CN111812073A - Optical control system of two-photon fluorescence immunoassay analyzer - Google Patents
Optical control system of two-photon fluorescence immunoassay analyzer Download PDFInfo
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Abstract
The invention relates to the technical field of optical control systems, in particular to an optical control system of a two-photon fluorescence immunoassay analyzer. The method is characterized in that: the installation bottom plate is provided with a two-photon laser generation module, a detection control module and an adjustment transmission module; a photomultiplier is arranged in the detection control module; the two-photon laser emitted by the two-photon laser generation module can be focused into the reaction cup through the detection control module and the adjustment transmission module, the two-photon laser excites a detection substance in the reaction cup to generate fluorescence, and the fluorescence can be returned to the photomultiplier of the detection control module through the adjustment transmission module to be measured. The two-photon laser generation module is arranged for generating two photons, and because the wavelength of the two photons is longer, the light with long wavelength is less influenced by scattering compared with the light with short wavelength, the light with long wavelength is easy to penetrate through a sample, the background interference fluorescence is reduced, and the accuracy of detection and analysis of equipment is improved.
Description
Technical Field
The invention relates to the technical field of optical control systems, in particular to an optical control system of a two-photon fluorescence immunoassay analyzer.
Background
In a general fluorescence phenomenon, because of the low photon density of the excitation light, one fluorescence molecule can only absorb one photon at the same time, and then emit one fluorescence photon through radiative transition, which is single photon fluorescence. At present, the fluorescence immunoassay analyzers at home and abroad are all single-photon fluorescence, and have the following defects for the single-photon fluorescence immunoassay analyzers: because the wavelength of the single photon is short, the single photon is greatly influenced by scattering and is not easy to penetrate through a sample; the single photon excited fluorescence background interferes the fluorescence intensity, seriously affects the detection and collection of the fluorescence excited by the specimen, and reduces the precision of the analysis result.
In addition, the laser beam of the existing analyzer has the problem of inaccurate focusing and positioning. In the actual use process, the laser beam cannot be accurately controlled to deflect and move a small displacement at a fixed directional distance, so that the aim of arranging the focus of the laser beam at the correct depth position of the liquid is fulfilled, and the accuracy and reliability of an analysis and detection result are reduced.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides an optical control system of a two-photon fluorescence immunoassay analyzer.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows: the utility model provides a two-photon fluorescence immunoassay appearance optical control system, includes mounting plate, its characterized in that: the installation bottom plate is provided with a two-photon laser generation module, a detection control module and an adjustment transmission module; a photomultiplier is arranged in the detection control module; the two-photon laser emitted by the two-photon laser generation module can be focused into the reaction cup through the detection control module and the adjustment transmission module, the two-photon laser excites a detection substance in the reaction cup to generate fluorescence, and the fluorescence can be returned to the photomultiplier of the detection control module through the adjustment transmission module to be measured.
Preferably, a scattering detector is further arranged in the detection control module, and the two-photon laser scattering light of the detection substance in the two-photon laser excitation reaction cup can be returned to the scattering detector of the detection control module for measurement through the adjustment transmission module; the resulting peaks in the measured scatter signal are used to determine when the scan has stopped and when to record the fluorescence signal measured at visible wavelengths.
Preferably, an up-shaped two-photon laser passage is arranged in the detection control module, the two-photon laser passage comprises a front passage and a rear passage, and the tail end of the front passage is vertically communicated with the middle part of the rear passage; the front end of the front passage is provided with a first inlet, the first inlet is connected with the two-photon laser generation module, the front part of the front passage is provided with an attenuator, the middle part of the front passage is vertically communicated with the front end of the front branch, the communication part of the front passage and the front branch is provided with a beam splitter, and the rear end of the front branch is communicated with a scattering detector; a dichroic mirror is arranged at the connection part of the front passage and the rear passage; one end of the back channel is communicated with the photomultiplier, the other end of the back channel is provided with a first outlet, and the first outlet is connected with the adjustment transmission module;
the two-photon laser emitted by the two-photon laser generating module can enter a front passage of the detection control module through the first inlet, and after passing through the attenuator and the beam splitter, the two-photon laser is reflected by the dichroic mirror and passes through the first outlet from a rear passage;
after entering a rear channel of the detection control module from the first outlet, the two-photon laser scattering light enters a scattering detector for detection after being reflected by the dichroic mirror and the beam splitter;
after the fluorescence enters the rear passage of the detection control module from the first outlet, the fluorescence can enter the photomultiplier for detection through the transmission of the dichroic mirror.
Preferably, the attenuator comprises an attenuator mirror frame, a light path attenuation channel is arranged in the attenuator mirror frame, and the attenuator mirror frame is arranged in the front passage; the gearbox motor is installed on the upper surface of the outer portion of the front passage through the bushing and the motor support, a motor shaft of the gearbox motor is downwards inserted into the front passage and connected with the attenuator mirror bracket, and the gearbox motor can drive the motor shaft and the attenuator mirror bracket to synchronously rotate so as to adjust the attenuation of incident light.
Preferably, the adjusting and transmitting module comprises a first lens frame, a second lens frame, a lifting control device and an objective lens; the first lens frame is connected with the second lens frame through a lifting control device, and the objective lens is arranged at the upper part of the second lens frame;
a second inlet is arranged on the side surface of the first mirror frame, a second outlet is arranged at the top of the first mirror frame, and a first reflector is arranged in the first mirror frame;
a third inlet is arranged on the bottom surface of the second mirror frame, a third outlet is arranged on the top of the second mirror frame, two groups of second reflecting mirrors are arranged in the second mirror frame, and the two groups of second reflecting mirrors form an angle of 45 degrees with the horizontal plane and are arranged oppositely; the two groups of second reflectors are respectively arranged on the two groups of piezoelectric ceramic control function groups, and the two groups of piezoelectric ceramic control function groups can respectively control the two groups of second reflectors to rotate and scan; the second outlet is connected with the third inlet through a telescopic lens cone; an objective lens is arranged at the upper end of the third outlet in a connecting mode, and a reaction cup is arranged above the objective lens;
the two-photon laser emitted from the first outlet can enter the first mirror frame through the second inlet, is emitted from the second outlet after being reflected by the first reflector, enters the third inlet through the telescopic lens cone, is emitted from the third outlet after being reflected by the two groups of second reflectors, and is emitted into the reaction cup through the objective lens.
Preferably, the piezoelectric ceramic control function group comprises a bushing seat, and the bushing seat is installed on the outer side surface of the second mirror frame; the second reflecting mirror is arranged on the mirror bracket, the mirror bracket is fixedly arranged at the inner end of the mounting shaft, and the outer end of the mounting shaft penetrates through the second mirror frame and penetrates out of the bushing seat; the middle part of the mounting shaft is fixed in the second mirror frame and/or the bushing seat through two groups of bearings, a push-turn plane is arranged on the mounting shaft between the two groups of bearings, and a spring-driven rotating device is arranged at the outer end of the mounting shaft; the spring driving rotating device comprises a spring lock, a driving spring and a spring positioning ring, the spring lock is fixed on the outer wall of the bushing seat, the spring positioning ring is fixed at the outer end of the mounting shaft, the driving spring is a torsion spring sleeved on the mounting shaft, one end of the torsion spring is fixed on the spring lock, and the other end of the torsion spring is inserted into the spring positioning ring; the spring-driven rotating device can apply unidirectional torque to the mounting shaft, so that the mounting shaft can rotate around the central axis thereof in a unidirectional way;
piezoelectric ceramics are fixed in the bushing seats through sleeves, and the telescopic ends of the piezoelectric ceramics are perpendicular to and in contact with the push-turn plane of the mounting shaft; the voltage can control the starting and stopping of the piezoelectric ceramic telescopic action and the telescopic amount, and the mounting shaft is pushed to rotate clockwise or anticlockwise around the central axis of the mounting shaft, so that the mirror frame rotates clockwise or anticlockwise together with the second reflecting mirror mounted on the mirror frame.
Preferably, the lifting control device comprises a stepping motor, the upper end of the stepping motor is connected with a threaded transmission sleeve, an inner threaded hole with an open upper end is formed in the threaded transmission sleeve, a lifting bolt is inserted into the inner threaded hole of the threaded transmission sleeve, the upper end of the lifting bolt is connected with one end of a lifting block, and one end of the lifting block is connected with the second mirror frame through a sliding mechanism.
Preferably, the sliding mechanism comprises a guide wheel mounting plate, a guide wheel and a guide rail, the inner side of the guide rail is mounted outside the second mirror frame through a screw, and the outer side of the guide rail is fixed with one end of the lifting block through a bolt; the guide wheel mounting plate is fixed on the first mirror frame through bolts, two pairs of guide wheels which are longitudinally arranged and symmetrically arranged are clamped on two sides of the guide rail, and the four guide wheels are mounted on the guide wheel mounting plate through screws;
when the lifting block goes up and down, the guide rail can be driven to move up and down under the clamping and guiding action of the guide wheel, and the second mirror frame is driven to go up and down synchronously.
Compared with the prior art, the invention has the following beneficial effects:
1. the two-photon laser generation module is arranged for generating two photons, and because the wavelength of the two photons is longer, the light with long wavelength is less influenced by scattering compared with the light with short wavelength, and the light with long wavelength is easy to penetrate through a specimen. One of the key features of two-photon excitation is that excitation occurs only in the apparent three-dimensional (3D) vicinity of the focal point. The minimum background fluorescence is generated outside the focal volume, i.e. in the surrounding sample medium and in the optical components. The excitation volume is very small (in the range of femtoliters) and is best suited for observing small sample volumes and structures.
2. The invention adopts the piezoelectric ceramic control function group to control the double-reflector (namely two groups of second reflectors) as the scanner, and has the advantages that: and controlling voltage by using a DA converter, controlling the expansion and contraction quantity of the piezoelectric ceramics through the change of the voltage, and then controlling two groups of second reflectors to start the scanning operation of searching particles. The scanning process can adopt a fixed mode scanning form, namely two groups of second reflectors rotate at a constant speed within a fixed angle range so as to realize the purpose of scanning scattered light. When the scattering signal reaches a preset threshold value, the scanning is finished, the photomultiplier starts to detect fluorescence and converts fluorescence photons into an analog signal, and the analyzer starts to measure the fluorescence signal from the particles and performs data comparison analysis. In the process, the piezoelectric ceramic control unit has the advantages of high reaction speed, good frequency stability, high precision, small volume, no moisture absorption, long service life and strong anti-interference performance. Because the deformation quantity generated by the piezoelectric ceramic under the action of the electric field is very small and is not more than one ten-million of the size of the piezoelectric ceramic, the micro deformation quantity can be used for accurately controlling the directional and fixed-distance deflection of the (two-photon) laser beam, and the accuracy of the experimental result of the analyzer is ensured.
3. The invention relates to an immunoassay analyzer which can detect a plurality of pathogens from the same sample and can continuously add the sample, and can carry out nine respiratory tract pathogen joint detections, namely, one sample detects and reports nine detection results. The specificity and the sensitivity are equivalent to the leading immunoassay method in the laboratory, the kit is very easy to use and is suitable for being used continuously for 7 days and 24 hours.
4. Antigen detection, shortening the window period, facilitating early diagnosis and timely treatment; the accuracy of performance index is better than that of immunochromatography, and the specificity is better than that of molecular diagnosis; the reaction volume is small, the reagent dosage is small, the waste liquid is small, and the cost is low; only a sample adding liquid path is provided, and the instrument is simple to maintain; is suitable for large, medium and small medical institutions.
5. Compared with a DFA detection method, the two-photon optical system detection method has the advantages of being good in overall consistency, high in speed, high in sensitivity, high in intelligent full automation degree and the like, the immune process is free of separation and cleaning, the operation is simple, manual operation is greatly reduced in the identification process, the cost and labor are saved, and the working efficiency is improved.
Drawings
FIG. 1 is an isometric view of a two-photon fluoroimmunoassay optical control system according to the present invention;
FIG. 2 is a schematic view of the installation and matching structure of the two-photon laser generation module and the detection control module;
FIG. 3 is a top view of the test control module (without the gearbox motor, motor mount and bushing);
FIG. 4 is a schematic side view of an adjusting transmission module;
FIG. 5 is a schematic view of the mounting structure of two second reflectors and two piezoelectric ceramic control function sets;
FIG. 6 is a schematic view of the spring-driven rotary device;
fig. 7 is a schematic structural diagram of an optical control system of a two-photon fluorescence immunoassay analyzer.
In the above figures: the thick solid line part is a two-photon laser line, the dotted line part is a scattering optical line, and the dot-dash line part is a fluorescent line.
Wherein the graphic symbols have the following meanings:
1. mounting a bottom plate;
2. a two-photon laser generating module;
3. a detection control module; 3.1, a first inlet; 3.2, an attenuator; 3.3, a beam splitter; 3.4, a dichroic mirror; 3.5, a photomultiplier tube; 3.6, a first outlet; 3.7, a gearbox motor; 3.8, a motor bracket; 3.9, lining; 3.10, a scatter detector; 3.11, anterior approach; 3.12, a rear path; 3.13, front supporting path;
4. adjusting the transmission module; 4.1, an objective lens; 4.2, a second spectacle frame; 4.3, a bushing seat; 4.4, a second reflector; 4.5, a first spectacle frame; 4.6, a first reflector; 4.7, a stepping motor; 4.8, a thread transmission sleeve; 4.9, lifting blocks; 4.10, lifting bolts; 4.11, a frame; 4.12, mounting a shaft; 4.13, bearings; 4.14, spring locking; 4.15, driving the spring; 4.16, a spring positioning ring; 4.17, piezoelectric ceramics; 4.18, sleeve; 4.19, rotating the plane in a pushing way; 4.20, guide rails; 4.21 guide wheel mounting plate; 4.22, a guide wheel;
5. a reaction cup.
Detailed Description
The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art.
The optical control system of the two-photon fluorescence immunoassay analyzer shown in fig. 1 comprises a mounting base plate 1, wherein a two-photon laser generation module 2, a detection control module 3 and an adjustment transmission module 4 are arranged on the mounting base plate 1. The two-photon laser generation module 12 may be a two-photon laser generator.
As shown in fig. 2 and 3, an upper-shaped two-photon laser path is provided in the detection control module 3, the two-photon laser path includes a front path 3.11 and a rear path 3.12, and the end of the front path 3.11 is vertically communicated with the middle of the rear path 3.12. The front end of the front passage 3.11 is provided with a first inlet 3.1, the first inlet 3.1 is connected with the two-photon laser generation module 2, the front part of the front passage 3.11 is provided with an attenuator 3.2, the middle part of the front passage 3.11 is vertically communicated with the front end of a front branch 3.13, the communication part of the front passage 3.11 and the front branch 3.13 is provided with a beam splitter 3.3, and the rear end of the front branch 3.13 is communicated with a scattering detector 3.10; a dichroic mirror 3.4 is arranged at the junction of the front and rear paths 3.11, 3.12; one end of the back passage 3.12 is communicated with the photomultiplier tube 3.5, the other end of the back passage 3.12 is provided with a first outlet 3.6, and the first outlet 3.6 is connected with the adjusting and transmitting module 4.
The attenuator 3.2 includes an attenuator mirror holder, and a light path attenuation channel is arranged in the attenuator mirror holder, and the attenuator mirror holder is arranged in the front path 3.11. A gearbox motor 3.7 is installed on the upper surface of the outer portion of the front passage 3.11 through a lining 3.9 and a motor support 3.8, a motor shaft of the gearbox motor 3.7 is downwards inserted into the front passage and connected with an attenuator mirror frame, and the gearbox motor 3.7 can drive the motor shaft and the attenuator mirror frame to synchronously rotate so as to adjust incident light attenuation.
As shown in fig. 3, the two-photon laser emitted by the two-photon laser generating module 2 can enter the front path 3.11 of the detection control module 3 through the first inlet 3.1, and after passing through the attenuator 3.2 and the beam splitter 3.3, the two-photon laser is reflected by the dichroic mirror 3.4 and sequentially passes through the rear path 3.12 to pass out of the first outlet 3.6. The two-photon laser scattering light enters a rear channel 3.12 of the detection control module 3 from a first outlet 3.6, is reflected by a dichroic mirror 3.4 and a beam splitter 3.3, and then enters a scattering detector 3.10 for detection. The fluorescence enters the rear passage 3.12 of the detection control module 3 from the first outlet 3.6, and then can enter the photomultiplier tube 3.5 for detection through the transmission of the dichroic mirror 3.4.
As shown in fig. 4, the adjusting and transmitting module 4 includes a first lens frame 4.5, a second lens frame 4.2, a lifting control device and an objective lens 4.1. The first lens frame 4.5 is connected with the second lens frame 4.2 through a lifting control device, and the objective lens 4.1 is installed at the upper part of the second lens frame 4.2. A second inlet is arranged on the side surface of the first lens frame 4.5, a second outlet is arranged on the top of the first lens frame, and a first reflector 4.6 is arranged in the first lens frame 4.5; set up the third entry in the bottom surface of second picture frame 4.2, set up the third export at the top of second picture frame 4.2, set up two sets of second mirrors 4.4 in second picture frame 4.2, two sets of second mirrors 4.4 all personally submit 45 degrees angles and relative setting with the level. The two groups of second reflectors are respectively arranged on the two groups of piezoelectric ceramic control function groups, and the two groups of piezoelectric ceramic control function groups can respectively control the two groups of second reflectors to rotate and scan. The second outlet is connected with the third inlet through a telescopic lens cone; an objective lens 4.1 is connected and arranged at the upper end of the third outlet, and a reaction cup 5 is arranged above the objective lens 4.1.
The two-photon laser emitted from the first outlet can enter the first lens frame 4.5 through the second inlet, is emitted from the second outlet after being reflected by the first reflector 4.6, enters the third inlet through the telescopic lens cone, is emitted from the third outlet after being reflected by the two groups of second reflectors 4.4, and is emitted into the reaction cup 5 through the objective lens 4.1.
As shown in fig. 4, 5 and 6, the piezoelectric ceramic control function group comprises a bushing seat 4.3, and the bushing seat 4.3 is installed on the outer side surface of the second lens frame 4.2. The second reflecting mirror 4.4 is installed on the mirror bracket 4.11, the mirror bracket 4.11 is fixedly arranged at the inner end of the installation shaft 4.12, and the outer end of the installation shaft 4.12 penetrates through the second mirror frame 4.2 and penetrates out of the bushing seat 4.3; the middle part of the mounting shaft 4.12 is fixed in the second spectacle frame 4.2 and/or the bushing seat 4.3 through two groups of bearings 4.13, a push-turn plane 4.19 is arranged on the mounting shaft 4.12 between the two groups of bearings 4.13, and the outer end of the mounting shaft 4.12 is provided with a spring-driven rotating device. As shown in fig. 6, the spring-driven rotating device includes a spring lock 4.14, a rotation driving spring 4.15 and a spring positioning ring 4.16, the spring lock 4.14 is fixed on the outer wall of the bushing seat 4.3, the spring positioning ring 4.16 is fixed on the outer end of the mounting shaft 4.12, the rotation driving spring 4.15 is a torsion spring sleeved on the mounting shaft 4.12, one end of the torsion spring is fixed on the spring lock 4.14, and the other end of the torsion spring is inserted into the spring positioning ring 4.16; the spring-driven rotating device can apply unidirectional torsion to the mounting shaft 4.12, so that the mounting shaft can rotate around the central axis thereof in a unidirectional way.
Piezoelectric ceramics 4.17 are fixed in the bushing seat 4.3 through a sleeve 4.18, and the telescopic end of the piezoelectric ceramics 4.17 is vertical to and contacts with the push-turn plane 4.19 of the mounting shaft 4.12; piezoelectric ceramics are fixed in the bushing seats through sleeves, and the telescopic ends of the piezoelectric ceramics are perpendicular to and in contact with the push-turn plane of the mounting shaft; the voltage can control the starting and stopping of the telescopic action of the piezoelectric ceramic 4.17 and the telescopic amount, and the mounting shaft 4.12 is pushed to rotate clockwise or anticlockwise around the central axis of the mounting shaft, so that the lens frame 4.11 and the second reflector 4.4 mounted on the lens frame rotate clockwise or anticlockwise. In the process, the spring-driven rotating device can play the roles of presetting torsion and driving reset.
The working principle of the piezoelectric ceramic control unit is as follows: the tip of the piezoelectric ceramic 4.17 is pressed against the push-turn plane 4.19 of the mounting shaft 4.12, the voltage is controlled by the DA converter, the extension and retraction of the piezoelectric ceramic 4.17 are controlled, the mounting shaft 4.12 is pushed to rotate clockwise or anticlockwise around the central axis of the mounting shaft, the lens frame 4.11 and the second reflector 4.4 mounted on the lens frame rotate clockwise or anticlockwise, and the light beam is deflected. (the principle of controlling the amount of expansion of piezoelectric ceramics is the prior art of maturity and is not described here in detail)
As shown in fig. 4, the lifting control device includes a stepping motor 4.7, the upper end of the stepping motor 4.7 is connected with a threaded transmission sleeve 4.8, an internal threaded hole with an open upper end is formed in the threaded transmission sleeve 4.8, a lower end of a lifting bolt 4.10 is inserted into the internal threaded hole of the threaded transmission sleeve 4.8, the upper end of the lifting bolt 4.10 is connected with one end of a lifting block 4.9, and the other end of the lifting block 4.9 is connected with the second spectacle frame 4.2 through a sliding mechanism. As shown in fig. 1 and 4, the sliding mechanism comprises a guide wheel mounting plate 4.21, a guide wheel 4.22 and a guide rail 4.20, the inner side of the guide rail 4.20 is mounted on the outer part of the second eye rim 4.2 through screws, and the outer side of the guide rail 4.20 is fixed with one end of the lifting block 4.9 through a bolt; the guide wheel mounting plate 4.21 is fixed on the first mirror frame 4.5 through bolts, two pairs of guide wheels 4.22 which are longitudinally arranged and symmetrically arranged are clamped at two sides of the guide rail 4.20, and the four guide wheels 4.22 are mounted on the guide wheel mounting plate 4.21 through screws; when the lifting block 4.9 goes up and down, the guide rail 4.20 can be driven to move up and down under the clamping and guiding action of the guide wheel 4.22, and the second mirror frame 4.2 is driven to synchronously go up and down. In the process of lifting the second lens frame, the telescopic lens barrel between the second outlet and the third inlet can be synchronously extended or shortened along with the change of the distance, so that the light transmission between the second outlet and the third inlet is always carried out in a closed passage. The telescopic lens barrel is of a telescopic sleeve type structure, belongs to mature optical equipment accessories, and the structure of the telescopic lens barrel is not described again.
Two-photon excitation route: the two-photon laser generation module 2 is opened firstly, the two-photon laser generation module 2 generates two-photon laser, the two-photon laser enters the front passage 3.11 of the adjustment transmission module through the first inlet 3.1, the two-photon laser sequentially penetrates through the optical path attenuation channel of the attenuator 3.2, is transmitted to the two-way mirror 3.4 from the beam splitter 3.3, is reflected by the two-way mirror 3.4 to penetrate out of the first outlet 3.6, enters the second inlet of the adjustment transmission module 4, passes through the first reflector 4.6 in the first mirror frame 4.5 to be reflected out of the second outlet, passes through the telescopic lens cone to enter the second mirror frame 4.2 through the third inlet, and reaches the objective lens 4.1 after twice reflection of the two groups of second reflectors 4.4. The two-photon laser penetrates through the objective lens and is transmitted upwards to the reaction cup 5, and when the two-photon laser beam strikes a particle solid phase in the reaction cup 5, the antigen and the antibody are compounded to generate fluorescence.
Fluorescence measurement route: the fluorescence downwards transmits from the objective lens 4.1, passes through the reflection of the two second reflectors 4.4, passes out of the second lens frame 4.2, enters the first lens frame 4.5, reaches the dichroic mirror 3.4 through the reflection of the first reflector 4.6 in the first lens frame 4.5, and the fluorescence is transmitted from the dichroic mirror 3.4 to the photomultiplier tube 3.5, so that the photomultiplier tube 3.5 measures the fluorescence.
Scatter detector measurement route: the two-photon laser scattering light passes through the objective lens 4.1 to be transmitted downwards, is reflected by the two second reflecting mirrors 4.4, penetrates out of the second lens frame 4.2 and enters the first lens frame 4.5, reaches the dichroic mirror 3.4 through the reflection of the first reflecting mirror 4.6 in the first lens frame 4.5, reaches the beam splitter 3.3 after being reflected by the dichroic mirror 3.4, is reflected to the scattering detector 3.10 from the beam splitter 3.3, and the scattering detector 3.10 measures the scattering light. The peak produced in the signal of the scattered light is used to determine when the scan has stopped and when the fluorescence signal measured at visible wavelengths is recorded: the piezoelectric ceramics 4.17 controls the two groups of second reflectors 4.4 to perform transverse scanning, and once the scattering signal exceeds a preset detection threshold value, the transverse scanning is stopped.
The overall measurement process of the invention is as follows: before the measurement, the detection plate with the cuvette 5 is moved over the objective 4.1. And (3) starting the two-photon laser generation module 2, enabling the objective lens 4.1 to move upwards, and when the focus of the two-photon laser is at the bottom of the reaction cup 5, reflecting the scattered light of the two-photon laser back to the scattering detector 3.10, and stopping the movement of the objective lens 4.1. When the scattering signal reaches a preset threshold value, the scanning is finished, the photomultiplier starts to detect fluorescence and converts fluorescence photons into an analog signal, and the analyzer starts to measure the fluorescence signal from the particles and performs data comparison analysis. After the measurement is finished, the objective 4.1 is driven back into position and is ready to start the next detection process.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. The comparison, analysis and calculation steps of the result by the analyzer belong to the known technology in the field, and are not the main innovation point of the patent, and are not described herein again. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. The utility model provides a two-photon fluorescence immunoassay appearance optical control system, includes mounting plate, its characterized in that: the installation bottom plate is provided with a two-photon laser generation module, a detection control module and an adjustment transmission module; a photomultiplier is arranged in the detection control module; the two-photon laser emitted by the two-photon laser generation module can be focused into the reaction cup through the detection control module and the adjustment transmission module, the two-photon laser excites a detection substance in the reaction cup to generate fluorescence, and the fluorescence can be returned to the photomultiplier of the detection control module through the adjustment transmission module to be measured.
2. The optical control system of a two-photon fluoroimmunoassay analyzer according to claim 1, wherein: a scattering detector is also arranged in the detection control module, and the two-photon laser scattering light of the detection substance in the two-photon laser excitation reaction cup can return to the scattering detector of the detection control module for measurement through the adjustment transmission module; the resulting peaks in the measured scatter signal are used to determine when the scan has stopped and when to record the fluorescence signal measured at visible wavelengths.
3. The optical control system of a two-photon fluoroimmunoassay analyzer according to claim 2, wherein: an upper-shaped two-photon laser passage is arranged in the detection control module, the two-photon laser passage comprises a front passage and a rear passage, and the tail end of the front passage is vertically communicated with the middle part of the rear passage; the front end of the front passage is provided with a first inlet, the first inlet is connected with the two-photon laser generation module, the front part of the front passage is provided with an attenuator, the middle part of the front passage is vertically communicated with the front end of the front branch, the communication part of the front passage and the front branch is provided with a beam splitter, and the rear end of the front branch is communicated with a scattering detector; a dichroic mirror is arranged at the connection part of the front passage and the rear passage; one end of the back channel is communicated with the photomultiplier, the other end of the back channel is provided with a first outlet, and the first outlet is connected with the adjustment transmission module;
the two-photon laser emitted by the two-photon laser generating module can enter a front passage of the detection control module through the first inlet, and after passing through the attenuator and the beam splitter, the two-photon laser is reflected by the dichroic mirror and passes through the first outlet from a rear passage;
after entering a rear channel of the detection control module from the first outlet, the two-photon laser scattering light enters a scattering detector for detection after being reflected by the dichroic mirror and the beam splitter;
after the fluorescence enters the rear passage of the detection control module from the first outlet, the fluorescence can enter the photomultiplier for detection through the transmission of the dichroic mirror.
4. The optical control system of a two-photon fluoroimmunoassay analyzer according to claim 3, wherein: the attenuator comprises an attenuator mirror frame, a light path attenuation channel is arranged in the attenuator mirror frame, and the attenuator mirror frame is arranged in the front passage; the gearbox motor is installed on the upper surface of the outer portion of the front passage through the bushing and the motor support, a motor shaft of the gearbox motor is downwards inserted into the front passage and connected with the attenuator mirror bracket, and the gearbox motor can drive the motor shaft and the attenuator mirror bracket to synchronously rotate so as to adjust the attenuation of incident light.
5. The optical control system of a two-photon fluoroimmunoassay analyzer according to claim 1, wherein: the adjusting and transmitting module comprises a first mirror frame, a second mirror frame, a lifting control device and an objective lens; the first lens frame is connected with the second lens frame through a lifting control device, and the objective lens is arranged at the upper part of the second lens frame;
a second inlet is arranged on the side surface of the first mirror frame, a second outlet is arranged at the top of the first mirror frame, and a first reflector is arranged in the first mirror frame;
a third inlet is arranged on the bottom surface of the second mirror frame, a third outlet is arranged on the top of the second mirror frame, two groups of second reflecting mirrors are arranged in the second mirror frame, and the two groups of second reflecting mirrors form an angle of 45 degrees with the horizontal plane and are arranged oppositely; the two groups of second reflectors are respectively arranged on the two groups of piezoelectric ceramic control function groups, and the two groups of piezoelectric ceramic control function groups can respectively control the two groups of second reflectors to rotate and scan; the second outlet is connected with the third inlet through a telescopic lens cone; an objective lens is arranged at the upper end of the third outlet in a connecting mode, and a reaction cup is arranged above the objective lens;
the two-photon laser emitted from the first outlet can enter the first mirror frame through the second inlet, is emitted from the second outlet after being reflected by the first reflector, enters the third inlet through the telescopic lens cone, is emitted from the third outlet after being reflected by the two groups of second reflectors, and is emitted into the reaction cup through the objective lens.
6. The optical control system of a two-photon fluoroimmunoassay analyzer according to claim 5, wherein: the piezoelectric ceramic control functional group comprises a bushing seat, and the bushing seat is arranged on the outer side surface of the second mirror frame; the second reflecting mirror is arranged on the mirror bracket, the mirror bracket is fixedly arranged at the inner end of the mounting shaft, and the outer end of the mounting shaft penetrates through the second mirror frame and penetrates out of the bushing seat; the middle part of the mounting shaft is fixed in the second mirror frame and/or the bushing seat through two groups of bearings, a push-turn plane is arranged on the mounting shaft between the two groups of bearings, and a spring-driven rotating device is arranged at the outer end of the mounting shaft; the spring driving rotating device comprises a spring lock, a driving spring and a spring positioning ring, the spring lock is fixed on the outer wall of the bushing seat, the spring positioning ring is fixed at the outer end of the mounting shaft, the driving spring is a torsion spring sleeved on the mounting shaft, one end of the torsion spring is fixed on the spring lock, and the other end of the torsion spring is inserted into the spring positioning ring; the spring-driven rotating device can apply unidirectional torque to the mounting shaft, so that the mounting shaft can rotate around the central axis thereof in a unidirectional way;
piezoelectric ceramics are fixed in the bushing seats through sleeves, and the telescopic ends of the piezoelectric ceramics are perpendicular to and in contact with the push-turn plane of the mounting shaft; the voltage can control the starting and stopping of the piezoelectric ceramic telescopic action and the telescopic amount, and the mounting shaft is pushed to rotate clockwise or anticlockwise around the central axis of the mounting shaft, so that the mirror frame rotates clockwise or anticlockwise together with the second reflecting mirror mounted on the mirror frame.
7. The optical control system of a two-photon fluoroimmunoassay analyzer according to claim 6, wherein: the lifting control device comprises a stepping motor, the upper end of the stepping motor is connected with a thread transmission sleeve, an inner thread hole with an open upper end is formed in the thread transmission sleeve, a lifting bolt is inserted into the inner thread hole of the thread transmission sleeve, one end of a lifting block is connected to the upper end of the lifting bolt, and one end of the lifting block is connected with the second mirror frame through a sliding mechanism.
8. The optical control system of a two-photon fluoroimmunoassay analyzer according to claim 7, wherein: the sliding mechanism comprises a guide wheel mounting plate, a guide wheel and a guide rail, the inner side of the guide rail is mounted outside the second mirror frame through a screw, and the outer side of the guide rail is fixed with one end of the lifting block through a bolt; the guide wheel mounting plate is fixed on the first mirror frame through bolts, two pairs of guide wheels which are longitudinally arranged and symmetrically arranged are clamped on two sides of the guide rail, and the four guide wheels are mounted on the guide wheel mounting plate through screws; when the lifting block goes up and down, the guide rail can be driven to move up and down under the clamping and guiding action of the guide wheel, and the second mirror frame is driven to go up and down synchronously.
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CN202010734701.3A CN111812073A (en) | 2020-07-28 | 2020-07-28 | Optical control system of two-photon fluorescence immunoassay analyzer |
CN202110189261.2A CN112748097A (en) | 2020-07-28 | 2021-02-19 | Optical control system of two-photon fluorescence immunoassay analyzer |
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JP2601698B2 (en) * | 1988-08-30 | 1997-04-16 | 株式会社小松製作所 | Laser scanner rotation detection mechanism |
JPH0588072A (en) * | 1991-09-30 | 1993-04-09 | Ntn Corp | Automatic focusing device |
JP3219123B2 (en) * | 1993-11-29 | 2001-10-15 | 株式会社デンソー | Two-dimensional optical scanning device and bar code reader using the same |
DE19801139B4 (en) * | 1998-01-14 | 2016-05-12 | Till Photonics Gmbh | Point Scanning Luminescence Microscope |
AUPS326102A0 (en) * | 2002-06-28 | 2002-07-18 | Clvr Pty Ltd | Scanning device and method of scanning an optical beam over a surface |
GB0303453D0 (en) * | 2003-02-14 | 2003-03-19 | Thermo Clinical Labsystems Oy | Automated sample analyzer and cuvette |
US20070239031A1 (en) * | 2006-02-15 | 2007-10-11 | Kye-Sung Lee | Systems and methods for performing simultaneous tomography and spectroscopy |
US8198604B2 (en) * | 2007-09-28 | 2012-06-12 | Trustees Of Boston University | System and method for providing enhanced background rejection in thick tissue with differential-aberration two-photon microscopy |
US8064059B2 (en) * | 2008-11-04 | 2011-11-22 | Alipasha Vaziri | Optical pulse duration measurement |
US9229207B2 (en) * | 2008-11-17 | 2016-01-05 | Femtonics Kft | Laser scanning microscope with focus-detecting unit |
DE102009029831A1 (en) * | 2009-06-17 | 2011-01-13 | W.O.M. World Of Medicine Ag | Apparatus and method for multi-photon fluorescence microscopy for obtaining information from biological tissue |
CN102637439B (en) * | 2012-05-07 | 2014-10-29 | 清华大学深圳研究生院 | Light path system applicable to three-dimensional two-photon fluorescence storage |
CN102998293B (en) * | 2012-12-20 | 2014-08-13 | 武汉大学 | Multichannel quantitative detection device and detection method of two-photon fluorescence optical tweezers |
CN104198458B (en) * | 2014-09-26 | 2017-02-22 | 哈尔滨工业大学 | Femtosecond laser two-photon fluorescent biological microimaging system and imaging method thereof |
US9939381B1 (en) * | 2017-04-07 | 2018-04-10 | Vidrio Technologies, Llc | Automated scanning path planner with path calibration for high frame rate multi photon laser scanning microscope with wide field of view |
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