Confocal Discrete Fluorescence Spectroscopy and Fluorescence Lifetime Detection Method and Device

A technology of fluorescence lifetime and fluorescence spectrum, which is applied in the direction of measuring devices, fluorescence/phosphorescence, and analytical materials, can solve problems such as inconsistent resolution of the detection system, collision of the objective lens with the sample to be tested, and surface contamination of the objective lens of the system to improve identification Speed ​​and identification accuracy, eliminating the limitation of measurement accuracy, and the effect of high measurement sensitivity

Active Publication Date: 2021-03-23
杨佳苗
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Problems solved by technology

[0004] However, in the process of scanning and imaging the surface excited fluorescence of the sample to be tested, when the surface of the sample to be tested is uneven, it is impossible to ensure that the spot size of the excitation beam at different positions on the surface of the sample to be tested is consistent, which leads to the detection system at different positions. The resolution cannot be kept consistent
Especially for some samples with large fluctuations, there is even the possibility that the objective lens collides with the sample to be measured during the sample scanning process due to the small working distance of the system measurement objective lens. In the end, not only the fluorescence distribution imaging on the sample surface cannot be obtained, but also the system will cause The surface of the objective lens is contaminated

Method used

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  • Confocal Discrete Fluorescence Spectroscopy and Fluorescence Lifetime Detection Method and Device
  • Confocal Discrete Fluorescence Spectroscopy and Fluorescence Lifetime Detection Method and Device
  • Confocal Discrete Fluorescence Spectroscopy and Fluorescence Lifetime Detection Method and Device

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Embodiment 1

[0041] The problem to be solved in this embodiment is to simultaneously scan the three-dimensional shape of the sample to be tested and analyze the spatial distribution of the tumor tissue in the sample to be tested, and judge the boundary information of the tumor tissue accordingly. In this embodiment, a confocal detection system is used to measure the three-dimensional shape, and the fluorescence lifetimes of the four wavelengths of 400 nm, 450 nm, 530 nm and 580 nm excited by the 355 nm wavelength pulsed laser in the sample are used to judge each Whether the scanning point is a tumor cell or not. Since the fluorescence signal of the sample is very weak, this embodiment uses a photomultiplier tube as a light intensity sensor for fluorescence detection to improve the fluorescence light intensity detection sensitivity of the system.

[0042] Figure 9 It is a specific implementation device for realizing confocal discrete fluorescence spectroscopy and fluorescence lifetime det...

Embodiment 2

[0052] Different from Embodiment 1, this embodiment utilizes the difference in spectral intensity of fluorescence to determine whether each point on the surface of the sample 7 to be tested is a tumor tissue or a normal tissue. Apparatus used and sample are identical with embodiment 1. In order to improve the stability of the fluorescence spectrum measurement, the present embodiment uses the light beam emitted by the continuous laser light source 2 to excite the sample 7 to be tested to generate fluorescence, and the measurement steps are as follows:

[0053] (a) Turn on the continuous laser light source 2, along x and y Move the sample 7 to be tested in the direction to the starting position of the transverse scan ( x 1 , y 1 ), then at that position along the z scan the sample 7 in the direction to be tested. Using the confocal detection system 10, it is measured that as the attached Figure 10 The confocal response curve shown I ( z ), and then according to the co...

Embodiment 3

[0059] Different from Example 1, as attached Figure 5 As shown, in order to improve the resolution of the detection beam, a converging lens and a pinhole are respectively added in front of all light intensity sensors. The pinhole is placed at the focus position of the converging lens, and the converging lens converges the fluorescent light beams of different wavelengths to perform spatial filtering through the pinhole. Therefore, the fluorescent signals detected by each light intensity sensor are the filtered fluorescent light intensity information, and these filtered fluorescent lights accurately correspond to the fluorescent signals excited by the focal point of the detection beam, and the fluorescent signals outside the focal point are effectively analyzed. shield.

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Abstract

The invention belongs to the technical field of chemical substance detection and relates to a confocal discrete fluorescence spectrum and fluorescence lifetime detection method and device. According to the method, a confocal object positioning technology and a discrete fluorescence spectrum and fluorescence lifetime detection technology are integrated; the problem about high-precision measurementof three-dimensional topography of a to-be-detected sample surface is solved with a confocal technology, the problem about high-sensitivity detection of fluorescence spectrum and fluorescence lifetimeof each point in the to-be-detected sample surface is solved with the discrete fluorescence spectrum and fluorescence lifetime detection technology, and distribution information of three-dimensionalhigh-resolution spatial substance composition is obtained. The confocal measurement technology and the discrete fluorescence substance composition detection technology are integrated for the first time, a fluorescence imaging system is ensured to have the same transverse resolution in each position of the to-be-detected sample surface, and finally, the measured fluorescence spectrum distribution and the three-dimensional topography correspond to each other accurately. The technology has broad application prospect in the fields of biology, medical science, material science and clinical medicaldiagnosis.

Description

technical field [0001] The invention belongs to the technical field of chemical substance detection. It can not only identify the components of substances with high sensitivity, but also detect the spatial distribution of the components of substances. It will play an important role in the research fields of biology, medicine, material science and clinical medical diagnosis. application. Background technique [0002] In the fields of biology, physics, chemistry and materials, spectral detection and analysis has become a basic measurement method for basic research due to its very high sensitivity, molecular specificity and non-contact measurement characteristics. Among them, fluorescence spectrum detection is a qualitative and quantitative analysis method that uses the characteristics and intensity of fluorescence produced by substances under ultraviolet light irradiation. Especially for most organic compounds, the fluorescence produced by them usually has strong specificity ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6458G01N2021/6421
Inventor 杨佳苗李静伟龚雷
Owner 杨佳苗
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