受激拉曼散射显微镜的髓鞘成像应用及连续波激光器搭建受激拉曼散射显微镜
发布时间：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)现象的相干拉曼散射成像技术,不需要额外染料分子或荧光蛋白标记,针对特定的化学键振动进行成像,具有非侵入性的优点,在生物学和医学成像领域有着非常重要的作用。经过二十年的发展,相干拉曼散射显微镜被广泛应用于生命科学和生物医学成像领域。分子振动成像赋予相干拉曼散射显微镜探测分子信息的优势,更便于检测疾病组织中分子组成成分的变化。病变组织分子组成成分为诊断疾病发生提供了重要信息,而相干拉曼散射显微镜正是探测这一过程最用力的工具。 髓鞘是神经系统最重要的组成成分之一,它保证动作电位沿着神经轴突以跳跃式传导方式传播,确保神经信号在长距离下快速高效的传导能力。髓鞘的发育和成熟,是神经系统正常工作的基础,因而髓鞘发育和成熟的研究对我们理解大脑的工作机制具有重要意义。在髓鞘相关疾病中,损伤的髓鞘妨碍或阻止了神经信号的传导,导致感觉、运动、认知等功能的障碍和缺陷,严重影响人类的正常生理健康和生活质量。因此脱髓鞘和髓鞘再生被广泛研究,以寻求能够治疗脱髓鞘疾病的方案和策略。目前研究髓鞘的成像手段有电子显微镜、经典组织形态学、荧光显微镜和核磁共振成像。电子显微镜的超高分辨率揭示了髓鞘的超微结构,为我们理解髓鞘提供了结构基础；组织形态学在形态学水平上为诊断正常和病变髓鞘提供了重要标准；荧光显微镜常常被用于研究髓鞘相关蛋白的功能；核磁共振成像是目前在体诊断髓鞘相关疾病最实用的工具。这几种髓鞘成像方法为理解髓鞘的生理机能,诊断髓鞘相关疾病提供了重要的手段,然而它们都具有各自的局限性：电子显微镜和组织形态学只能观察固定的样本,不能进行活体在位观察；荧光显微镜需要对髓鞘蛋白进行荧光探针标记,而人们很难保证外源性的荧光探针整合后不会影响体内内源性髓鞘蛋白的正常功能；核磁共振成像是目前可以进行活体在位检查的工具,然而其成像分辨率十分有限,不能进行超微层面的观察。探测分子振动的能力保证了SRS显微镜可以对髓鞘进行无标记观察,避免了分子探针对髓鞘正常功能的干扰,非线性效应使得SRS显微镜可以进行三维层析成像,并且具有很好的成像分辨率。我们应用模式生物非洲爪蟾蝌蚪,利用其发育期身体透明的特征,避免了暴露神经系统手术操作的潜在损伤和干扰,使用SRS显微镜,我们分别观察了髓鞘的形成过程、郎飞氏节的成熟过程,以及脱髓鞘过程。我们的工作阐述了新的动物模型和成像工具用于研究髓鞘发育和髓鞘相关疾病。 目前常用的SRS显微镜需要两束空问上共线的超快激光光源作为激发光源,而超快激光器的昂贵价格大大地限制了SRS显微镜在普通生物学实验室和医学实验室的应用。为了降低SRS显微镜的成本,我们使用成本低廉的连续波激光器作为激发光源,搭建了连续波激光受激拉曼散射(cw-SRS)显微镜。在搭建cw-SRS显微镜前,我们首先搭建了cw-SRS光谱检测装置。两个连续波半导体激光器被用作激发光源,其中一束波长可调的激光器作为泵浦(pump)光束,其波长范围为765-781nm,另一束中心波长为982nm激光器作为斯托克斯(Stokes)光束。斯托克斯光束以5.4MHz的频率进行电压调制,泵浦光束发生受激拉曼损耗(stimulated Raman loss, SRL)过程,其信号频率和斯托克斯光束的调制频率一致。泵浦光和斯托克斯光在空间上完全共线,由10mm的透镜聚焦在样本上,从样本上透射出的泵浦光被光电二极管检测,斯托克斯光被滤光镜滤除。光电二极管的输出信号包含了泵浦光自身的光强和SRL信号,泵浦光光强为直流信号,SRL为交流信号,其频率为5.4MHz。用锁相放大器将SRL信号拾取出来,经放大后送至数据采集系统。进行光谱采集时,使用Lab View程序控制泵浦激光器的波长范围,波长从766nm扫描至776nm,扫描步进为0.6nm,对应的拉曼频谱为2700～2870cm-1,不同波长对应的SRL信号由数据采集系统采集,并绘制成SRS光谱输出。我们用橄榄油、甲醇和环乙烷作为样本,分别取得了SRL光谱,和自发拉曼光谱完全一致。由于斯托克斯激光器直接被电压进行调制,获取的SRL信号具有很高的背景,为了消除背景,我们改用双调制模式检测和频信号,避开斯托克斯激光器电压调制引起的背景。我们将泵浦光束以0.8MHz进行调制,斯托克斯光束以4.6MHz进行调制,光电二极管检测5.4MHz的和频信号。由于和频信号的频率和斯托克斯光束的调制频率不同,双调制模式完全去除了背景噪声。之后我们将两束激光送入激光扫描显微镜中,对橄榄油和脂肪肝切片进行显微成像,并取得了cw-SRS图像。和皮秒脉冲激光器相比,连续波激光器激发的SRL信号弱103左右,这是因为连续波激光的能量比脉冲激光器的峰值能量弱103。连续波激光对生物组织的光损伤很小,理论上可以通过提高激发光的能量来提高SRS信号的强度。由于连续波激光器的低廉价格,cw-SRS显微镜大大降低了传统SRS显微镜的成本,这将开拓SRS显微镜在生物学和医学研究领域的更多应用。
[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
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：Q-336;R310
本文编号：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
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：Q-336;R310
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