The molecular vibration imaging gives the advantages of the coherent Raman scattering microscope to detect the molecular information, and is more convenient for detecting the change of the molecular composition component in the disease tissue. The composition of the molecular composition of the diseased tissue provides important information for the diagnosis of the disease, and the coherent Raman scattering microscope is the most powerful tool for detecting the process. Myeloid is one of the most important components of the nervous system. It can ensure that the action potential is spread in a jump-type conduction mode along the nerve axon, so as to ensure the rapid and efficient conduction of the nerve signal at long distance. The development and maturation of the pulpitis is the basis of the normal work of the nervous system, so the research of the development and maturation of the pulpitis is of great importance to us to understand the working mechanism of the brain The sense, movement, cognition, and other functional obstacles and defects, which seriously affect the normal physical health and life of the human being. Quality. Therefore, the defibrination and the regeneration of the pulp are widely studied in order to find a solution to be able to treat the defibrination disorder. The present study on the imaging means of the pulpitis has an electron microscope, a classical tissue morphology, a fluorescence microscope, and a nuclear magnetic resonance. The ultra-high resolution of the electron microscope revealed the ultrastructure of the pulpitis, which provided a structural basis for us to understand the medullary canal; the morphology of the tissue provided an important criterion for the diagnosis of normal and pathological changes in the morphology of the medullary canal; the fluorescence microscope was often used to study the related protein of the pulp Functional; nuclear magnetic resonance imaging is the most practical in vivo in the diagnosis of myeloid-related diseases. The use of these methods is an important means to understand the physiological function of the medullary canal and to diagnose the related diseases of the pulpitis. However, they all have their respective limitations: the electron microscope and the tissue morphology can only observe the fixed sample and can not carry out the in-vivo position. It is difficult to ensure that the external fluorescent probe does not affect the normal function of the endogenous myeloid protein in the body after the fluorescent probe is fully integrated, and the nuclear magnetic resonance imaging can be carried out in the in-vivo examination at present. The tool, however, is very limited in imaging resolution and cannot be ultra-micro It is observed that the ability of detecting the vibration of the molecule ensures that the SRS microscope can carry out no-mark observation on the medullary canal, so that the interference of the molecular probe on the normal function of the pulp is avoided, the non-linear effect enables the SRS microscope to carry out three-dimensional tomography, and has good imaging score. We applied the model organism to make the tadpole of the Xenopus laevis, take advantage of the characteristics of the body to be transparent during the development period, avoid the potential damage and interference of the operation of the exposed nervous system, and use the SRS microscope. We respectively observe the forming process of the pulp and the maturation of the Kronfly's section. The process, as well as the defibrination. The process. Our work sets forth new animal models and imaging tools for the study of the development of the pulpitis and the correlation of the pulp At present, the commonly used SRS microscope needs two superfast laser light sources which are co-linear on an empty question as the excitation light source, and the expensive price of the ultrafast laser greatly limits the SRS microscope in the general biological laboratory and the medicine In order to reduce the cost of the SRS microscope, we use the low-cost continuous wave laser as the excitation light source to set up the continuous wave laser stimulated Raman scattering (cw-SR). S) Microscope. Before setting up the cw-SRS microscope, we first set up the cw-SRS light The two continuous-wave semiconductor lasers are used as the excitation light source, one of which is used as a pump beam, the wavelength range of which is 765-781 nm, and the other of the central wavelength is 982nm laser as Stokes (Stok es) the stokes beam is subjected to a voltage modulation at a frequency of 5.4 mhz, the pump beam generating an stimulated raman loss (srl) process, a signal frequency and a Stokes beam modulation, the pump light and the stokes light are completely collinear in space, the pump light transmitted from the sample is focused on the sample, the pump light transmitted from the sample is detected by the photodiode, and the stokes light is the output signal of the photodiode comprises the light intensity of the pump light itself and the SRL signal, the pump light is strong as a direct current signal, the SRL is an AC signal, and the frequency is 5 .4 MHz. The SRL signal is picked up by the phase-locked amplifier and sent to the number after amplification According to the acquisition system, when the spectrum acquisition is performed, the wavelength range of the pump laser is controlled by using the Lab View program, the wavelength is scanned from 766 nm to 776 nm, the scanning step is 0.6 nm, the corresponding Raman spectrum is 2700-2870cm-1, and the SRL signals corresponding to different wavelengths are collected by the data acquisition system and are drawn into the SR S-spectral output. We used olive oil, methanol and cycloethane as samples to obtain the SRL spectrum and the spontaneous Raman light, respectively. The spectrum is exactly the same. Because the Stokes laser is directly modulated by the voltage, the obtained SRL signal has a high background. In order to eliminate the background, we use the double modulation mode detection and frequency signal to avoid the Stokes laser voltage modulation. The background is caused. We modulate the pump beam at 0.8 MHz, the Stokes beam is modulated at 4.6 MHz, and the photodiode detects 5.4 MHz because the frequency of the sum frequency signal and the modulation frequency of the stokes beam are different, the double modulation mode is completely removed, background noise. After that, we sent two laser beams into a laser scanning microscope for microscopic imaging of olive oil and fatty liver slices and obtained cw- The SRS image. Compared with the picosecond pulse laser, the SRL signal excited by the continuous wave laser is about 103, because the energy of the continuous wave laser is less than the peak energy of the pulse laser. The light damage of the biological tissue by the continuous wave laser is very small, and the SRS can be improved by increasing the energy of the excitation light theoretically. The intensity of the signal. Due to the low price of the continuous wave laser, the cw-SRS microscope greatly reduces the cost of the conventional SRS microscope, which will open up the SRS microscope in the fields of biology and medicine
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：Q-336;R310
本文編號(hào)：2478733
[Abstract]:A coherent Raman scattering imaging technique based on coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) phenomenon does not require an additional dye molecule or a fluorescent protein mark, Has the advantages of non-invasive property, and plays a very important role in the field of biology and medical imaging. With the development of twenty years, the coherent Raman scattering microscope is widely used in the field of life science and biomedical imaging. The molecular vibration imaging gives the advantages of the coherent Raman scattering microscope to detect the molecular information, and is more convenient for detecting the change of the molecular composition component in the disease tissue. The composition of the molecular composition of the diseased tissue provides important information for the diagnosis of the disease, and the coherent Raman scattering microscope is the most powerful tool for detecting the process. Myeloid is one of the most important components of the nervous system. It can ensure that the action potential is spread in a jump-type conduction mode along the nerve axon, so as to ensure the rapid and efficient conduction of the nerve signal at long distance. The development and maturation of the pulpitis is the basis of the normal work of the nervous system, so the research of the development and maturation of the pulpitis is of great importance to us to understand the working mechanism of the brain The sense, movement, cognition, and other functional obstacles and defects, which seriously affect the normal physical health and life of the human being. Quality. Therefore, the defibrination and the regeneration of the pulp are widely studied in order to find a solution to be able to treat the defibrination disorder. The present study on the imaging means of the pulpitis has an electron microscope, a classical tissue morphology, a fluorescence microscope, and a nuclear magnetic resonance. The ultra-high resolution of the electron microscope revealed the ultrastructure of the pulpitis, which provided a structural basis for us to understand the medullary canal; the morphology of the tissue provided an important criterion for the diagnosis of normal and pathological changes in the morphology of the medullary canal; the fluorescence microscope was often used to study the related protein of the pulp Functional; nuclear magnetic resonance imaging is the most practical in vivo in the diagnosis of myeloid-related diseases. The use of these methods is an important means to understand the physiological function of the medullary canal and to diagnose the related diseases of the pulpitis. However, they all have their respective limitations: the electron microscope and the tissue morphology can only observe the fixed sample and can not carry out the in-vivo position. It is difficult to ensure that the external fluorescent probe does not affect the normal function of the endogenous myeloid protein in the body after the fluorescent probe is fully integrated, and the nuclear magnetic resonance imaging can be carried out in the in-vivo examination at present. The tool, however, is very limited in imaging resolution and cannot be ultra-micro It is observed that the ability of detecting the vibration of the molecule ensures that the SRS microscope can carry out no-mark observation on the medullary canal, so that the interference of the molecular probe on the normal function of the pulp is avoided, the non-linear effect enables the SRS microscope to carry out three-dimensional tomography, and has good imaging score. We applied the model organism to make the tadpole of the Xenopus laevis, take advantage of the characteristics of the body to be transparent during the development period, avoid the potential damage and interference of the operation of the exposed nervous system, and use the SRS microscope. We respectively observe the forming process of the pulp and the maturation of the Kronfly's section. The process, as well as the defibrination. The process. Our work sets forth new animal models and imaging tools for the study of the development of the pulpitis and the correlation of the pulp At present, the commonly used SRS microscope needs two superfast laser light sources which are co-linear on an empty question as the excitation light source, and the expensive price of the ultrafast laser greatly limits the SRS microscope in the general biological laboratory and the medicine In order to reduce the cost of the SRS microscope, we use the low-cost continuous wave laser as the excitation light source to set up the continuous wave laser stimulated Raman scattering (cw-SR). S) Microscope. Before setting up the cw-SRS microscope, we first set up the cw-SRS light The two continuous-wave semiconductor lasers are used as the excitation light source, one of which is used as a pump beam, the wavelength range of which is 765-781 nm, and the other of the central wavelength is 982nm laser as Stokes (Stok es) the stokes beam is subjected to a voltage modulation at a frequency of 5.4 mhz, the pump beam generating an stimulated raman loss (srl) process, a signal frequency and a Stokes beam modulation, the pump light and the stokes light are completely collinear in space, the pump light transmitted from the sample is focused on the sample, the pump light transmitted from the sample is detected by the photodiode, and the stokes light is the output signal of the photodiode comprises the light intensity of the pump light itself and the SRL signal, the pump light is strong as a direct current signal, the SRL is an AC signal, and the frequency is 5 .4 MHz. The SRL signal is picked up by the phase-locked amplifier and sent to the number after amplification According to the acquisition system, when the spectrum acquisition is performed, the wavelength range of the pump laser is controlled by using the Lab View program, the wavelength is scanned from 766 nm to 776 nm, the scanning step is 0.6 nm, the corresponding Raman spectrum is 2700-2870cm-1, and the SRL signals corresponding to different wavelengths are collected by the data acquisition system and are drawn into the SR S-spectral output. We used olive oil, methanol and cycloethane as samples to obtain the SRL spectrum and the spontaneous Raman light, respectively. The spectrum is exactly the same. Because the Stokes laser is directly modulated by the voltage, the obtained SRL signal has a high background. In order to eliminate the background, we use the double modulation mode detection and frequency signal to avoid the Stokes laser voltage modulation. The background is caused. We modulate the pump beam at 0.8 MHz, the Stokes beam is modulated at 4.6 MHz, and the photodiode detects 5.4 MHz because the frequency of the sum frequency signal and the modulation frequency of the stokes beam are different, the double modulation mode is completely removed, background noise. After that, we sent two laser beams into a laser scanning microscope for microscopic imaging of olive oil and fatty liver slices and obtained cw- The SRS image. Compared with the picosecond pulse laser, the SRL signal excited by the continuous wave laser is about 103, because the energy of the continuous wave laser is less than the peak energy of the pulse laser. The light damage of the biological tissue by the continuous wave laser is very small, and the SRS can be improved by increasing the energy of the excitation light theoretically. The intensity of the signal. Due to the low price of the continuous wave laser, the cw-SRS microscope greatly reduces the cost of the conventional SRS microscope, which will open up the SRS microscope in the fields of biology and medicine
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：Q-336;R310
【共引文獻(xiàn)】
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