飞秒激光显微成像及CARS光谱探测研究

发布时间：2018-01-12 05:15

本文关键词：飞秒激光显微成像及CARS光谱探测研究　出处：《南昌航空大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着科技的发展,人类希望观察到一个更加微观的世界,不仅研究生物组织或者细胞的结构形态,还要了解生物最基本的组成成分——分子或者原子。近年来,与此相关技术取得了很大成就,如对生物组织或者细胞的显微成像和物质分子检测等,其中飞秒激光双光子荧光显微成像技术和相干反斯托克斯拉曼散射(CARS)光谱探测技术作为新兴的生物组织显微成像和分子结构检测的重要手段,以其独特的优势吸引了很多研究着的研究。飞秒激光双光子荧光显微成像技术作为生物组织和细胞显微成像的有效手段之一,其激发光源选用了近红外的飞秒激光,不仅双光子荧光波长远离了激发波长,实现了暗场成像大大提高了信噪比,还对生物样品的光损伤小、穿透能力强、可以获得较大成像深度,相对于单光子荧光共焦显微成像这是其重要的优点。本文理论上推导了双光子的激励过程,分析了双光子荧光空间分布强度,继而相比于单光子共焦显微成像分析其优势。在理论研究基础上,利用锁模钛宝石激光器搭建了一套飞秒激光双光子荧光显微成像系统,自行编写了系统控制软件,设计了信号扫描采集系统。本文通过实验测量了Rhodamine B溶液在不同浓度下的双光子荧光光谱,总结了双光子荧光光谱在Rhodamine B溶液浓度不断增大的情况下,荧光光谱特征峰产生的非线性红移规律,其中心波长随着Rhodamine B溶液浓度增大向最大荧光发射波长610nm慢慢靠近。通过一系列实验,研究了样品与物镜焦点之间距离对成像效果的影响,印证了理论推导结果,即只有在焦点附近才能产生双光子荧光过程。本文对Rhodamine B形成的微结构样品进行了显微成像,并就其成像结果进行了分析研究。本文基于相干反斯托克斯拉曼散射(coherent Anti-stokes Raman scattering,CARS)技术对分子结构检查进行了研究。在理论研究了CARS的三阶非线性过程之后,采用时间分辨方法抑制CARS光谱信号中非共振背景噪声。为了获取比较完整分子振动模式对应的CARS光谱,利用飞秒脉冲抽运光子晶体光纤(Photonic Crystal Fiber,PCF)产生超连续谱激光作为激励光源,并以此搭建了超连续谱激发时间分辨CARS系统。在此系统中,锁模钛宝石激光器输出的飞秒脉冲一分为二,其中一部分飞秒脉冲抽运光子晶体光纤产生超连续谱激光,作为CARS过程的泵浦光和斯托克斯光;另一部分飞秒脉冲通过适当的时间延迟作为CARS过程的探针光。本文通过实验测量了二甲基亚砜分子振动范围在500cm-1-3500 cm-1内的CARS光谱信息,并讨论了温度对CARS光谱信号的影响。
[Abstract]:With the development of science and technology, human beings want to observe a more microscopic world, not only to study the structure and morphology of biological tissues or cells, but also to understand the most basic components of biology-molecules or atoms. Great achievements have been made in related techniques, such as microscopic imaging of biological tissues or cells and detection of substances and molecules. Among them, femtosecond laser two-photon fluorescence microscopy and coherent anti-Stokes Raman scattering (CARSs) spectrum detection techniques are important means of tissue microscopic imaging and molecular structure detection. The femtosecond laser two-photon fluorescence microscopy is one of the effective methods for tissue and cell microimaging. The excitation light source is femtosecond laser with near infrared. The two-photon fluorescence wavelength is far away from the excitation wavelength, and the dark field imaging greatly improves the SNR, and the light damage to biological samples is small, and the penetration ability is strong. Compared with single photon fluorescence confocal microscopy, the imaging depth can be obtained, which is an important advantage. In this paper, the two-photon excitation process is derived theoretically, and the intensity of two-photon fluorescence spatial distribution is analyzed. On the basis of theoretical research, a femtosecond laser two-photon fluorescence microscopy imaging system is built by using mode-locked Ti: sapphire laser. The system control software was written and the signal scanning acquisition system was designed. The two-photon fluorescence spectra of Rhodamine B solution at different concentrations were measured experimentally. In this paper, the nonlinear redshift of the characteristic peaks of two-photon fluorescence spectra with the increasing concentration of Rhodamine B solution is summarized. The center wavelength is approaching to the maximum fluorescence emission wavelength of 610nm with the increase of Rhodamine B solution concentration. A series of experiments have been carried out. The influence of the distance between the sample and the focus of the objective lens on the imaging effect is studied, and the theoretical results are verified. That is, only near the focal point can produce the two-photon fluorescence process. In this paper, the microstructure of Rhodamine B formed by microscopic imaging. The imaging results are analyzed and studied. This paper is based on coherent anti-Stokes Anti-stokes Raman scattering. After studying the third-order nonlinear process of CARS in theory. The time-resolved method is used to suppress the non-resonant background noise of the CARS spectral signal. In order to obtain the CARS spectrum corresponding to the complete molecular vibration mode. Photonic Crystal fiber PCFs are generated by femtosecond pulse pumped photonic crystal fiber (Photonic Crystal fiber) as excitation light source. In this system, the femtosecond pulse output from the mode-locked Ti: sapphire laser is divided into two parts. One part of femtosecond pulse pumped photonic crystal fiber produces supercontinuum spectrum laser, which is used as pump light and Stokes light in CARS process. Another part of femtosecond pulse is used as probe light of CARS process by proper time delay. The vibrational range of dimethyl sulfoxide molecule is measured in 500cm-1-3500 experimentally. CARS spectral information in cm-1. The influence of temperature on CARS spectral signal is discussed.
【学位授予单位】：南昌航空大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TN249

【相似文献】