WO2017193759A1 - 生物光子光谱检测系统及检测方法 - Google Patents

生物光子光谱检测系统及检测方法 Download PDF

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WO2017193759A1
WO2017193759A1 PCT/CN2017/080342 CN2017080342W WO2017193759A1 WO 2017193759 A1 WO2017193759 A1 WO 2017193759A1 CN 2017080342 W CN2017080342 W CN 2017080342W WO 2017193759 A1 WO2017193759 A1 WO 2017193759A1
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biophotonic
spectrum
stripe
detecting
slit
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French (fr)
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戴甲培
王卓
李泽华
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中南民族大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2803—Investigating the spectrum using photoelectric array detector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0243—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows having a through-hole enabling the optical element to fulfil an additional optical function, e.g. a mirror or grating having a throughhole for a light collecting or light injecting optical fiber
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2823—Imaging spectrometer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/255—Details, e.g. use of specially adapted sources, lighting or optical systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J2003/006—Fundamentals or review articles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2803—Investigating the spectrum using photoelectric array detector
    • G01J2003/282—Modified CCD or like
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/068—Optics, miscellaneous

Definitions

  • the invention relates to a biophotonic spectrum detection of organisms (individuals, tissues, cells and molecules, etc.) by using super-weak photon radiation signals, and realizes biophotonic spectrum imaging, and belongs to the field of biological and biomedical technology imaging. This technique can also be applied to other fields where long-term imaging is required for weak-light spectral analysis.
  • Biophotonics is an abbreviation for biological ultra-weak luminescence. In essence, it has no difference from the light usually referred to.
  • the spectrum is 200-800 nm, which is ubiquitous in various living organisms such as microorganisms, plants and animals. Biophotonic activity can sensitively reflect the physiological state of an organism.
  • biophotons can be understood as the energy released by biomolecules from high energy state to low energy state transition.
  • the biological system has the function of metabolism. It is a typical open system, and there is an eternal exchange of matter, energy and information with the external environment.
  • the outside world constantly pumps the dissipated biological system in a state away from heat balance. High-energy biomass molecules are unstable and must transition to low-energy states. In this process, biophotons are radiated, and molecules returning to low-energy states transition to high-energy states under external influence. External pumping and photon radiation occur together to achieve a dynamic balance.
  • biophotons Because the internal electrons of different living substances have different movements of electrons, they emit different biophotons. These biophotons are separated by a dispersion system (such as a prism or a grating), and a pattern in which the dispersed monochromatic light is sequentially arranged in accordance with the wavelength (or frequency) is called a spectrum. Studying the luminescence of different substances and absorbing light has important theoretical and practical significance. Therefore, the spectral characteristics of biophotons help us to conduct in-depth research and can be widely used in life science research, medical clinical examination, agricultural production, food safety and environmental protection.
  • a dispersion system such as a prism or a grating
  • a biophotonic imaging system (UBIS) was constructed with EM-CCD as the core, which can detect the intensity information of biophotons in different time and space.
  • EM-CCD as the core
  • a new technique and method is needed to detect the spectral characteristics of biophotons.
  • the invention adds a spectroscopic subsystem consisting of a slit and a grating on the basis of an EM-CCD-based biophotonic imaging system (UBIS), and the improved biophotonic spectrum detecting system can sensitively detect the time of the biophoton. Spatial information, intensity and spectral properties of biophotons.
  • UIS EM-CCD-based biophotonic imaging system
  • the biophotonic spectrum detection system is composed of a splitting subsystem and an imaging subsystem.
  • the imaging subsystem consists of a hood, a high light transmission lens, an EMCCD imaging device and its controller, a computer and a coolant circulation pump, and the hood is connected to the hood. Between the lens and the sample stage of the EMCCD imaging device, it is used to shield the effect of photoluminescence in the dark box on imaging.
  • the high light transmission lens is used for imaging, and the coolant circulation pump is used to ensure the operation of the EMCCD imaging device in a low temperature environment.
  • the light splitting subsystem is composed of a slit and a grating.
  • the slit is placed above the sample, so that the biological photon of the sample passes through the slit and becomes a line light source, and the line light source illuminates the grating and is formed by the multi-slit diffraction to form an equal interval.
  • the aligned bright stripes, the imaging subsystem records the biophotonic image and analyzes and calculates the biophotonic spectrum. Each stripe distance is related to the wavelength of the biophotonic.
  • the spectroscopic subsystem allows biophotons to form bright stripes of equal spacing according to different wavelengths.
  • the grating is an optical element consisting of a number of parallel slits of equal width and equal spacing.
  • the detection method of the biophotonic spectrum detecting system is characterized in that the method comprises the following steps: (1) detecting preparation and imaging subsystem preheating; (2) laser calibration; (3) LED light source reliability verification; (4) Sample detection; (5) image processing; (6) data analysis.
  • Step (2) The laser calibration is specifically: the biophoton detection system is calibrated by using at least three different wavelengths of laser light, and the laser light source of each wavelength needs to be separately calibrated under normal light intensity and biophoton intensity respectively.
  • the reliability verification of the LED light source is specifically: after the laser is calibrated to the biophotonic detection system, the biophotonic detection system is used to detect four different colors of blue, green, yellow and red after maintaining the experimental conditions consistent with the laser calibration conditions.
  • the normal intensity of the LED light source and the spectrum of the biophoton intensity are used to verify the biophotonic detection system. System reliability.
  • the sample detection and image processing are specifically: placing the prepared test sample in the middle of the imaging field and ensuring the level of the slit position. After detecting the biophotonic spectrum image of the sample, the biophotonic spectrum image is first converted into the TIF format. After the matlab algorithm goes to the bright spot, the gray value of each pixel of the biophotonic spectrum image is extracted and saved in Microsoft Excel, and then the gray value of each line is added to obtain the gray value sum distribution curve.
  • the data analysis is specifically: according to the biophotonic spectrum image, the position of the pixel point of the edge of the zero-order stripe and the first-order stripe is determined by the computer Andor Solis (Solis version 4.27.30001.0, Harbor., Northern Ireland) software, and then combined with the sum of the gray values. The data of the distribution curve is confirmed to obtain the pitches ⁇ Lmin, ⁇ Lc, and ⁇ Lmax of the zero-order stripes and the first-order stripes. Wherein, the center of the stripe is determined by the two edges of the stripe, as shown in Figure 2c.
  • ⁇ Lmin is the distance from the nearest edge of the first-order stripe to the center of the zero-order stripe
  • ⁇ Lc is the distance from the center of the first-order stripe to the center of the zero-order stripe
  • ⁇ Lmax is the distance from the farthest edge of the first-order stripe to the center of the zero-order stripe. The distance is represented by the pixel difference.
  • the sample, the spectroscopic subsystem and part of the imaging subsystem device are placed in a dark box composed of an inner layer of lead steel sheets to isolate external light and cosmic rays to protect the imaging process. The influence of external light.
  • the invention provides a biophotonic spectrum detecting system, which has the following characteristics:
  • a biophotonic spectroscopy detection system consisting of a biophotonic imaging system (UBIS) with a EM-CCD as the core and a spectroscopic subsystem can detect the spectral characteristics of the ultra-weak luminescent radiation of the living body;
  • UIS biophotonic imaging system
  • EM-CCD EM-CCD
  • each device is independent of each other, easy to replace, diverse in selection, and has good scalability and upgrade potential;
  • FIG. 1 is a schematic structural view of a biophotonic spectrum detecting system of the present invention
  • Figure 2a is a laser calibration spectrum diagram of the present invention
  • 2b is a spectrum diagram of the LED light source of the present invention.
  • 2c is a schematic diagram of spectral image analysis of the present invention.
  • Figure 2d is a fitting curve of the normal intensity laser calibration of the present invention.
  • 2e is a fitting curve of the biophoton intensity laser calibration of the present invention
  • 2f is a comparative diagram of spectral detection of the LED light source of the present invention.
  • Figure 3a is a 532 nm laser spectrum of the present invention.
  • Figure 3b is a graph showing the distribution of the gray value of the 532 nm laser spectrum of the present invention.
  • Figure 4a is a biophotonic spectrum of a mouse brain slice of the present invention.
  • Fig. 4b is a graph showing the results of spectroscopic analysis of mouse brain slices of the present invention.
  • the biophotonic spectrum detecting system of the present invention is composed of a splitting subsystem and an imaging subsystem 1.
  • the imaging subsystem consists of a hood, a high light transmission lens, an EMCCD imaging device and its controller, a computer and
  • the coolant circulation pump is composed of a hood connected between the lens of the EMCCD imaging device and the sample stage for shielding the influence of photoluminescence in the dark box on the imaging.
  • the high transmittance lens is used for imaging, and the coolant circulation pump is used to secure the EMCCD.
  • the imaging device operates in a low temperature environment, characterized in that the spectroscopic subsystem is composed of a slit 3 and a grating 2, and the slit 3 is placed above the sample, so that the biological photon of the sample passes through the slit to become a line source, and The line source illuminates the grating 2 and forms a bright stripe arranged at equal intervals after multi-slit diffraction.
  • the imaging subsystem 1 records the biophoton image and analyzes and calculates the biophotonic spectrum.
  • the grating is an optical element consisting of a number of parallel slits of equal width and equal spacing.
  • biophotonic spectroscopy detection system of the present invention to form a photon imaging device using an X-10375 EM-CCD of ANDOR, a GT13-12 transmission grating 2 and a slit 1 of Thorlabs. Subsystem, preliminary detection and study of glutamate-induced biophotonic spectra of mouse brain slices.
  • Test materials Adult Kunming mice from the Hubei Provincial Experimental Animal Public Service Center.
  • Test preparation Before starting the test, first turn on the air conditioner to maintain the laboratory room temperature at around 25 °C, then clean the brain slice perfusion system with ultrapure water, and turn on the EM-CCD and low temperature coolant circulation pump.
  • the EM-CCD is preheated in a working environment of -95 °C.
  • the biophotonic detection system is calibrated using three different wavelengths of laser light at 405 nm, 532 nm and 650 nm. Moreover, the laser source needs to be calibrated under normal intensity and biophoton intensity, respectively.
  • the calibration results are shown in Fig. 2a.
  • the upper graph shows the laser spectrum at normal intensity
  • the lower graph shows the laser spectrum at attenuated to the biophoton intensity.
  • the spectral image includes a zero-order stripe (the brightest strip in the middle) and two first-order strips (the relatively bright strips on both sides).
  • the two first-order fringes show a clear tendency to move away from the zero-order fringes, and the spectral results of the laser calibration at different intensities are consistent.
  • the spectral results of the LED source are shown in Figure 2b.
  • the top panel shows the spectrum of the LED source at normal intensity.
  • the bottom panel shows the spectrum of the LED source that is attenuated to the biophoton intensity.
  • mice were sacrificed by decapitation, the whole brain was quickly taken out, placed in an ACSF of 0 to 4 ° C, and then a sagittal brain slice of 1 to 2 mm thickness was cut with a vibrating slicer.
  • Open perfusion system The brain slices of mice immersed in ACCF at 0-4 °C were placed at room temperature for 1 h, and then the brain sections were transferred to a perfusion system.
  • the perfusate is contained in a glass bottle and is supplied with a mixture of 95% O2+5% CO2.
  • the perfusate was perfused through a peristaltic pump into a perfusion tank containing a brain slice in a dark box at a flow rate of 5 ml/min.
  • Start detection imaging First, the EM-CCD takes a positioning photo of the brain slice under normal lighting conditions, then closes the black box, and after avoiding the external light interference in the dark for 30 minutes, the glutamic acid is added to start real-time imaging. The time is 60s and lasts for 120 minutes.
  • Biophotonic Spectral Detection Adding the spectroscopic subsystem and adjusting the position, mainly to maintain the slit level. Then continue real-time imaging, the exposure time is 1500s, and the glutamic acid is eluted and re-dosed during the imaging process. Biophotonic spectral images of the maintenance period (50 min), the elution period (25 min), and the refill period (25 min) of glutamate-induced biophotonic radiation were taken and superimposed.
  • Image processing After obtaining a spectral image of glutamic acid-induced brain photobiophoton radiation, image processing was performed using the 532 nm laser spectral image of Fig. 3a as an example. The first is to convert the spectral image into TIF format. After the matlab algorithm is used to brighten the spot, the gray value of each pixel of the image is extracted and saved in Microsoft Excel, and then the gray values of each line are added. A gray value sum sum distribution curve is obtained. The scatter plot drawn is shown in Figure 3b.
  • edges of the zero-order stripes and the first-order stripes are determined by visual observation and data analysis. Finally, the fringe spacing and the wavelength of the biophotons are calculated.
  • the biophotonic spectrum of 50 mM glutamate-induced mouse brain slices was detected using a calibrated biophotonic spectroscopy system while ensuring that other experimental conditions were consistent.
  • the results are shown in Figure 4a.
  • the biophotonic spectral range of these brain slices can be calculated by data analysis and measuring the stripe spacing ⁇ Lmin, ⁇ Lc, ⁇ Lmax, as shown in Fig. 4b.

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Abstract

一种生物光子光谱检测系统及检测方法，生物光子光谱检测系统是在超弱生物光子成像系统的基础上，加入由狭缝(3)和光栅(2)组成的分光子系统。光栅(2)是由大量等宽等间距的平行狭缝构成的光学元件，利用多缝衍射原理使光发生色散。根据多缝衍射原理，待检测样品所发出的超弱生物光子通过狭缝(3)后形成线光源，线光源照射光栅后经过多缝衍射形成等间距排列的明条纹，其距离与生物光子的波长相关。成像子系统(1)用于图像记录和分析。实验检测证明，生物光子光谱检测系统灵敏度高，可以检测生物超弱发光的光谱，同时具有操作简便、扩展性好、功能丰富等特点，可广泛应用于生命科学研究、医学临床检查、农业生产、食品安全与环境保护等领域中。

Description

生物光子光谱检测系统及检测方法 技术领域
本发明涉及一种利用超弱生物光子辐射信号，对生物(个体、组织、细胞和分子等)进行生物光子光谱检测，实现生物光子光谱成像，属于生物和生物医学技术成像领域。此技术也可应用到其它领域需要长时间成像进行弱光光谱分析的领域。
背景技术：
生物光子是生物超弱发光的简称，在本质上，与通常所指的光没有差异，谱段为200-800nm，普遍存在于微生物、植物和动物等各种生命体中。生物光子活动可以灵敏地反映有机体的生理状态。
从分子物理学的观点来看，生物光子可以理解为生物分子从高能态向低能态跃迁过程中释放出来的能量。生物系统具有新陈代谢的功能，它是一个典型的开放系统，与外界环境存在着永恒的物质、能量、信息的交换。外界不断的泵浦耗散的生物系统，使之处于一种远离热平衡的状态。高能生命物质分子不稳定，必须向低能态跃迁，在此过程中辐射出生物光子，而回到低能态的分子在外界作用下又跃迁到高能态。外界泵浦和光子辐射相伴发生，达到一个动态平衡。
因为不同生命物质的原子内部电子的运动情况不同，所以它们辐射出的生物光子也不同。这些生物光子经过色散系统(如棱镜、光栅)分光后，被色散开的单色光按波长(或频率)大小而依次排列形成的图案，被称为光谱。研究不同物质的发光和吸收光的情况，有重要的理论和实际意义。因此，生物光子的光谱特性有助于我们进行深入研究，可广泛应用于生命科学研究、医学临床检查、农业生产、食品安全和环境保护等领域。
在20世纪20年代，苏联生物学家Gurwitsch首次使用生物探测器检测到生物光子辐射。但是这种生物探测器在当时没有受到足够的重视，而且因为生物光子强度太弱，受到当时技术的局限，没有出现更灵敏的生物光子检测手段。直至1955年，Colli等人才使用当时刚发明的光电倍增管(PMT)最先对植物的生物光子进行直接检测。随着电子和信息通信技术的飞速发展，光子检测器也在不断地更新换代，随后出现了光导电检测器、光敏晶体管和电荷耦合元件(CCD)。其中，电子倍增-电荷耦合元件(EM-CCD) 具有量子效率高、信噪比高、体积小和寿命长的优点，广泛应用于生物光子检测领域。
以EM-CCD为核心构建了一套生物光子成像系统(UBIS)，可以检测到生物光子在不同时间和空间上的强度信息。但是，为了对生物光子的性质进行更深入的研究，还需要一种新的技术和方法来检测生物光子的光谱特性。
发明内容：
本发明在以EM-CCD为核心的生物光子成像系统(UBIS)的基础上，加入由狭缝和光栅组成的分光子系统，改进后的生物光子光谱检测系统可以灵敏地检测生物光子的时间和空间信息、生物光子的强度和光谱特性。
生物光子光谱检测系统，由分光子系统和成像子系统两部分组成，成像子系统由遮光罩、高透光镜头、EMCCD成像器件及其控制器、计算机和冷却液循环泵组成，遮光罩连接于EMCCD成像器件的镜头和样品台间，用于屏蔽暗箱内的光致发光对成像的影响，高透光镜头用于成像，冷却液循环泵用于保障EMCCD成像器件在低温环境下工作，其特征在于：所述分光子系统由狭缝和光栅组成，狭缝放置在样品上方，使样品的生物光子透过狭缝后成为一个线光源，且该线光源照射光栅后经过多缝衍射形成等间距排列的明条纹，成像子系统记录生物光子图像并分析计算出生物光子光谱。各条纹距离与生物光子的波长相关。分光子系统使得生物光子根据不同的波长而形成等间距不同的明条纹。
所述的光栅是由若干等宽等间距的平行狭缝构成的光学元件。
利用所述的生物光子光谱检测系统的检测方法，其特征在于包括如下步骤：(1)检测准备及成像子系统预热；(2)激光标定；(3)LED光源可靠性验证；(4)样品检测；(5)图像处理；(6)数据分析。
步骤(2)激光标定具体为：采用至少3种不同波长的激光来对生物光子检测系统进行标定，而且，每种波长的激光光源需要分别在正常光强度下和生物光子强度下分别进行标定。
LED光源可靠性验证具体为：在完成激光对生物光子检测系统标定后，在保持实验条件与激光标定条件一致的情况下，使用生物光子检测系统检测蓝色、绿色、黄色和红色四种不同颜色LED光源的正常强度和生物光子强度的光谱，来验证该生物光子检测系 统的可靠性。
样品检测及图像处理具体为：将准备好的检测样品放置在成像视野中间，并保证狭缝位置水平，检测获得样品的生物光子光谱图像后，首先是将生物光子光谱图像转换为TIF格式，经过matlab算法去亮斑后，提取出生物光子光谱图像每一个像素点上的灰度值，并保存在Microsoft Excel中，然后将每一行的灰度值相加得到灰度值总和分布曲线。
数据分析具体为：根据生物光子光谱图像通过计算机Andor Solis(Solis version 4.27.30001.0,Belfast.,Northern Ireland)软件确定零级条纹和一级条纹的边缘所在的像素点位置，然后结合灰度值总和分布曲线的数据来确认，得到零级条纹和一级条纹的间距△Lmin、△Lc、△Lmax。其中，条纹的中心通过条纹的两个边缘来确定，如图2c所示。△Lmin为一级条纹最近边缘到零级条纹中心的距离，△Lc为一级条纹中心到零级条纹中心的距离，△Lmax为一级条纹最远边缘到零级条纹中心的距离。距离由像素差表示。最后根据激光标定拟合的线性方程计算出该样品的生物光子光谱范围。
本发明的生物光子光谱检测过程中，样品、分光子系统和部分成像子系统器件需放置于由外层钢板内层铅板组成的暗箱中，以隔绝外界光线和宇宙射线，使成像过程免受外界光的影响。
本发明的技术特点：
本发明提供了一种生物光子光谱检测系统，具有以下特点：
(1)以EM-CCD为核心的生物光子成像系统(UBIS)和分光子系统构成的生物光子光谱检测系统，可以实现对生物体超弱发光辐射的光谱特性检测；
(2)灵敏度高：以英国ANDOR公司的X-10375型EM-CCD为光子成像器件，并配合暗箱等相关的元件，可以达到很好的信噪比，而且待检测样品与分光子系统直接接触，减少了生物光子检测过程中的损耗，从而更灵敏的检测到生物光子信号；
(3)操作简便：根据多缝衍射原理，以狭缝和光栅组成的分光子系统原理简单，操作方便；
(4)扩展性好：该系统中，各器件相互独立，更换方便，选择多样，具有很好的扩展性和升级潜力；
(5)功能丰富：根据检测条件可以调整不同的实验参数，可广泛应用于生命科学研究、医学临床检查、农业生产、食品安全与环境保护等领域中的生物光子光谱检测。
附图说明
图1为本发明的生物光子光谱检测系统结构示意图；
图2a为本发明的激光标定光谱图；
图2b为本发明的LED光源检测光谱图；
图2c为本发明的光谱图像分析示意图；
图2d为本发明的正常强度激光标定的拟合曲线图；
图2e为本发明的生物光子强度激光标定的拟合曲线图；
图2f为本发明的LED光源光谱检测对比图；
图3a为本发明的532nm激光光谱图；
图3b为本发明的532nm激光光谱图的灰度值总和分布曲线图；
图4a为本发明的小鼠脑片的生物光子光谱图；
图4b为本发明的小鼠脑片的光谱分析结果图。
具体实施方式
为了更好地理解本发明，下面结合实施例进一步阐明本发明的具体内容、操作流程和实际性能。但本发明的内容不仅仅局限于下面的实施例，本领域技术人员可以对本发明作各种改进、升级和拓展等，这些等价形式同样在本申请所列权利要求书限定范围之内。
如图1所示，本发明的生物光子光谱检测系统，由分光子系统和成像子系统1两部分组成，成像子系统由遮光罩、高透光镜头、EMCCD成像器件及其控制器、计算机和冷却液循环泵组成，遮光罩连接于EMCCD成像器件的镜头和样品台间，用于屏蔽暗箱内的光致发光对成像的影响，高透光镜头用于成像，冷却液循环泵用于保障EMCCD成像器件在低温环境下工作，其特征在于：所述分光子系统由狭缝3和光栅2组成，狭缝3放置在样品上方，使样品的生物光子透过狭缝后成为一个线光源，且该线光源照射光栅2后经过多缝衍射形成等间距排列的明条纹，成像子系统1记录生物光子图像并分析计算出生物光子光谱。所述的光栅是由若干等宽等间距的平行狭缝构成的光学元件。
相关研究提示，中枢神经系统中可能存在有生物光子活动并具有信号传递的功能。基于此，本发明人利用本发明所述的生物光子光谱检测系统，以ANDOR公司的X-10375型EM-CCD为光子成像器件，Thorlabs公司的GT13-12型透射光栅2和狭缝1组成分光 子系统，初步检测和研究了小鼠脑片谷氨酸诱导的生物光子光谱。
实施例：小鼠脑片谷氨酸诱导的生物光子光谱
一、检测材料：成年昆明小鼠，来自湖北省实验动物公共服务中心。
二、检测步骤：
(1)检测准备：在开始检测之前，首先要打开空调，使实验室室温维持在25℃左右，然后使用超纯水清洗脑片灌流系统，并开启EM-CCD和低温冷却液循环泵，使EM-CCD处于-95℃的工作环境中进行预热。
(2)激光标定：采用405nm、532nm和650nm 3种不同波长的激光来对生物光子检测系统进行标定。而且，激光光源需要分别在正常强度下和生物光子强度下进行标定。
标定结果如附图2a所示，上图为正常强度下的激光光谱，下图为减弱到生物光子强度下的激光光谱。从图中可以看出，光谱图像包括一个零级条纹(中间最亮的条纹)和两个一级条纹(两边相对较亮的条纹)。随着激光波长的增加，两个一级条纹相对于零级条纹呈现明显的远离趋势，而且不同强度下激光标定的光谱图结果一致。
(3)LED光源可靠性验证：完成激光对生物光子检测系统标定后，在保持其他实验条件一致的情况下，使用该系统检测蓝色、绿色、黄色和红色四种不同颜色LED光源的正常强度和生物光子强度的光谱，如图2f所示，以验证该生物光子检测系统的可靠性。
LED光源的光谱结果如附图2b所示，上图为正常强度下的LED光源光谱，下图为减弱到生物光子强度下的LED光源光谱。
(4)检测材料的制备：小鼠在断头处死后，迅速将其全脑取出，放置在0～4℃的ACSF中，然后用振动切片机切取一个1～2mm厚度的矢状大脑切片。
(5)打开灌流系统：将浸泡在0～4℃ACSF的小鼠大脑切片放置在室温下1h，然后，把大脑切片转移到灌流系统中。灌流液装在一个玻璃瓶中，并通有95％O2+5％CO2的混合气。灌流液通过蠕动泵灌注到暗箱中装有脑片的灌流槽里，流速为5ml/min。
(6)开始检测成像：首先是EM-CCD在正常光照条件下拍摄脑片的定位照，然后关闭暗箱，在避光30分钟以避免外界光的干扰后，加入谷氨酸开始实时成像，曝光时间为60s，持续120min。
(7)生物光子光谱检测：加入分光子系统并调整位置，主要是保持狭缝水平。然后继续实时成像，曝光时间为1500s，在成像过程中对谷氨酸进行洗脱和再加药处理，拍 摄出谷氨酸诱导的生物光子辐射时维持期(50min)、洗脱期(25min)和再加药期(25min)的生物光子光谱图像，并进行叠加处理。
(8)图像处理：在得到谷氨酸诱导的脑片生物光子辐射的光谱图像后，以附图3a的532nm激光光谱图像为例进行图像处理。首先是将该光谱图像转换为TIF格式，经过matlab算法去亮斑后，提取出该图像每一个像素点上的灰度值，并保存在Microsoft Excel中，然后将每一行的灰度值相加得到灰度值总和分布曲线。绘制的散点图如附图3b所示。
(9)数据分析：根据以上图像和数据，我们可以确定如附图2c所示的零级条纹和一级条纹的上下边缘。首先计算机Andor Solis(Solis version 4.27.30001.0,Belfast.,Northern Ireland)软件通过视觉来确定图像边缘所在的像素点，然后结合灰度值总和分布曲线的数据来确认，得到零级条纹和一级条纹的间距△Lmin、△Lc、△Lmax。条纹的边缘通常选择相邻灰度值总和相差最大的那个像素点。
通过以上的分析方法，我们就可以计算出△Lmin、△Lc、△Lmax，而激光的波长已知，这样就能够得到条纹间距△L与波长λ的线性回归方程，并进行相关性检验。如图2d、图2e所示，分别是正常强度下和生物光子强度下的激光标定拟合曲线。
通过视觉观察和数据分析确定出零级条纹和一级条纹的边缘。最后计算出条纹间距和生物光子的波长。
三、检测结果：
在保证其他实验条件一致的情况下，使用标定过的生物光子光谱检测系统检测50mM谷氨酸诱导的小鼠脑片的生物光子光谱，结果如附图4a所示，从图中能够看到明显的一条零级条纹和两条一级条纹。根据前面介绍的数据分析方法，通过数据分析，测量出条纹间距△Lmin、△Lc、△Lmax后就能计算出这些脑片的生物光子光谱范围，如附图4b所示。
以上所述，仅是用以说明本发明的具体实施案例而已，并非用以限定本发明的可实施范围，举凡本领域熟练技术人员在未脱离本发明所指示的精神与原理下所完成的一切等效改进、升级和拓展等改变或修饰，仍应由本发明权利要求的范围所覆盖。

Claims (7)

  1. 生物光子光谱检测系统，由分光子系统和成像子系统两部分组成，成像子系统由遮光罩、高透光镜头、EMCCD成像器件及其控制器、计算机和冷却液循环泵组成，遮光罩连接于EMCCD成像器件的镜头和样品台间，用于屏蔽暗箱内的光致发光对成像的影响，高透光镜头用于成像，冷却液循环泵用于保障EMCCD成像器件在低温环境下工作，其特征在于：所述分光子系统由狭缝和光栅组成，狭缝放置在样品上方，使样品的生物光子透过狭缝后成为一个线光源，且该线光源照射光栅后经过多缝衍射形成等间距排列的明条纹，成像子系统记录生物光子图像并分析计算出生物光子光谱。
  2. 根据权利要求1所述的生物光子光谱检测系统，其特征在于：所述的光栅是由若干等宽等间距的平行狭缝构成的光学元件。
  3. 利用权利要求1或2所述的生物光子光谱检测系统的检测方法，其特征在于包括如下步骤：(1)检测准备及成像子系统预热；(2)激光标定；(3)LED光源可靠性验证；(4)样品检测；(5)图像处理；(6)数据分析。
  4. 根据权利要求3所述的生物光子光谱检测系统的检测方法，其特征在于：步骤(2)激光标定具体为：采用至少3种不同波长的激光来对生物光子检测系统进行标定，而且，每种波长的激光光源需要分别在正常光强度下和生物光子强度下分别进行标定。
  5. 根据权利要求3所述的生物光子光谱检测系统的检测方法，其特征在于：LED光源可靠性验证具体为：在完成激光对生物光子检测系统标定后，在保持实验条件与激光标定条件一致的情况下，使用生物光子检测系统检测蓝色、绿色、黄色和红色四种不同颜色LED光源的正常强度和生物光子强度的光谱，来验证该生物光子检测系统的可靠性。
  6. 根据权利要求3所述的生物光子光谱检测系统的检测方法，其特征在于样品检测及图像处理具体为：将准备好的检测样品放置在成像视野中间，并保证狭缝位置水平，检测获得样品的生物光子光谱图像后，首先是将生物光子光谱图像转换为TIF格式，经过matlab算法去亮斑后，提取出生物光子光谱图像每一个像素点上的灰度值，并保存在Microsoft Excel中，然后将每一行的灰度值相加得到灰度值总和分布曲线。
  7. 根据权利要求6所述的生物光子光谱检测系统的检测方法，其特征在于：数据分 析具体为：根据生物光子光谱图像通过计算机Andor Solis软件确定零级条纹和一级条纹的边缘所在的像素点位置，然后结合灰度值总和分布曲线的数据来确认，得到零级条纹和一级条纹的间距△Lmin、△Lc、△Lmax。其中，条纹的中心通过条纹的两个边缘来确定，△Lmin为一级条纹最近边缘到零级条纹中心的距离，△Lc为一级条纹中心到零级条纹中心的距离，△Lmax为一级条纹最远边缘到零级条纹中心的距离，距离由像素差表示，最后根据激光标定拟合的线性方程计算出该样品的生物光子光谱范围。
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