WO2017088656A1 - 一种荧光寿命测量方法及装置 - Google Patents

一种荧光寿命测量方法及装置 Download PDF

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WO2017088656A1
WO2017088656A1 PCT/CN2016/105048 CN2016105048W WO2017088656A1 WO 2017088656 A1 WO2017088656 A1 WO 2017088656A1 CN 2016105048 W CN2016105048 W CN 2016105048W WO 2017088656 A1 WO2017088656 A1 WO 2017088656A1
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秦海燕
彭笑刚
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浙江大学
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    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

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  • the invention relates to a fluorescence lifetime measuring method and apparatus for detecting the life measurement and imaging of emitted light caused by irradiation of excitation light.
  • TCSPC time-dependent single photon counting
  • This method uses a photomultiplier tube or an avalanche photodiode as a detector, and then cooperates with the point.
  • the second is to use Time-gated technology, using an enhanced charge-coupled device (ICCD) camera as a detector, each light pulse is delayed after a certain period of time to collect the fluorescent signal, and the process is repeated several times to delay different times.
  • ICCD enhanced charge-coupled device
  • the fluorescence lifetime is measured, and the wide-field imaging method is used to perform fluorescence lifetime imaging.
  • both techniques must use a pulsed laser as the light source, with photomultiplier tubes (or avalanche photodiodes) and enhanced charge coupled device cameras as detectors, which are very expensive.
  • the two methods are required to periodically drive the detector according to the illumination of the excitation light source, and the optical path is complicated, and the adjustment and processing are time-consuming.
  • the present invention provides a fluorescence lifetime measuring method and apparatus.
  • a fluorescence lifetime measuring method comprising the following steps:
  • the fluorescent sample is excited by the excitation light source, and then starts to emit light;
  • the imaging device collects the fluorescence image, and records the acquisition time; and obtains a series of fluorescent images;
  • the excitation light source is a pulse light source or a continuous light source controlled by a switch;
  • the controlled continuous light source is turned off by the switch after exciting the fluorescent sample.
  • the fluorescent sample to be tested is composed of a plurality of fluorescent materials, and after the sample is excited, different fluorescent materials respectively form a fluorescent image.
  • a fluorescence lifetime measuring device comprising:
  • a sample loading device for placing or moving a fluorescent sample to be tested
  • An excitation device is provided with an excitation light source for generating excitation light to excite the sample to be tested;
  • An imaging device for performing image collection and storage of fluorescence emitted by the sample to be tested
  • a data processing device for calculating a fluorescence lifetime based on an image acquired in the imaging device.
  • the apparatus further includes adjustment means for adjusting the relative position of the excitation means and the sample means, and the relative position of the imaging means to the sample means such that a fluorescent image is obtained on the imaging means.
  • the fluorescence lifetime measuring and imaging apparatus stores a fluorescent signal photograph at a certain speed, which allows a normal high-speed camera to be used instead of a photomultiplier tube or an enhanced charge coupled device camera to obtain a change in intensity of light occurring in a short period of time, thereby obtaining The fluorescence lifetime of the sample.
  • the entire device is simple, low cost, easy to operate, and enables fast and high-throughput real-time online measurements.
  • Figure 1 is a fluorescence attenuation diagram of the fluorescent substance to be tested No. 1;
  • Figure 2 is a graph showing the attenuation fit of the No. 1 fluorescent substance to be tested
  • Figure 3 is a fluorescence attenuation diagram of the fluorescent substance to be tested No. 2;
  • Figure 4 is a graph showing the attenuation fit of the No. 2 fluorescent substance to be tested
  • Figure 5 is a fluorescence attenuation diagram of the fluorescent substance to be tested No. 3;
  • Figure 6 is a graph showing the attenuation fit of the No. 3 fluorescent substance to be tested.
  • Figure 7 is a fluorescence attenuation diagram of the fluorescent substance to be tested No. 4.
  • Figure 8 is a graph showing the attenuation fit of the No. 4 fluorescent substance to be tested.
  • Figure 9 is a fluorescence attenuation diagram of the fluorescent substance to be tested No. 5;
  • Figure 10 is a graph showing the attenuation fit of the No. 5 fluorescent substance to be tested.
  • the substrate with the No. 1 fluorescent substance to be tested was fixed on a sample holder, and the sample was irradiated with a pulse light source at a pulse repetition frequency of 1 kHz.
  • the high-speed camera continuously shoots the fluorescent signal with an exposure time of 50 us per frame.
  • the fluorescence signal corresponding to the repetitive frequency of the excitation pulse appears, and the fluorescence signal changes from strong to weak.
  • a total of 20 photos of continuous changes from strong to weak fluorescent signals were randomly selected.
  • the corresponding position of the fluorescent substance in the photograph is taken, as shown in Fig. 1.
  • the sum of the gray values of all the pixels in each frame is plotted against time, and the fluorescence attenuation curve is obtained, as shown in FIG. 2 .
  • the substrate with the No. 2 fluorescent substance to be tested was fixed on a sample holder, and the sample was irradiated with a pulse light source at a pulse repetition frequency of 1 kHz.
  • the high-speed camera continuously shoots the fluorescent signal with an exposure time of 50 us per frame.
  • the fluorescence signal corresponding to the repetitive frequency of the excitation pulse appears, and the fluorescence signal changes from strong to weak.
  • a total of 20 photos of continuous changes from strong to weak fluorescent signals were randomly selected.
  • the corresponding position of the fluorescent substance in the photograph is taken, as shown in FIG. Then, the sum of the gray values of all the pixels in each frame is plotted against time, that is, a fluorescence decay curve is obtained, as shown in FIG.
  • ⁇ i is the lifetime of the ith fluorescence decay channel. Average fluorescence lifetime can be passed Calculated. Specifically, as shown in Fig. 4, the lifetime of the first fluorescence decay channel of the sample No. 2 is 136 ⁇ s; the lifetime of the second fluorescence decay channel is 1046 ⁇ s; and the average fluorescence lifetime is 839 ⁇ s.
  • the substrate with the No. 3 fluorescent substance to be tested is fixed on the sample holder, and the sample is irradiated with a pulse light source, and the operation is performed according to the method of Embodiment 1 (the exposure time per frame is 100 ⁇ s, the continuous shooting pulse source is excited, and the repetition frequency is 500 ⁇ s), a fluorescence decay image as shown in Fig. 5 was obtained. Since the pulse repetition frequency of the excitation light source is high, the fluorescence signal of the substance to be tested is not attenuated to a lower value, and the next excitation pulse has arrived.
  • the multi-segment fluorescence decay time series can be used for fitting, and the fitting result is shown in FIG. 6.
  • the obtained fluorescence lifetime is averaged to be 200 ⁇ s.
  • the substrate with the No. 4 fluorescent substance to be tested is fixed on the sample holder, and the sample is irradiated with a pulse light source, and the operation is performed according to the method of Embodiment 1 (the exposure time per frame is 50 ⁇ s, the continuous shooting pulse source is excited, and the repetition frequency is 250 ⁇ s), a fluorescence decay image as shown in Fig. 7 was obtained. Since the pulse repetition frequency of the excitation light source is high, the fluorescence signal of the substance to be tested is not attenuated to a lower value, and the next excitation pulse has arrived.
  • the multi-segment fluorescence decay time series can be used for fitting, and the fitting result is shown in FIG. 8.
  • the obtained fluorescence lifetime is 147 ⁇ s.
  • the fluorescence lifetimes of a plurality of fluorescent samples to be tested are simultaneously tested, and the time-series fluorescence images taken after the excitation pulse irradiation of the No. 5 sample are as shown in FIG. 9 according to the method described in Embodiment 1, as shown in FIG.
  • Different fluorescent substances have different fluorescence intensity decay rates.
  • the gray values of the regions of the photograph corresponding to the three substances are selected, and their gray values are plotted against time, respectively, to obtain a fluorescence decay curve. Then they were fitted with a single exponential or multi-exponential decay function.
  • the fluorescence lifetimes of the three samples A, B and C were 36.6 ⁇ s, 69.0 ⁇ s and 281.8 ⁇ s, respectively.
  • the gradation value of each pixel position of the shooting area is plotted with time and is fitted by a single exponential or multi-exponential decay function, that is, the fluorescence lifetime of each pixel position in the shooting area is obtained.
  • the gray value or pseudo color is used to indicate the fluorescence lifetime of each pixel position, and the fluorescence lifetime image of the region is reconstructed. This technology can be extended to anti-counterfeiting applications.

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  • Physics & Mathematics (AREA)
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  • Analytical Chemistry (AREA)
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Abstract

一种荧光寿命测试的方法和装置，所述装置包括载样装置、激发装置、成像装置和数据处理装置，将荧光样品放置在样品装置上，利用激发装置的光源来激发样品的荧光，然后关掉光源利用成像装置实时对样品所发荧光进行成像，得到系列荧光强度随时间变化图像，利用该系列图像即可以计算出荧光寿命。该装置简单，成本低，易操作，可以实现快速和高通量实时在线测量。

Description

一种荧光寿命测量方法及装置 技术领域
发明涉及一种荧光寿命测量方法及装置，用于检测有激发光的照射引起的发射光的寿命测量和成像的技术领域。
背景技术
目前，荧光寿命的测量和成像主要有两种方法：一是使用时间相关单光子计数(TCSPC)技术对荧光寿命进行测量，这种方法采用光电倍增管或雪崩光电二极管作为探测器，再配合点扫描方法，进行荧光寿命成像。二是使用时间门(Time-gated)技术，用增强型电荷耦合器件(ICCD)相机作为探测器，每个光脉冲激发后延迟一定时间收集荧光信号，多次反复此过程延迟不同的时间，对荧光寿命进行测量，再配合宽场成像方法，进行荧光寿命成像。
然而，这两种技术均必须使用脉冲激光作为光源，搭配光电倍增管(或雪崩光电二极管)和增强型电荷耦合器件相机作为探测器，价格十分昂贵。另外采用这两种方法还需根据激发光源的照射来定时驱动探测器件，光路复杂，调整及处理耗时。
发明内容
为了解决上述问题，本发明提供一种荧光寿命测量方法及装置。
本发明的目的是通过以下技术方案实现的：一种荧光寿命测量方法，该方法包括以下步骤：
(1)用激发光源激发待测荧光样品；
(2)荧光样品受激发光源激发后，开始发光；成像装置采集荧光图像，并记录采集时间；获得一系列荧光图像；
(3)数据处理，将荧光图像转换成灰度图后，读取灰度值；根据灰度值以及对应的采集时间，输出荧光衰减曲线，得到荧光寿命。
进一步地，所述激发光源为脉冲光源或由开关控制的连续光源；所述由开 关控制的连续光源在激发荧光样品后，通过开关关闭。
进一步地，待测荧光样品由多种荧光材料组成，样品受到激发后，不同种荧光材料分别形成荧光图像。
一种荧光寿命测量装置，所述装置包括：
载样装置，用于放置或者移动要待测荧光样品；
激发装置，设置有激发光源，用于产生激发光以激发待测样品；
成像装置，用于对待测样品所发荧光进行图像采集并存储；
数据处理装置，用于根据成像装置中所采集的图像计算荧光寿命。
进一步地，所述装置还包括调整装置，用于调整激发装置与样品装置的相对位置，以及成像装置与样品装置的相对位置，以使得在成像装置上获得荧光图像。
根据本发明，荧光寿命测量和成像设备以一定的速度存储荧光信号照片，这允许以普通高速相机而不是光电倍增管或者增强型电荷耦合器件相机获得短时段内发生的光的强度变化，从而得到样品的荧光寿命。整个装置简单，成本低，易操作，可以实现快速和高通量实时在线测量。
附图说明
图1为1号待测荧光物质的荧光衰减图；
图2为1号待测荧光物质的衰减拟合图；
图3为2号待测荧光物质的荧光衰减图；
图4为2号待测荧光物质的衰减拟合图；
图5为3号待测荧光物质的荧光衰减图；
图6为3号待测荧光物质的衰减拟合图；
图7为4号待测荧光物质的荧光衰减图；
图8为4号待测荧光物质的衰减拟合图；
图9为5号待测荧光物质的荧光衰减图；
图10为5号待测荧光物质的衰减拟合图。
具体实施方式
实施例1
把带有1号待测荧光物质的基片固定在样品架上，用一脉冲光源照射样品，脉冲重复频率为1kHz。高速相机对荧光信号进行连续拍摄，每帧曝光时间为50us。所拍摄的系列照片中，即出现与激发脉冲重频相对应的荧光信号，荧光信号从强到弱的周期变化。从其中任意选取从强到弱的荧光信号连续变化的照片共20张。截取照片中荧光物质相应的位置，如图1所示。再把每帧中所有像素的灰度值的总和对时间作图，即得到荧光衰减曲线图，如图2所示。
然后用单指数衰减函数I(t)＝Ibg+A·exp(-t/τ)对数据点进行拟合，其中，Ibg为背景光强，τ为待测物质的荧光寿命。拟合得到1号待测物质的荧光寿命值为149μs。
实施例2
把带有2号待测荧光物质的基片固定在样品架上，用一脉冲光源照射样品，脉冲重复频率为1kHz。高速相机对荧光信号进行连续拍摄，每帧曝光时间为50us。所拍摄的系列照片中，即出现与激发脉冲重频相对应的荧光信号，荧光信号从强到弱的周期变化。从其中任意选取从强到弱的荧光信号连续变化的照片共20张。截取照片中荧光物质相应的位置，如图3所示。再把每帧中所有像素的灰度值的总和对时间作图，即得到荧光衰减曲线图，如图4所示。
首先用单指数衰减函数I(t)＝Ibg+A·exp(-t/τ)对数据点进行拟合，其中，Ibg为背景光强，τ为待测物质的荧光寿命。发现该样品不能用单指数衰减函数拟合，用多(双)指数衰减函数
Figure PCTCN2016105048-appb-000001
对数据点进行拟合。τi为第i个荧光衰减通道的寿命。平均荧光寿命可通过
Figure PCTCN2016105048-appb-000002
计算 得。具体见图4，2号待测物质的第1个荧光衰减通道的寿命为136μs；第2个荧光衰减通道的寿命为1046μs；平均荧光寿命为839μs。
实施例3
把带有3号待测荧光物质的基片固定在样品架上，用一脉冲光源照射样品，按照实施例1的方法进行操作(每帧曝光时间为100μs，连续拍摄脉冲光源激发，重频为500μs)，得到如图5所示的荧光衰减图像。由于激发光源的脉冲重复频率较高，致使待测物质荧光信号未衰减到较低值，下一激发脉冲已到达。
这种情况下，可以用多段荧光衰减时间序列进行拟合，拟合结果如图6所示，采用实施例2中的计算公式，得到的荧光寿命取平均值为200μs。
实施例4
把带有4号待测荧光物质的基片固定在样品架上，用一脉冲光源照射样品，按照实施例1的方法进行操作(每帧曝光时间为50μs，连续拍摄脉冲光源激发，重频为250μs)，得到如图7所示的荧光衰减图像。由于激发光源的脉冲重复频率较高，致使待测物质荧光信号未衰减到较低值，下一激发脉冲已到达。
这种情况下，可以用多段荧光衰减时间序列进行拟合，拟合结果如图8所示，采用实施例2中的计算公式，得到的荧光寿命取平均值为147μs。
实施例5
本实施例同时测试多种待测荧光样品的荧光寿命，按照实施例1所述的方法，对5号样品进行激发脉冲照射后，拍摄到的时间序列荧光图片，如图9所示，三种不同荧光物质的荧光强度衰减速度不同。对三种物质对应的照片的区域的灰度值选取出来，分别用它们的灰度值对时间作图，即得到荧光衰减曲线。然后分别对它们用单指数或者多指数衰减函数进行拟合，如图10所示，得到A、B、C三种待测样品的荧光寿命分别为36.6μs,69.0μs,281.8μs。
对拍摄区域的每一个像素位置的灰度值随时间的变化曲线都画出来，并用单指数或者多指数衰减函数拟合，即得到拍摄区域每一个像素位置的荧光寿命。 再用灰度值或者伪彩色，表示每一个像素位置的荧光寿命，重构出该区域的荧光寿命成像图。该技术可以扩展到防伪应用。

Claims (5)

  1. 一种荧光寿命测量方法，其特征在于，该方法包括以下步骤：
    (1)用激发光源激发待测荧光样品；
    (2)荧光样品受激发光源激发后，开始发光；成像装置采集荧光图像，并记录采集时间；获得一系列荧光图像；
    (3)数据处理，将荧光图像转换成灰度图后，读取灰度值；根据灰度值以及对应的采集时间，输出荧光衰减曲线，得到荧光寿命。
  2. 根据权利要求1所述的方法，其特征在于，所述激发光源为脉冲光源或由开关控制的连续光源；所述由开关控制的连续光源在激发荧光样品后，通过开关关闭。
  3. 根据权利要求1所述的方法，其特征在于，待测荧光样品由多种荧光材料组成，样品受到激发后，不同种荧光材料分别形成荧光图像。
  4. 一种实现权利要求1所述方法的装置，其特征在于，所述装置一般包括：
    载样装置，用于放置或者移动待测荧光样品；
    激发装置，设置有激发光源，用于产生激发光以激发待测样品；
    成像装置，用于对待测样品所发荧光进行图像采集并存储；
    数据处理装置，用于根据成像装置中所采集的图像计算荧光寿命。
  5. 根据权利要求4所述的装置，其特征在于，所述装置还包括调整装置，用于调整激发装置与样品装置的相对位置，以及成像装置与样品装置的相对位置，以使得在成像装置上获得荧光图像。
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