CN110108677B - Biological delay luminescence detection system - Google Patents
Biological delay luminescence detection system Download PDFInfo
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- CN110108677B CN110108677B CN201910375707.3A CN201910375707A CN110108677B CN 110108677 B CN110108677 B CN 110108677B CN 201910375707 A CN201910375707 A CN 201910375707A CN 110108677 B CN110108677 B CN 110108677B
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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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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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
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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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
Abstract
The invention provides a biological delay luminescence detection system, which comprises: an excitation light path; the detection collection optical path includes: a photon collection optical path and a single photon detector; the photon collecting light path is used for receiving the laser output by the excitation light path and irradiating the laser to the object to be tested, and the object to be tested emits photons after being irradiated by the laser; the single photon detector is connected with the photon collection light path and is used for collecting photons output by the photon collection light path. In the invention, the single photon detector with very good control performance is adopted, the gating response time is short, the measurement delay luminescence can be started immediately after excitation, and the photon acquisition accuracy is ensured. Compared with the existing PMT detection mode, the single photon detector does not need a huge cooling device, a high-voltage control circuit and the like, so that the whole system can be miniaturized, and the time resolution function can be realized.
Description
Technical Field
The invention relates to the technical field of biological delay luminescence, in particular to a biological delay luminescence detection system.
Background
Biological photons refer to ultra-weak photons with a wavelength range of 200nm-800nm that are biologically emitted. These photons carry information about the composition and structure of the biomolecules, which are highly sensitive to changes inside the biological system and to external environmental influences. There are two theories on the mechanism of photon generation, one is metabolic oxygen radical luminescence, the other is quantum coherence theory of biological photons, both theories can explain some related phenomena, but the biological photon emission is not completely explained. In summary, various molecules and atoms in the interior of the living organism interact with the photons, so that the biological photons have a wide molecular spectrum, and the changes of basic biological processes such as metabolism, gene expression and the like can cause the relevant changes of the emission of the biological photons, so that the biological photons are closely related to the life state.
Currently, a photomultiplier (Photomultiplier Tube, or PMT) is mostly adopted to collect biological ultra-weak luminescence (Ultraweak Photon Emission, or UPE) and delayed luminescence (Delay Luminescence, or DL), and a large photosensitive cathode and a low dark count rate of the PMT are suitable for collecting UPE. Because of poor PMT control performance, PMTs must be turned off when the excitation beam is on, so as not to permanently destroy the PMT by too strong a reflected light, and a mechanical or electronic shutter is currently used to block the emitted light during excitation. However, the response time of these shutters is above 1ms, especially the delayed lighting time of the human body is very short, and after a decay of 1ms the intensity is already below the initial 1/3, so that the device is only suitable for measuring plants with long delayed lighting times. If the first dynode of the PMT is used to control whether the PMT receives photons, the accuracy can be up to 10us, however commercial PMTs do not expose the dynode to user control and must be tailored to be adequate. PMTs require relatively bulky cooling devices to achieve low dark count rates, and because of the cooling devices, the circuitry must be external to the cooling system, and thus the control circuitry counter, etc., needs to be contained within another module, and therefore are bulky in construction.
In addition, referring to fig. 1, the existing excitation photon system mostly adopts a dark box or a cavity type, a light source 2 'and a PMT3' are arranged at the top of the dark box 1', a sample box 4' is arranged inside, and light rays 5 'emitted by the light source irradiate the sample box 4', but the excitation photon system is suitable for measuring plant, cell and other in-vitro biological substances, and is not suitable for exciting the living organism DL. The optical fiber is suitable for collecting biological photons of various parts of living organisms, but if excitation light and the biological photons pass through the same optical fiber (or a bundle of optical fibers), fluorescence of the excited optical fiber can be collected, and the signal to noise ratio is reduced. Referring to fig. 2, if the bio-photon is collected by using a plurality of optical fibers, wherein the excitation light 6 'is in the center and the periphery includes a plurality of bio-photon collecting optical fibers 7', the generation of fluorescence can be avoided, but the area of the collected signal is greatly reduced, which also results in lower signal to noise ratio. Furthermore, biological photons associated with biological effects are associated at various sites, even different sites, of a biological individual. The biological photons have weak correlations in terms of time and space, of parameters such as intensity, luminescence time, or strong correlations in terms of time, even entangled photons,it is difficult for existing systems to detect the association event. In addition, DL is much weaker than the fluorescence emitted photons, e.g., about 100 to several thousand photons per second per degree solid angle per mm for human luminescence 2 Therefore, a large photosensitive area is required, but generally the detector noise is proportional to the photosensitive area, and if the area of the photosensitive area is increased, the detector noise is also increased.
Disclosure of Invention
In view of the above, the invention provides a bio-delay luminescence detection system, which aims to solve the problem that the response time of bio-delay luminescence collected by a photomultiplier is long at present.
The invention provides a biological delay luminescence detection system, which comprises: an excitation light path; the detection collection optical path includes: a photon collection optical path and a single photon detector; the photon collecting light path is used for receiving the laser output by the excitation light path, irradiating the laser to the object to be tested, and outputting photons emitted by the object to be tested after the laser irradiation; the single photon detector is connected with the photon collection light path and is used for collecting photons output by the photon collection light path.
Further, in the above bio-delay luminescence detection system, the photon collection optical path includes: the converging device is used for receiving the laser output by the excitation light path, irradiating the laser to the object to be tested, and converging and outputting photons emitted by the object to be tested after the irradiation of the laser; and the focusing device is used for receiving and focusing the photons output by the collecting and converging device and outputting the focused photons to the single photon detector.
Further, in the above bio-delay luminescence detection system, the converging device includes: the collector is provided with a cavity, a through hole is formed in the wall surface of the collector, and the collector is used for enabling laser output by the excitation light path to irradiate an object to be tested through the through hole and the opening of the collector; the at least one photon converging mirror is arranged in the cavity at intervals along the emitting direction of photons emitted by the object to be tested, and the at least one photon converging mirror is used for converging the photons emitted by the object to be tested after passing through the at least one photon converging mirror.
Further, in the above-described bio-delay luminescence detection system, the wall surface of the collector is provided with a protruding portion, the through hole is obliquely penetrated through the protruding portion and the wall surface of the collector, and the through hole is inclined in the direction of the laser light output from the excitation light path.
Further, in the bio-delay luminescence detection system, the focusing assembly includes: the first end of the first optical fiber is positioned in the cavity and used for receiving photons output by the at least one photon converging mirror, and the second end of the first optical fiber is positioned outside the cavity; the first focusing device is connected with the second end of the first optical fiber and is used for receiving photons output by the first optical fiber; the first end of the second optical fiber is connected with the first focusing device, the second end of the second optical fiber is connected with the single photon detector, and the second optical fiber is used for receiving photons output by the first focusing device and outputting the photons to the single photon detector.
Further, in the above bio-delay luminescence detection system, the excitation light path includes: a laser; the leveling focusing assembly is used for receiving laser emitted by the laser, leveling and focusing the laser, and outputting the laser after leveling and focusing to the photon collecting light path.
Further, in the above bio-delay luminescence detection system, the leveling focusing assembly includes: the leveling assembly is used for receiving and leveling laser emitted by the laser; the second concentrator is used for receiving and focusing the laser output by the leveling component; and one end of the third optical fiber is connected with the second concentrator, and the third optical fiber is used for receiving the laser output by the second concentrator and outputting the laser to the photon collection optical path.
Further, in the above bio-delay luminescence detection system, the leveling assembly includes: the first reflecting mirror is obliquely arranged and is used for receiving laser emitted by the laser; and the second reflector is obliquely arranged, and the oblique direction of the second reflector is opposite to that of the first reflector, and the second reflector is used for receiving the laser reflected by the first reflector and reflecting the laser to the second concentrator.
Further, in the above bio-delay luminescence detection system, the leveling assembly includes: the first reflecting mirror is obliquely arranged and receives laser emitted by the laser; the beam splitter is obliquely arranged, the oblique direction of the beam splitter is opposite to that of the first reflector, and the beam splitter is used for receiving the laser reflected by the first reflector and splitting the laser into at least two beams of beam splitting laser; the second concentrators, the detection and collection light paths and the beam splitting lasers are equal in number, the focusers receive the beam splitting lasers in one-to-one correspondence, and the detection and collection light paths receive the lasers focused and output by the second focusers in one-to-one correspondence.
Further, in the above bio-delay luminescence detection system, the excitation light path further includes: the light spot shaper is arranged between the laser and the leveling focusing assembly and is used for receiving laser emitted by the laser and outputting the laser to the leveling focusing assembly.
Further, the bio-delay luminescence detection system further comprises: the circuit control system is respectively connected with the excitation light path and the single photon detector, and is used for controlling the excitation light path to output laser and the single photon detector to collect photons synchronously.
Further, the bio-delay luminescence detection system further comprises: and the coincidence counter is connected with the single photon detector and is used for receiving and counting photons output by the single photon detector.
Further, the bio-delay luminescence detection system further comprises: the upper computer is connected with the coincidence counter and is used for receiving and recording data output by the coincidence counter.
Further, in the bio-delay luminescence detection system, the single photon detector is a single photon avalanche diode detector.
In the invention, the single photon detector comprises the counter and the gate control circuit, the acquisition circuit of the SPAD can be opened when required, the precision can reach 2us, the response time is short, the invention is not only suitable for plants, but also particularly suitable for organisms with very short delayed luminescence, the photon can be acquired when the delayed luminescence attenuation degree is not large, and the photon acquisition accuracy is ensured. Meanwhile, the single photon detector can obtain lower dark count rate without a huge cooling device, and can be miniaturized, so that the size of the whole biological delay luminescence detection system is reduced.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to designate like parts throughout the figures. In the drawings:
FIG. 1 is a schematic diagram of an excitation photonic system according to the prior art;
FIG. 2 is a schematic diagram of another excitation photonic system according to the prior art;
FIG. 3 is a schematic diagram of a bio-delay luminescence detection system according to an embodiment of the present invention;
fig. 4 is a schematic structural diagram of a bio-delay luminescence detection system according to an embodiment of the present invention, which has a plurality of detection collection optical paths.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.
Referring to fig. 3, a preferred structure of the bio-delay luminescence detection system according to the present embodiment is shown. As shown, the system includes: the excitation light path 1 and the detection and collection light path comprise a photon collection light path 2 and a single photon detector 3. The excitation light path 1 outputs laser, the photon collection light path 2 receives the laser output by the excitation light path 1, and irradiates the laser on the object 4 to be tested, for example, a finger of a person, the object 4 to be tested emits photons after being irradiated by the laser, and the photon collection light path 2 outputs the photons emitted by the object 4 to be tested. The single photon detector 3 is in communication with the photon collection optical path 2, so as to collect photons output from the photon collection optical path 2, and in implementation, the single photon detector 3 is a single photon avalanche diode detector (SPDA).
The single photon detector 3 is internally provided with a counter and a gating circuit, the precision can reach 2us, the control performance is very good, the gating response time is short, the delayed luminescence can be measured immediately after excitation, the single photon detector is not only suitable for plants, but also particularly suitable for organisms with very short delayed luminescence, photons can be collected when the attenuation degree of the delayed luminescence is not large, and the photon collection accuracy is ensured. Meanwhile, the single photon detector 3 can obtain lower dark count rate without a huge cooling device and does not need a high-voltage control circuit and other structures, and the single photon detector 3 can be miniaturized, so that the size of the whole biological delay luminescence detection system is reduced.
The photon collection optical path 2 includes: a converging means 21 and a focusing means 22. The converging device 21 receives the laser output by the excitation light path 1, irradiates the laser onto the object 4 to be tested, emits photons after the object 4 to be tested is irradiated by the laser, and then converges the photons by the converging device 21, and then outputs the converged photons by the converging device 21. The focusing device 22 receives the photons output from the focusing device 21, focuses the photons, and outputs the focused photons to the single photon detector 3. The laser output by the excitation light path 1 is converged by the converging device 21 and then focused by the focusing device 22, so that the photon collecting efficiency of the single photon detector 3 is improved.
The converging means 21 comprises: an open-ended collector 211 and at least one photon collection mirror 214. The object 4 to be tested is attached to an opening of the collector 211, which is a data collection port, and the collector 211 has a cavity 212, so that the collector 211 becomes a camera bellows with the cavity 212. The wall surface of the collector 211 is provided with a through hole 213, and the laser output by the excitation light path 1 enters the cavity 212 through the through hole 213 and directly irradiates the object 4 to be tested only through the opening of the collector 211. After the laser irradiates the object 4 to be tested, the object 4 to be tested emits photons, at least one photon converging mirror 214 is arranged at intervals along the emitting direction of the photons, each photon converging mirror 214 is located in the cavity 212, and the photons are converged after passing through each photon converging mirror 214. The focusing device 22 is connected with the single photon detector 3, and photons output by the photon converging mirror 214 are output to the single photon detector 3 through the focusing device 22. In specific implementation, a photon collecting mirror 214 is disposed in the cavity 212, the photon collecting mirror 214 has a preset focal length, the distance from the opening of the collector 211 to the photon collecting mirror 214 is 2 times of the preset focal length, and meanwhile, the focal length from the photon collecting mirror 214 to the input end of the focusing device 22 is 2 times of the preset focal length, so as to form a 4f imaging system, f is the preset focal length, that is, a double focal length imaging method is used in the cavity 212 of the collector 211, so that photons are collected. Photon collection mirror 214 may be an optical lens.
The wall surface of the collector 211 is provided with a protruding portion 216, and the through hole 213 penetrates the protruding portion 216 and the wall surface of the collector 211 obliquely so as to be communicated with the cavity 212, and at the same time, the oblique direction of the through hole 213 is consistent with the direction of the output of the laser light output by the excitation light path 1, so that the laser light is ensured to be transmitted in a straight line in the through hole 213. The photon collecting light path adopts a microscopic objective shrinking imaging system, namely a 4f imaging system, the aperture angle of a photon collecting light emitting surface can be increased by 20-60 times, the area of the photon collecting light emitting surface is increased by 400-3600 times, the aperture angle of the collected photons reaches 2-5 degrees, and the signal to noise ratio is greatly improved.
The focusing device 22 includes: a first optical fiber 221, a first focuser 222, and a second optical fiber 223. The first end (lower end shown in fig. 3) of the first optical fiber 221 is located in the cavity 212, and the first end of the first optical fiber 221 is provided with a probe 224 for detecting photons, and the second end (upper end shown in fig. 3) of the first optical fiber 221 is located outside the cavity 212, that is, the first optical fiber 221 is penetrating through the cavity 212, and the focal length from the photon collecting mirror 214 to the probe 224 is 2 times of the preset focal length. The first focusing device 222 is connected to a second end of the first optical fiber 221, a first end (lower end shown in fig. 3) of the second optical fiber 223 is also connected to the first focusing device 222, and a second end (upper end shown in fig. 3) of the second optical fiber 223 is connected to the single photon detector 3. The photons output by the photon converging mirror 214 are transmitted to the first focusing device 222 through the first optical fiber 221, the photons are focused by the first focusing device 222, and the photons are transmitted to the single photon detector 3 through the second optical fiber 223 after being focused. The first focuser 222 may focus the photons, thereby improving the collection efficiency of the photons. In specific implementation, the first focusing device 222 may be a first microscope lens with a magnification of 40 times, or may be replaced by an optical lens system according to actual application requirements, so as to achieve a converging effect of photons of 6cm-10 cm; the first optical fiber 221 may be a liquid optical fiber with a core diameter of 6cm-10cm, and the liquid optical fiber is matched with the 4f imaging system, so that the area of collecting photons of the object 4 to be tested can be controlled according to the selected core size of the liquid optical fiber. A filter may be disposed between the first focusing device 222 and the second optical fiber 223, so as to obtain photons with specific wavelengths, and a spectrometer or a monochromator may be used to replace the filter, so as to calibrate the wavelength of photons with delayed luminescence.
The excitation light path 1 includes: a laser 11 and a leveling focus assembly 12. The laser 11 emits laser light, the leveling and focusing assembly 12 receives the laser light, levels the laser light, focuses the leveled laser light, and outputs the focused laser light to the collector 211 of the photon departure system. Leveling the laser by the leveling focusing assembly 12 can ensure that the laser is in a horizontal state before being focused by the leveling focusing assembly 12, and meanwhile, the leveling focusing assembly 12 focuses the laser before the laser is injected into the collector 211, so that the laser can be amplified and focused, and the photon collection efficiency can be further improved. In principle, a laser 11 emitting laser light of any wavelength can be used, but for delayed luminescence of an animal body, the signal-to-noise ratio of the light collected by the single photon detector 3 is optimal, and the light with the wavelength of 400nm-532nm is considered to be absorbed by the animal body more easily and conducted to deeper layers of skin, while the light with the wavelength of 400nm-532nm is more easily excited to atoms of epidermal cells on the surface of the skin, so that the same laser energy, the stimulated radiation signal of the light with the wavelength of 400nm-532nm is stronger and the signal-to-noise ratio is better. In addition, the laser 11 emits continuous laser light, and the laser 11 is a semiconductor laser.
The leveling focus assembly 12 includes: a leveling assembly 121, a second concentrator 122, and a third optical fiber 123. Wherein the leveling assembly 121 receives the laser light emitted from the laser 11 and levels the laser light. The leveled laser light is output to the second concentrator 122, and the second concentrator 122 focuses the leveled laser light. The first end (upper end shown in fig. 3) of the third optical fiber 123 is connected to the second concentrator 122, and the second end (lower end shown in fig. 3) of the third optical fiber 123 corresponds to the through hole 213 of the collector 211, and the focused laser light is injected into the cavity 212 of the collector 211 through the third optical fiber 123 and the through hole 213. The leveling assembly 121 may ensure that the laser light is incident on the second concentrator 122 in a horizontal manner. In practical implementation, the second focusing device 122 may be a second microscope lens with a magnification of 40 times, or may be replaced by an optical lens system according to practical application requirements, so as to achieve the effect of photon collection.
The leveling assembly 121 includes: a first mirror 124 and a second mirror 125. The first mirror 124 and the second mirror 125 are both disposed obliquely, and the oblique directions of the two are opposite. The laser light emitted from the laser 11 is reflected by the first mirror 124, reflected by the second mirror 125, and reflected by the second mirror 125 to the second concentrator 122. The laser 11 emits laser light, and the laser light is twice reflected by the first mirror 124 and the second mirror 125, so that the horizontality of the laser light can be sufficiently ensured.
Referring to fig. 4, in order to achieve multi-path coincidence detection of photons, the second mirror 125 in the leveling assembly 121 may be replaced with a beam splitter 126, with the beam splitter 126 and the first mirror 124 having opposite inclinations. The laser light emitted by the laser 11 is reflected by the first reflecting mirror 124 and is reflected to the beam splitter 126, and the beam splitter 126 can split the laser light into at least two split laser light beams. Meanwhile, the second concentrators 122, the detection and collection optical paths and the beam splitting lasers are equal in number, the leveling and focusing assemblies 12 receive the beam splitting lasers in a one-to-one correspondence mode, meanwhile, the second concentrators 122 focus the received beam splitting lasers and output the focused beam splitting lasers, the detection and collection optical paths receive the lasers output by the second concentrators 122 in a one-to-one correspondence mode so as to realize multi-optical-path coincidence detection, photon emission of two or more relevant parts of organisms, particularly animals can be collected at the same time, the correlation between the space and time of the photons is measured, whether the photons correlated in time are in a quantum entangled state is evaluated, and therefore the correlation of photons at different detection points is determined, and the photon arrival time (picosecond magnitude) is accurately detected. In specific implementation, the biological delay luminescence detection system can realize coincidence detection of two paths, four paths and even more paths. Compared with the traditional PMT for collecting photons, when the coincidence detection of multiple light paths is performed, the SPAD is used as a detector, and independent measurement among all light paths is easier to control, so that the collected photon spectrum range is more matched with biological photons.
The excitation light path 1 further includes: the spot shaper 13, the spot shaper 13 is disposed between the laser 11 and the first mirror 124 of the leveling component 121, the laser emitted by the laser 11 is simply shaped by the spot shaper 13 and modulated into a near-circular spot, so as to avoid that the types of the selected lasers 11 are different, which may result in different shapes of the laser spots output by the laser 11, and the shaped laser is output to the first mirror 124 of the leveling focusing component 12. In a specific implementation, the spot shaper 13 may be a diaphragm, and the spot shaper 13 may be connected in series to perform spot shaping. Of course, if the laser 11 has strictly internally shaped the laser spot, the spot shaper 13 in the optical path is not required. The laser 11 may be a continuous laser that emits continuous laser light.
The bio-delayed luminescence detection system further comprises: the first output end of the circuit control system 5 is connected with the laser 11 of the excitation light path 1, the second output end of the circuit control system 5 is connected with the single photon detector 3, the circuit control system 5 can control the laser pulse width by adjusting the electric pulse signal, and meanwhile, the detection gate width of the single photon detector 3 is triggered synchronously, so that the synchronism of laser triggering and single photon acquisition is ensured. When the laser 11 is a semiconductor laser, the control system 5 is used to realize the emission of pulse laser by controlling the electric pulse of the continuous laser, so that the instantaneous power of the laser pulse is the same under different pulse widths. If the instantaneous power of the laser pulse is required to increase along with the decrease of the laser pulse width or smaller laser pulse width is required to be achieved, the laser 11 can be replaced by a laser with adjustable Q according to the actual requirement, and the Q adjustment refers to a pulse laser pulse width modulation mode. However, for detection of certain applications, due to the principle of pumping the Q-switched laser, the instantaneous power of the output pulsed laser of the Q-switched laser may be varied when the pulse width of the laser is varied, so that a chopper may be provided between the diaphragm and the first mirror 124, so that the instantaneous power of the output pulsed laser remains unchanged when the pulse width of the Q-switched laser is varied. Because the control performance is good, the circuit control system 5 can control the laser triggering of the laser 11 and the photon collection of the single photon detector 3 to be synchronously carried out, the time precision of the photon excitation and the photon collection can be greatly improved, the control precision of 1 microsecond can be achieved at present, and the data collection speed is high: multiple data can be acquired by one excitation, and 100 times of single excitation acquisition can be realized at present. The single photon detector 3 can be controlled by a circuit control system 5 through current, so that the single photon detector 3 does not count when the pulse laser is excited, and the single photon detector 3 starts counting when the pulse laser is stopped, namely, the photon collection light path 2 is closed before the next pulse laser comes, and laser pumping and photon collection are alternately performed.
The bio-delayed luminescence detection system further comprises: the coincidence counter 6 is connected with the single photon detector 3, and can receive photons output by the single photon detector 3 and count the received photons. If the system has coincidence detection of multiple light paths, the coincidence counter 6 is a multi-channel coincidence counter, the single photon detector 3 of each detection collection light path is connected to one multi-channel coincidence counter, the multi-channel coincidence counter can accurately measure the photon quantity of each channel in a set time, and the coincidence circuit can measure the accurate time (ps magnitude) of photon arrival of each channel in a set time window, so that the detection system can accurately measure the delayed luminescence curve of a sample and accurately obtain the spatial and temporal correlation of the photons.
The bio-delayed luminescence detection system further comprises: the upper computer 7, the upper computer 7 is connected with the coincidence counter 6, can receive the data that coincidence counter 6 exports, and record this data. In the specific implementation, the upper computer 7 is a computer system.
The optical path of the bio-delay luminescence detection system provided in this embodiment is:
the semiconductor laser is used for emitting continuous laser with the wavelength of 400nm, the laser power is in the order of tens of milliwatts, after the light spot shaping is carried out through the diaphragm, the first reflecting mirror 124 and the second reflecting mirror 125 are used for carrying out light path leveling, and the laser is ensured to be emitted into the second micro lens in a horizontal mode. The laser light is focused by a second microscope with a magnification of 40 times, then guided to a third optical fiber 123, and irradiated onto the object 4 to be tested through the third optical fiber 123. The delayed luminescence of the object 4 to be tested is carried out in the cavity 212 of the collector 211, photons emitted by the object 4 to be tested are led out through the liquid optical fiber after being converged by the photon converging mirror 214, focused through the first micro lens with the magnification of 40 times, then led into the single photon detector 3 through the second optical fiber 223, the single photon detector 3 is used for collecting photons, then counted and matched through the coincidence counter 6, and finally data which is coincident with the counter 6 is led into the computer system for data recording. The laser 11 and the single photon detector 3 are controlled by a circuit control system 5 to ensure the synchronism of laser triggering and photon collection.
When the device works, the upper computer 7 sets working parameters, then the circuit control system 5 is started, and the circuit control system 5 controls the laser 11 (or other light sources) of the excitation light path 1 to emit pulse light beams so as to excite biological samples at a plurality of data acquisition ports; then in a very short time (microsecond) after the excitation light is stopped, the SPDA and the multichannel coincidence counter of each detection and collection light path perform photon counting and coincidence judgment, and continuously store the collected photon counting data and coincidence counting data in different time into the upper computer 7, and store the data into a magnetic disk of the upper computer 7 after the data collection is finished.
In conclusion, the single photon detector 3 is internally provided with the counter and the gate control circuit, the acquisition circuit of the SPAD can be opened when needed, the accuracy can reach 2us, the response time is short, the single photon detector is not only suitable for plants, but also particularly suitable for organisms with very short delayed luminescence, such as human bodies, the photons can be acquired when the delayed luminescence attenuation degree is not large, and the photon acquisition accuracy is ensured. Meanwhile, the single photon detector 3 can obtain a lower dark count rate without a huge cooling device, and the single photon detector 3 can be miniaturized, so that the size of the whole biological delay luminescence detection system is reduced. Compared with the traditional photodiode (or avalanche diode), the enhancer (ICCD) and the fluorescence detection system, the system provided by the embodiment has higher quantum efficiency, lower dark count rate can be obtained by adopting the single photon detector 3, and the photon collection light path adopts a 4f imaging system, so that the signal to noise ratio is better.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. A bio-delayed luminescence detection system, comprising:
an excitation light path (1);
a probe collection optical path, the probe collection optical path comprising: a photon collection optical path (2) and a single photon detector (3); wherein, the liquid crystal display device comprises a liquid crystal display device,
the photon collection light path (2) is used for receiving the laser output by the excitation light path (1) and irradiating the laser to the to-be-detected
A test object (4), and outputting photons emitted by the object (4) to be tested after being irradiated by the laser;
the single photon detector (3) is connected with the photon collection optical path (2) and is used for collecting the photon collection optical path
(2) The output photons;
a circuit control system (5), wherein the circuit control system (5) is respectively connected with the excitation light path (1) and the single photon detection
The circuit control system (5) is used for controlling the excitation light path (1) to output laser and the single photon probe to be connected with the detector (3)
The photon collection of the detector (3) is synchronously carried out;
a coincidence counter (6), the coincidence counter (6) being connected to the single photon detector (3) for receiving and counting
A photon conforming to the output of the single photon detector (3);
the photon collecting light path (2)
Comprising the following steps:
a converging device (21), wherein the converging device (21) is used for receiving the laser output by the excitation light path (1) and making the laser
Irradiating the object to be tested (4), and converging and outputting light emitted by the object to be tested (4) after the irradiation of the laser
A seed;
a focusing device (22), wherein the focusing device (22) is used for receiving and focusing and collecting the light output by the focusing device (21)
A sub-and outputting the focused photons to the single photon detector (3);
the converging device (21) comprises:
a collector (211) with one end open, the collector (211) having a cavity (212), the collector (211) having walls
The surface is provided with a through hole (213), and the collector (211) is used for enabling the laser output by the excitation light path (1) to pass through the through hole
(213) And an opening of the collector (211) is irradiated to the object (4) to be tested;
at least one photon collection mirror (214), at least one of the photon collection mirrors (214) emitting along the object (4) to be tested
The emitting direction of the emitted photons is arranged in the cavity (212) at intervals, and at least one photon converging mirror (214) is used for enabling
Photons emitted by the object to be tested (4) are converged after passing through at least one photon converging mirror (214).
2. The bio-delayed luminescence detection system of claim 1, wherein,
the collector (211) has a convex portion (216) on its wall surface, and the through hole (213) is formed obliquely through the convex portion
A portion (216) and a wall surface of the collector (211), and the through hole (213) outputs laser light along the excitation light path (1)
Is inclined in the direction of (a).
3. The bio-delayed luminescence detection system according to claim 1, wherein the focusing means (22) comprises
The method comprises the following steps: a first optical fiber (221), a first end of the first optical fiber (221) is positioned in the cavity (212) and is used for receiving the optical fiber
At least one photon output by the photon converging mirror (214), the second end of the first optical fiber (221) is positioned in the cavity
(212) An outer part;
a first focussing means (222), said focussing means (222) being connected to a second end of said first optical fibre (221) for
-receiving photons output by the first optical fiber (221);
a second optical fiber (223), a first end of the second optical fiber (223) is connected with the first focusing device (222), and the second optical fiber
A second end of two optical fibers (223) is connected with the single photon detector (3), and the second optical fibers (223) are used for receiving the optical fibers
And the first focusing device (222) outputs photons to the single photon detector (3).
4. The bio-delayed luminescence detection system according to claim 1, wherein the excitation light path (1) comprises:
a laser (11);
a leveling focusing assembly (12), wherein the leveling focusing assembly (12) is used for receiving the laser light emitted by the laser (11) and
leveling and focusing the laser light, and outputting the leveled and focused laser light to the photon collection optical path (2).
5. The bio-delay luminescence detection system according to claim 4, wherein the leveling focus assembly (12)
Comprising the following steps: -a levelling assembly (121), the levelling assembly (121) being adapted to receive and level the laser light emitted by the laser (11);
a second focussing means (122), said second focussing means (122) being arranged to receive and focus the output of said levelling assembly (121)
Laser;
a third optical fiber (123), one end of the third optical fiber (123) is connected with the second concentrator (122), the third
An optical fiber (123) for receiving the laser light output from the second focusing device (122) and outputting the laser light to the photon collection light
A road (2).
6. The bio-delayed luminescence detection system of claim 5, wherein the leveling assembly (121) comprises
The method comprises the following steps: a first mirror (124), the first mirror (124) being disposed obliquely, and the first mirror (124) being configured to
Receiving laser light emitted by the laser (11);
a second reflecting mirror (125), the second reflecting mirror (125) being disposed obliquely, and the second reflecting mirror (125) being disposed obliquely
The second mirror (125) is used for receiving the light beam passing through the first mirror (124)
A mirror (124) reflects the laser light and reflects the laser light to the second focussing means (122).
7. The bio-delayed luminescence detection system of claim 5, wherein the leveling assembly (121) comprises
The method comprises the following steps: a first mirror (124), the first mirror (124) being disposed obliquely, and the first mirror (124) receiving
-a laser light emitted by said laser (11);
a beam splitter (126), wherein the beam splitter (126) is obliquely arranged, and the oblique direction of the beam splitter (126) is equal to that of the beam splitter
The first reflecting mirror (124) is inclined in opposite directions, and the beam splitter (126) is used for receiving the beam reflected by the first reflecting mirror (124)
The laser beam is emitted and divided into at least two beam splitting lasers;
the second focusing device (122), the detection and collection optical path and the beam splitting laser have the same number, and each focusing device
Each beam splitting laser is received in a one-to-one correspondence, and each detection and collection light path is received in a one-to-one correspondence with the second focusing device
(122) And collecting the output laser.
8. The bio-delay luminescence detection system according to claim 4, wherein the excitation light path (1) further comprises
The method comprises the following steps: a light spot shaper (13), wherein the light spot shaper (13) is arranged on the laser (11) and the leveling focusing assembly
(12) The spot shaper (13) is used for receiving the laser beam emitted by the laser (11) and outputting the laser beam to
The leveling focus assembly (12).
9. The bio-delayed luminescence detection system of any of claims 1-8, further comprising:
the upper computer (7) is connected with the coincidence counter (6) and is used for receiving and recording the coincidence counter
The data output by the counter (6).
10. The bio-delayed luminescence detection system according to any of the claims 1-8, wherein,
the single photon detector (3) is a single photon avalanche diode detector.
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| CN111728584A (en) * | 2020-05-13 | 2020-10-02 | 新绎健康科技有限公司 | Device and method for determining qi obtaining effect of target acupuncture points |
| CN112763464A (en) * | 2020-12-18 | 2021-05-07 | 新绎健康科技有限公司 | Spectrum measurement system and method for biological delayed luminescence |
| WO2024079304A1 (en) * | 2022-10-14 | 2024-04-18 | Oulun Yliopisto | Optical apparatus and method for measuring a pluraltiy of targets using a single photon avalanche detector |
| CN116539599B (en) * | 2023-07-04 | 2024-03-26 | 科美诊断技术股份有限公司 | Photo-excitation chemiluminescence detection device, control method and photo-excitation chemiluminescence analyzer |
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