CN1166939C - Multiphoton excitation fluorescence method based on Q-switched pulsed laser - Google Patents
Multiphoton excitation fluorescence method based on Q-switched pulsed laser Download PDFInfo
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Abstract
Description
技术领域technical field
本发明属于一种基于调Q脉冲激光实现多光子激发荧光的一种方法。可用于生物和医学等学科的研究。The invention belongs to a method for realizing multi-photon excitation of fluorescence based on Q-switched pulse laser. It can be used for research in disciplines such as biology and medicine.
背景技术Background technique
由于光毒性小，穿透性好和高分辨率等优点，多光子激发荧光显微系统(MPE)已成为生物医学研究的重要工具。Due to the advantages of low phototoxicity, good penetrability and high resolution, multiphoton excited fluorescence microscopy (MPE) has become an important tool in biomedical research.
多光子激发是一种已经被广泛使用的技术，尤其是在生物医学等领域的研究中，多光子激发已成为一种必不可少的技术手段。国外已经在这方面申请了多项专利，例如美国专利No.5523573，美国专利No.5777732，美国专利No.5034613，美国专利No.5952192，美国专利No.6154310。在这些专利中或者使用锁模超短(皮秒或飞秒)脉冲激光作为激发光源，或者使用连续激光作为激发光源。锁模超短(皮秒或飞秒)脉冲激光器技术操作都非常复杂，这给非光学专业的用户带来极大困难，而且锁模超短(皮秒或飞秒)脉冲激光器尤其是飞秒脉冲价格非常昂贵。连续光作为多光子激发源需要非常高的激发平均功率，高平均功率的高散射和高热吸收使得基于连续光的多光子激发荧光激发系统难以成为实用系统，而且至今没有连续光三光子激发荧光的报道。Multiphoton excitation is a technology that has been widely used, especially in the research of biomedicine and other fields, multiphoton excitation has become an indispensable technical means. Foreign countries have applied for multiple patents in this regard, such as US Patent No. 5,523,573, US Patent No. 5,777,732, US Patent No. 5,034,613, US Patent No. 5,952,192, and US Patent No. 6,154,310. In these patents, either a mode-locked ultrashort (picosecond or femtosecond) pulse laser is used as the excitation light source, or a continuous laser is used as the excitation light source. The operation of mode-locked ultrashort (picosecond or femtosecond) pulse laser technology is very complicated, which brings great difficulties to users who are not specialized in optics, and mode-locked ultrashort (picosecond or femtosecond) pulse lasers especially femtosecond Pulse is very expensive. Continuous light as a multiphoton excitation source requires very high excitation average power. The high scattering and high thermal absorption of high average power make it difficult for a continuous light-based multiphoton excitation fluorescence excitation system to become a practical system, and so far there is no continuous light three-photon excitation fluorescence system. reports.
发明内容Contents of the invention
本发明的目的是针对现有基于超短脉冲激光或连续激光的多光子激发荧光系统的问题和不足，提出一种基于调Q脉冲激光的多光子激发荧光方法。The purpose of the present invention is to propose a multi-photon excitation fluorescence method based on Q-switched pulse laser for the problems and deficiencies of the existing multi-photon excitation fluorescence system based on ultrashort pulse laser or continuous laser.
为了在既能获得高荧光强度的同时，又不产生明显的热效应和减少光散射对测量的影响，激发光必须具有高峰值功率和低平均功率的特点，这就要求脉冲激光器具有低重复频率，调Q脉冲激光器显然是最佳的选择。In order to obtain high fluorescence intensity without producing obvious thermal effects and reducing the influence of light scattering on the measurement, the excitation light must have the characteristics of high peak power and low average power, which requires the pulse laser to have a low repetition rate. A Q-switched pulsed laser is clearly the best choice.
本发明所说的基于调Q脉冲激光的多光子激发荧光方法原理是，使样品分子(或基团)同时吸收两个或多个光子后跃迁到激发态，然后跃迁回到基态并发射荧光(或磷光)，激发光采用调Q脉冲激光。The principle of the multi-photon excitation fluorescence method based on Q-switched pulsed laser in the present invention is to make the sample molecule (or group) absorb two or more photons at the same time and then transition to an excited state, then transition back to the ground state and emit fluorescence ( or phosphorescence), the excitation light is Q-switched pulsed laser.
实现多光子激发荧光的步骤为：1)采用调Q技术获得高峰值功率的脉冲激光，脉冲宽度为纳秒量级；2)利用调Q脉冲激光激发用荧光探针标记的或能够产生自发荧光的生物样品，产生多光子激发荧光。The steps to achieve multi-photon excitation of fluorescence are: 1) Using Q-switching technology to obtain pulsed laser with high peak power, the pulse width is on the order of nanoseconds; 2) Using Q-switched pulsed laser to excite the fluorescent probe-labeled or capable of producing autofluorescence biological samples, resulting in multiphoton-excited fluorescence.
本发明所说的基于调Q脉冲激光的多光子激发荧光方法中，其调Q激光器结构简单，价格较低，性能稳定，光束质量高，而且操作方便。调Q脉冲激光的脉宽为纳秒量级或更宽，而且其重复频率很容易调节。与锁模激光器的高重复频率相比，利用调Q激光器的低重复频率特性可以获得高峰值功率和低平均功率的脉冲激光。这正是多光子激发研究尤其是生物活体的多光子激发研究所必须的。In the multiphoton excitation fluorescence method based on Q-switched pulsed laser in the present invention, the Q-switched laser has simple structure, low price, stable performance, high beam quality and convenient operation. The pulse width of the Q-switched pulsed laser is on the order of nanoseconds or wider, and its repetition rate is easy to adjust. Compared with the high repetition frequency of the mode-locked laser, the pulsed laser with high peak power and low average power can be obtained by using the low repetition frequency characteristic of the Q-switched laser. This is exactly what is necessary for multiphoton excitation research, especially for living organisms.
具体实施方式Detailed ways
本发明所说的基于调Q脉冲激光的多光子激发荧光方法中，所说的激发脉冲激光是由调Q脉冲激光器产生的。In the Q-switched pulse laser-based multiphoton excitation fluorescence method of the present invention, the excitation pulse laser is generated by a Q-switched pulse laser.
所说的调Q脉冲激光器可以是固体激光器，也可以染料激光器。例如YAG激光器，钛宝石激光器和若丹明激光器等激光器。The said Q-switched pulsed laser can be a solid-state laser or a dye laser. For example, lasers such as YAG laser, titanium sapphire laser and Rhodamine laser.
所说的调Q脉冲激光器的脉宽是纳秒量级或更宽。The pulse width of the Q-switched pulsed laser is nanosecond order or wider.
所说的调Q脉冲激光器的重复频率可从1Hz到1MHz进行调节。The repetition frequency of said Q-switched pulsed laser can be adjusted from 1 Hz to 1 MHz.
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| JP6075963B2 (en) * | 2012-03-26 | 2017-02-08 | オリンパス株式会社 | Fluorescence observation method and fluorescence observation apparatus |
| US10706536B2 (en) | 2015-07-20 | 2020-07-07 | Min Xu | Photon structure and chemometrics pathologic system |
| CN106568754A (en) * | 2016-11-06 | 2017-04-19 | 浙江大学 | Optical system used for measuring liquid sample multiphoton fluorescence spectrum |
| CN108051413A (en) * | 2017-11-30 | 2018-05-18 | 百色学院 | A kind of light activated photoluminescence spectra measuring system of pulse |
| CN111537444B (en) * | 2020-05-25 | 2021-07-06 | 南京航空航天大学 | A laser ultrasonic nondestructive testing method and system with virtual control of repetition frequency |
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