Turntable and device for measuring fluorescent and phosphorescent service life at high flux
阅读说明：本技术 一种高通量测量荧光磷光寿命的转盘和装置 (Turntable and device for measuring fluorescent and phosphorescent service life at high flux ) 是由 朱泽策 于 2018-06-22 设计创作，主要内容包括：一种用于高通量测量荧光磷光寿命的转盘和装置,可以用于高通量的荧光磷光寿命测量,测量方法是将激发光聚焦于一束,照射与转盘边缘一处,当转盘受电机驱动转动时,转盘上的光通道可将照射在转盘边缘的激发光引入到样品槽中,用于激发样本,各样品槽的样本被激发后在空间旋转,使不同延迟时间的发光信号在空间上分离,通过照相机拍照分析可获得样本的荧光磷光寿命。该方法实现了使用连续光源同时测量多个样本的荧光磷光寿命；由于激发光具有较高功率密度,可以测量低浓度样本和弱发光的样本；将激发光限制在光通道内部传输,可显著降低光源散射背景。此外该方法不需要昂贵的脉冲光源、高速探测器和复杂的锁相控制,具有成本低、仪器简单便携的特点。(A rotating disc and a device for measuring the fluorescence and phosphorescence life at high flux can be used for measuring the fluorescence and phosphorescence life at high flux, the measuring method is that excitation light is focused on one beam and irradiates the edge of the rotating disc, when the rotating disc is driven by a motor to rotate, an optical channel on the rotating disc can lead the excitation light irradiating the edge of the rotating disc into sample grooves for exciting samples, the samples in each sample groove rotate in space after being excited, so that light-emitting signals with different delay times are separated in space, and the fluorescence and phosphorescence life of the samples can be obtained through camera shooting analysis. The method realizes the simultaneous measurement of the fluorescence and phosphorescence lifetimes of a plurality of samples using a continuous light source; because the exciting light has higher power density, a low-concentration sample and a sample with weak luminescence can be measured; the exciting light is limited to be transmitted inside the light channel, and the scattering background of the light source can be obviously reduced. In addition, the method does not need expensive pulse light sources, high-speed detectors and complex phase-locked control, and has the characteristics of low cost and simple and portable instrument.)
1. A rotating disk for measuring fluorescence and phosphorescence lifetime, characterized by:
the turntable is provided with a plurality of sample grooves, the distances from the sample grooves to the circle center of the turntable are different, each sample groove is connected with one optical channel, all the optical channels are distributed on the turntable, and each optical channel is independent and does not interfere with each other;
the other end of the optical channel is connected with the edge of the turntable and used for introducing exciting light irradiated on the edge of the turntable into the optical channel and exciting a sample in the sample groove;
the medium constituting the optical channel is selected from air, optical fiber, and capillary.
2. The rotary pan of claim 1, wherein: the optical channel medium is a multimode optical fiber.
3. The rotary pan of claim 1, wherein: the medium of the light channel is air, and the inner wall of the light channel is provided with a reflecting film.
4. The rotary pan of claim 1, wherein: the optical channel medium is a transparent capillary tube, and the capillary tube is simultaneously used as a sample groove.
5. A fluorescence and phosphorescence lifetime measuring apparatus, characterized in that: comprising the turntable of any one of claims 1-4, further comprising a motor, a light source, a camera;
the turntable is driven by the motor shaft, the light emitted by the light source is irradiated on a certain point of the edge of the turntable, and the lens of the camera is parallel to the turntable and is used for shooting the image of the turntable.
6. The life measuring device according to claim 5, wherein: and a condensing lens is also arranged between the light source and the turntable and used for focusing light of the light source to a certain point on the edge of the turntable.
7. The life measuring device according to claim 5, wherein: the light source is selected from a laser diode or an LED light source.
8. The life measuring device according to claim 5, wherein: the optical channel medium is a capillary; when the liquid sample is measured, the liquid sample is placed in the capillary tube, and can be centrifuged firstly and then detected, so that the integration of centrifugal separation and service life detection is realized.
9. The life measuring device according to claim 5, wherein: an imaging lens is arranged between the camera and the turntable and is used for magnifying the image of the turntable and then taking a picture.
10. The life measuring device according to claim 5, wherein: the front of the camera lens also comprises a filter used for filtering the exciting light.
Technical Field
The invention relates to a method for measuring the lifetime of photoluminescence and the manufacture and application of related instruments and devices. Belongs to the field of optical instrument manufacture and instrument analysis.
Background
The characteristics of substance such as light absorption, photoluminescence, chemiluminescence and the like are applied to the field of analysis and detection. The principle is that the change of certain factors in the environment can cause the change of the absorbance and the luminous intensity of a substance, and the change of certain factors is judged by detecting the change of the light intensity. However, in actual measurement, whether the absorbance or the luminous intensity is measured, the measurement has a great relationship with the sensitivity of the detector; in the detection of photoluminescence, the intensity of excitation light directly affects the intensity of fluorescence and phosphorescence. In addition, many factors such as the light collection efficiency of the instrument, the interference of ambient light, and the light emission of impurities affect the measurement result. Thus, the same sample will have different results measured by different instruments.
In order to eliminate the above errors, the standard sample and the sample to be measured are often compared under the same measurement conditions to eliminate the error of the instrument, or a working curve is prepared to realize the quantitative analysis of the sample to be measured. In addition, due to factors such as aging of the instrument (for example, weakening of a light source) and the like, the instrument is often required to be repeatedly corrected, which directly causes a tedious operation process, increases human errors and reduces repeatability of measurement results. On the other hand, most of the devices depending on the detection are large-sized devices, which are inconvenient for point-of-care testing (POCT). Although in recent years, many portable instruments and devices are developed, and even many detections can be realized on intelligent hardware such as a mobile phone, the nature of the detected light intensity is not changed, so that the problem that the instruments need to be corrected still exists.
Time-resolved luminescence detection has also been used in many applications in analytical detection (see patents CN201210215872.0, CN201610166288.9, CN 201610416029.7). The method can remove background fluorescence interference with short service life and has high signal-to-noise ratio. However, the nature of the light intensity is still detected, and a calibration instrument is still required.
Another time-resolved detection is to measure the lifetime of fluorescence or phosphorescence, i.e., the lifetime of an excited state, and determine a change in a certain factor in the environment by detecting the lifetime of the luminescence. One method of detecting luminescence lifetime is: exciting a sample by using a pulse light source, and detecting the change of the luminous intensity of the sample along with time through a detector; and analyzing the curve of the light intensity with the time decay to obtain the length of the service life. In a single exponential decay curve, the length of the lifetime is defined as the time required for the decay to the original 1/e (ref. anal. chem., 1990, 62, 270A-277A; Pure appl. chem. 2014, 86, 1969-.
The lifetime of the excited state is an inherent property of the molecule, and in most cases, the lifetime of the excited state is independent of the intensity of the excitation light, so that the lifetime values of the same sample measured in different instruments are substantially identical, and the instrument does not need to be calibrated independently. Compared with the detection light intensity, the detection light intensity has the advantages that the errors caused by instrument differences can be eliminated by taking the service life of the excited state as the detection basis, and the detection light intensity has very high reliability.
Existing lifetime detection has been applied in fluorescence lifetime imaging (refer to anal. chem., 2017, 89, 8104-. However, the lifetime detection has not been widely applied to the analysis and detection of substances, and the main reason is that the existing lifetime detection instruments are expensive, such as pulse light sources, time-dependent single photon counters, stripe cameras, and other detectors (refer to chinese patents CN201410353200.5, CN201310694918.6, and CN 201310027775.3). On the other hand, the devices for measuring the service life are difficult to be small and portable, which limits the application and development of service life detection.
In addition, in the existing lifetime detection, one measurement can only detect the lifetime of one sample, and the method cannot be used for high-flux fluorescence lifetime detection. This is mainly because the existing lifetime measuring detectors, such as single photon counters, fringe cameras, etc., have difficulty in distinguishing different samples from each other in the spatial domain. In recent years, special image sensors, such as single photon avalanche diode (SADP) arrays, phase recordable CMOS sensors, etc. (references m. Gersbach, et al, proc. SPIE2010, 7780, 77801H; r. Franke, g.a. Holst, proc. SPIE 2015, 9328, 93281K) have been developed, which can measure the fluorescence lifetime of a plurality of pixels simultaneously and have a fast imaging speed in global fluorescence lifetime imaging. However, these detectors also need to be used in conjunction with a pulsed light source, phase lock control.
The inventor discloses a method for measuring lifetime by using an image sensor in a previous patent (cn201711110647. x), wherein an excited sample is driven by a motor to rotate in space, so that light-emitting signals with different delay times are separated in space, time domain information is converted into space domain information for detection, and synchronous measurement of fluorescence lifetime of a plurality of samples is realized. The method does not need pulse and phase-locked control and has ultralow cost. However, in high throughput lifetime measurements, to achieve multi-spot excitation, multiple laser spots or diverging LED light is required to be focused on the turntable. This aspect can cause scattering interference; on the other hand, for samples requiring strong light excitation, the scattered laser or LED beam directly reduces the excitation light optical power density, making it difficult to detect low-concentration or trace samples. Although the use of multiple lasers to excite light point-to-point can ensure excitation intensity, it can add complexity and cost to the instrument.
Disclosure of Invention
The patent is an important improvement of the patent (cn201711110647. x), and a motor is used for driving an excited sample to rotate in space, so that light-emitting signals with different delay times are separated in space, and time domain information is converted into space domain information for detection. The improvement of the patent lies in that the excitation light is focused on one beam, and the excitation light beam excites the sample from the direction parallel to the rotation plane of the sample, so that the rotation tracks of all samples can be intersected with the excitation light beam, namely, the sample can be excited by the excitation light in the rotation process. The method only uses one light source, not only ensures the energy density of the light beam, but also can be used for measuring the fluorescence lifetime with high flux, and has significant progress compared with the prior patent.
To achieve the above object, the present patent discloses a turntable for measuring fluorescence and phosphorescence lifetime, which is characterized in that:
the turntable is provided with a plurality of sample grooves, the distances from the sample grooves to the circle center of the turntable are different, each sample groove is connected with one optical channel, all the optical channels are distributed on the turntable, and each optical channel is independent and does not interfere with each other;
the other end of the optical channel is connected with the edge of the turntable and used for introducing exciting light irradiated on the edge of the turntable into the optical channel and exciting a sample in the sample groove;
the medium constituting the optical channel is selected from air, optical fiber, and capillary.
In a preferred turntable, the optical channel medium is a multimode optical fiber.
In another preferred embodiment, the light tunnel medium is air, and the light tunnel has a reflective film on its inner wall.
In another preferred embodiment, the optical channel medium is a transparent capillary, and the capillary serves as a sample well.
Based on above-mentioned various carousel, this patent discloses a fluorescence phosphorescence life-span measuring device, its characterized in that:
comprises any one of the turntables, a motor, a light source and a camera,
the turntable is driven by the motor shaft, the light emitted by the light source is irradiated on a certain point of the edge of the turntable, and the lens of the camera is parallel to the turntable and is used for shooting the image of the turntable.
In a preferred life measuring device, a condenser lens is further included between the light source and the turntable for focusing light from the light source to a point on the edge of the turntable. Therefore, the utilization rate of exciting light can be improved, and a fluorescence signal can be enhanced.
A preferred lifetime measuring device, characterized by: the light source is selected from a laser diode or an LED light source.
A preferred lifetime measuring device, characterized by: the optical channel medium is a capillary; when the liquid sample is measured, the liquid sample is placed in the capillary tube, and can be centrifuged firstly and then detected, so that the integration of centrifugal separation and service life detection is realized.
In another preferred life measuring device, an imaging lens is further included between the camera and the turntable for magnifying the image of the turntable and then taking a picture.
Preferably, in all the above measuring devices, a filter is further included in front of the camera lens for filtering the excitation light.
All of the above devices can be used to detect fluorescent phosphorescent lifetimes.
The principle of measuring the fluorescence lifetime by using the various devices is as follows:
the light emitted by the light source irradiates at a certain position of the edge of the turntable, and when the turntable rotates, the light enters each light channel of the turntable in turn and excites the sample in the sample groove at the tail end of the light channel to emit light,
because the rotating disc is continuously rotated, the sample in the sample groove can be excited only when the tail end of the optical channel is rotated to the illumination area; with the continuous rotation of the turntable, the tail end of the optical channel rotates out of the illumination area, the sample is not excited by illumination any more, but the luminescence of the sample can be attenuated along with the time; if the speed of the rotating disk is constant, the sample emits light to form an arc on the rotating disk, and the arc length is proportional to the delay time.
Because the turntable is provided with a plurality of optical channels, a plurality of samples can be excited to form a plurality of light arcs, if the turntable is photographed, images of the light arcs emitted by all the samples can be recorded, and each light arc represents a curve of the light-emitting attenuation of one sample along with time;
according to the rotating speed of the turntable, the delay time of each point on the light arc can be calculated;
the relative luminous intensity of each point on the light arc can be calculated according to the brightness or gray value of the image pixel points;
thus, a curve of the luminous intensity of the sample decaying along with time is obtained, and the service life can be obtained by fitting the curve.
Such a method can obtain the luminescence lifetime of a plurality of samples through only one photo, and thus is a high-throughput lifetime measurement.
The advantage of this method over the method in the previous patent (cn201711110647. x) is that:
the light of a single light source is focused on one point, the power density of exciting light is ensured while high-flux measurement is carried out, a plurality of light sources are not needed, the instrument is obviously simplified, and the cost is reduced;
because the exciting light has higher power density, a low-concentration sample and a sample with weak luminescence can be measured;
the exciting light is limited in the light channel for transmission, so that the scattering background of the light source can be obviously reduced;
the multi-mode optical fiber is adopted to transmit the exciting light, so that each sample can be excited by light with approximately equal intensity, and the test of a plurality of parallel samples is facilitated.
Compared with the existing fluorescence and phosphorescence life measuring device, the method and the device disclosed by the patent realize the measurement of the life of the excited state by using a steady-state light source, and have the following remarkable advantages:
expensive pulse light sources and detectors are not needed, phase-locked control is not needed, and the cost of service life measurement is greatly reduced;
all parts can be miniaturized and can be made into portable micro equipment, so that the instant detection and application are facilitated;
the luminous service life of a plurality of samples can be detected simultaneously, the flux and the efficiency of detection are improved, and the system error is favorably reduced.
Drawings
Fig. 1 shows a life measuring device, in which 101 is a turntable, 102 is a sample cell, 103 is an optical channel, 104 is a motor, 105 is a light source, and 106 is a camera.
Fig. 2 shows a lifetime measuring apparatus, wherein 201 is a turntable, 202 is a sample chamber, 203 is an optical channel, 204 is a light blocking ring, 205 is a motor, 206 is a light source, 207 is a reflector, 208 is a lens, 209 is a lens, 210 is a filter, and 211 is a mobile phone.
Fig. 3 is a schematic top view of a turntable, where 301 is a sample cell and 302 is an optical channel.
Fig. 4 is a schematic side and top view of a turntable, 401 is a turntable, 402 is a transparent capillary, 403 is a sample addition slot, and 404 is a light through hole.
Fig. 5 is a schematic side and top view of a turntable, 501 is the turntable, 502 is a capillary tube, and 503 is a light through hole.
Detailed Description
The present invention will be further illustrated by the following examples for the purpose of illustrating the principles of the present invention and its advantages, which are intended to facilitate a better understanding of the contents of the present invention, but which are not intended to limit the scope of the present invention in any way.
EXAMPLE 1, turntable and Life measuring apparatus
As shown in fig. 1,
In a working state, the turntable rotates at a constant speed, the laser is started, samples in all the sample grooves are excited by the laser once when the turntable rotates for one circle, fluorescence or phosphorescence of the samples are released in the rotating process to form light arcs, the rotating radiuses of all the sample grooves are different, therefore, the light arcs of all the samples cannot be overlapped, the light arcs are photographed by the
The light-emitting lifetime can be obtained by processing the image, and the processing method is consistent with the patent (cn201711110647. x), and specifically comprises the following steps:
the gray value of the pixel point of the light arc is in direct proportion to the luminous intensity and can be used as the relative luminous intensity,
under the condition of uniform rotation, the arc length of the light arc is in direct proportion to the delay time, the delay time of each pixel point can be calculated by knowing the rotating speed of the motor,
the gray-scale value of the pixel point is plotted against the delay time to obtain a luminous attenuation curve, and the luminous life value can be obtained by fitting the curve according to a formula and a method known in the art.
In this embodiment, the optical channel may also use an optical fiber.
According to the same principle, the number of the sample grooves and the number of the optical channels can be increased on the rotating disc, and the simultaneous measurement of more samples can be realized.
Compared with the method of the prior patent (CN201711110647. X), the method has the advantages that the excitation light energy is concentrated at one position, the power density is higher, samples with lower concentration and weaker luminescence can be excited, the samples are excited in turn each turn, the utilization rate of the excitation light is improved, the intensity of the excitation light received by each sample groove is approximately equal, and the samples can be compared. In addition, the optical channel is embedded in the turntable, so that scattered light interference is greatly reduced.
EXAMPLE 2A turntable and a life measuring device
As shown in fig. 2, a sample groove 202 is formed on a turntable 201, the rotation radius of each sample groove is different, each sample groove is connected with an optical channel 203, the optical channel is a multimode optical fiber and leads to the edge of the turntable, light emitted by a light source 206 is reflected by a reflector 207 and then focused on the edge of the turntable 201 by a lens 208; 204 is a light blocking ring for blocking the scattering of the exciting light; the turntable 201 is driven to rotate by a motor 205; the lens 209, the optical filter 210 and the mobile phone 211 form a photographing system for photographing the arc.
The method and principle of the device for measuring lifetime are consistent with the embodiment example 1.
The lens 209 can amplify the partial image of the turntable, namely, the light arc is amplified and imaged, and because the length of the light arc represents the delay time, the amplified light arc is recorded by more pixel points at the same rotating speed, so that the time resolution is higher, and the shorter luminous life can be measured.
The filter 210 may filter out scattered light and light of non-measured wavelengths, enhancing the signal-to-noise ratio of the light arc imaging.
The whole device can be miniaturized, and is convenient to use a mobile phone to take pictures.
EXAMPLE 3A turntable
The top view of the main structure of the rotating disk is shown in fig. 3, wherein 301 is a sample groove, 302 is an optical channel, and the optical channel is composed of an optical fiber and connects the sample groove and the edge of the rotating disk, so that light at the edge of the rotating disk can be guided into the sample groove. This rotary disk can be used for life measurement instead of the rotary disks in the embodiment examples 1 and 2.
The turntable gives full play to the flexibility advantage of the optical fiber, and can establish a nonlinear optical path between any point on the turntable and the edge of the turntable.
EXAMPLE 4A turntable
As shown in fig. 4, the
During the rotation of the turntable, the liquid sample is limited at the turning angle of the capillary due to the centrifugal force; the upper layer of the disk is opaque, but has a plurality of light-transmitting
This rotary disk can be used for life measurement instead of the rotary disks in the embodiment examples 1 and 2.
The embodiment gives full play to the waveguide characteristic and the capillary principle of the capillary, and simultaneously utilizes the centrifugal force.
The method can be used for centrifugal separation and life measurement of the mixture.
EXAMPLE 5A turntable
As shown in fig. 5, the
This rotary disk can be used for life measurement instead of the rotary disks in the embodiment examples 1 and 2.
In the turntable, the capillary tube is used as a sample groove and is also used for guiding light; and the rotating disc can generate centrifugal force when rotating, so that different components of some samples can be separated in the U-shaped capillary tube, and the purpose of separation and detection is achieved.
Like embodiment 4, this embodiment makes full use of the waveguide characteristics and capillary principle of the capillary tube, and also utilizes centrifugal force.
The method can be used for centrifugal separation and life measurement of the mixture.
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