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Split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection method and device

A differential confocal and fluorescence lifetime technology, which is applied in spectrometry/spectrophotometry/monochromator, measuring device, fluorescence/phosphorescence, etc., can solve the problem that the spot size of the excitation beam cannot be guaranteed and the resolution of the detection system cannot be guaranteed. Problems such as inability to maintain consistency, failure to obtain fluorescence distribution imaging on the sample surface, etc., to achieve high identification speed and identification accuracy, convenient detection process, and helpful for precise positioning

Active Publication Date: 2020-10-30
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 objective lens surface is contaminated
In addition, when detecting weak fluorescence signals, on the excitation light path, strong light will not only excite the fluorescence of the sample, but also stimulate the autofluorescence of the optical components, which will interfere with the experimental results and reduce the signal-to-noise ratio of the system

Method used

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  • Split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection method and device
  • Split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection method and device
  • Split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection method and device

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Embodiment 1

[0042] 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 split-pupil differential confocal detection system is used to measure three-dimensional topography, using the fluorescence lifetimes of four wavelengths of 400 nm, 450 nm, 530 nm and 580 nm excited from the sample by a pulsed laser with a wavelength of 355 nm To judge whether each scanning point is a tumor cell. 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. Figure 9 It is a specific implementation device for realizing split-pupil differential confocal discrete fluorescence spectroscopy ...

Embodiment 2

[0053] Different from Example 1, this example uses discrete fluorescence spectroscopy to determine whether each point on the surface of the sample 8 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 fluorescence spectrum measurement, in this embodiment, the light beam emitted by the continuous laser light source 2 is used to excite the sample 8 to generate fluorescence, and the measurement steps are as follows.

[0054] (a) Turn on the continuous laser light source 2, along x with y Move the sample 8 to be tested in the direction to the starting position of the transverse scanning ( x 1 , y 1 ), then at that position along the z Scan the sample 8 to be tested in the direction. Using the split-pupil differential confocal detection system 12, it is measured that the attached Figure 10 The differential confocal response curve FES shown ( z ), and then according to the diffe...

Embodiment 3

[0060] 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. By adoption of design of spectral pupil, the interference on the result by autofluorescence of an optical component in anexcitation light path can be effectively shielded and the signal to noise ratio of the system is increased. In addition, a differential confocal object surface positioning technology, a discrete fluorescence spectrum and a fluorescence lifetime measuring technology are fused; and high-precision measurement of three-dimensional shape of a to-be-tested sample is realized by utilizing a differentialconfocal technology, and high-sensitivity detection of the fluorescence spectrum and fluorescence lifetime of each point of the surface of the to-be-tested sample is realized by utilizing a discrete fluorescence spectrum and fluorescence lifetime detection technology, so that three-dimensional high-resolution spatial material component distribution information is acquired. Furthermore, in the process of measuring the fluorescence information of the surface of the sample, various different discrete fluorescence detection means are used, and a user can select to use ether fluorescence spectrum detection or fluorescence lifetime detection according to the chemical characteristic of the to-be-tested substance. Wide application prospect in the fields of biology, medicine, material science and clinical medicine diagnosis is achieved.

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 method for qualitative and quantitative analysis of substances by using 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 st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01J3/44G01J3/02
CPCG01J3/0216G01J3/0262G01J3/0291G01J3/4406G01N21/6458
Inventor 杨佳苗李静伟龚雷
Owner 杨佳苗
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