CN110361365A - A kind of scanning fluoroscopic imaging device and the portable QPCR device using it - Google Patents
A kind of scanning fluoroscopic imaging device and the portable QPCR device using it Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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Abstract
The embodiment of the invention provides a kind of scanning fluoroscopic imaging device and using its portable QPCR device, the scanning fluoroscopic imaging device includes the laser set gradually along primary optic axis line, the first optical filter, pellicle mirror and dichroscope；Along 2-D vibration mirror, dichroscope, the second optical filter, convergent lens and the second signal detection device vertically set gradually with the second optical axis of primary optic axis line；2-D vibration mirror, the F-theta lens, microlens array set gradually along the third optical axis vertical with the second optical axis.Portable QPCR device provided in an embodiment of the present invention applies the scanning fluoroscopic imaging device, so that the QPCR device average power consumption is lower than 250W, plant bulk is only 24cm × 16cm × 32cm, weight is less than 10kg, and the time for completing the imaging of 384 Sample Scans only needs 1s, it can be achieved that the miniaturization of real-time quantitative PCR device and high-throughput detection.
Description
Technical Field
The invention relates to the technical field of fluorescence imaging, in particular to a scanning fluorescence imaging device and a portable QPCR device using the same.
Background
Real-time Quantitative PCR (QPCR) has become one of the most commonly used DNA and mRNA quantification techniques, and is the most commonly used pathogen and virus detection technique at present. However, the general commercial QPCR instrument is large in size and high in power consumption, and the transfer and handling of the instrument are also limited, so that the requirement for rapid transfer deployment of regional medical assistance personnel for spreading infectious diseases cannot be met; most of the existing miniaturized equipment has low flux and cannot cope with large-scale screening and mixed infection.
Currently available QPCR devices are generally used to detect several types of well plates including sub-96 well, 384 well, 1536 well, etc. Devices with 96 wells and below typically use a linear array of diode sensors for well-by-well scanning, taking 96 wells as an example, the single scan time for the entire plate is about 4 seconds. When the flux reaches 384 holes, the distance between the holes is very small and limited by the volume of the diode sensor, and a corresponding number of sensors cannot be put down in a single row, so that the scanning mode of the linear array diode sensor array is not suitable for the orifice plates with 384 holes or more. The existing high-flux fluorescence imaging system usually adopts a breadth fluorescence imaging system, a CCD is adopted as a sensor, single image imaging only needs dozens of milliseconds, but the CCD sensor needs a matched lens with a larger caliber, and the working distance and the rear intercept of the lens are longer, so that the whole optical structure of the existing breadth imaging system has a larger volume. Preheating is carried out for about 30 minutes after starting is needed to ensure the stability of the light source, the power consumption of the light source is up to hundreds of watts, and the principle problems lead to the difficulty of miniaturization and low power consumption of high-flux equipment; and various optical device structures have high requirements on installation accuracy and shock resistance, and are not suitable for low-cost portable movement. In view of the above problems, there is a need to develop a fluorescence imaging device that enables a miniaturized QPCR apparatus to achieve high throughput.
Disclosure of Invention
An object of an embodiment of the present invention is to provide a scanning fluorescence imaging device and a portable QPCR device using the same, so as to achieve miniaturization and high-throughput detection of the QPCR device. The specific technical scheme is as follows:
the invention firstly provides a scanning fluorescence imaging device which is used for detecting the fluorescence intensity of a fluorescence sample and comprises a laser, a first optical filter, a semi-transparent mirror, a dichroic mirror, a two-dimensional vibrating mirror, an F-theta lens, a micro-lens array, a converging lens, a second optical filter, a first signal detection device and a second signal detection device;
the laser, the first optical filter, the semi-transparent mirror and the dichroic mirror are sequentially arranged along a first optical axis;
the two-dimensional galvanometer, the dichroic mirror, the second optical filter, the converging lens and the second signal detection device are sequentially arranged along a second optical axis perpendicular to the first optical axis; the incident direction of the two-dimensional galvanometer and the reflecting surface of the dichroic mirror form an included angle of 45 degrees;
the two-dimensional galvanometer, the F-theta lens and the micro-lens array are sequentially arranged along a third optical axis which is vertical to the second optical axis;
the first signal detection device is used for receiving first exciting light emitted by the laser and reflected by the semi-transparent mirror and determining the intensity of the first exciting light;
the second signal detection device is used for receiving the first fluorescence emitted by the fluorescence sample and converged by the converging lens and determining the fluorescence intensity of the first fluorescence; and a correction value of the fluorescence intensity of the first fluorescence is determined based on the intensity of the first excitation light and the fluorescence intensity of the first fluorescence.
Optionally, the first signal detection device comprises a photosensor; the photoelectric sensor is used for receiving the first exciting light, performing photoelectric conversion on the first exciting light and determining an electric signal corresponding to the intensity of the first exciting light;
the second signal detection device comprises a photomultiplier and a signal processing module;
the photomultiplier is used for receiving the first fluorescence and performing photoelectric conversion on a fluorescence signal of the first fluorescence to obtain an electric signal corresponding to the fluorescence intensity of the first fluorescence;
the information processing module is used for determining a correction value of the fluorescence intensity of the first fluorescence according to the electric signal corresponding to the intensity of the first excitation light and the electric signal corresponding to the fluorescence intensity of the first fluorescence.
Optionally, the scanning fluorescence imaging device provided by the invention further includes an aperture, and the aperture is disposed between the converging lens and the second signal detection device and is used for improving the signal-to-noise ratio of the fluorescence signal of the first fluorescence converged by the converging lens.
The invention also provides another scanning fluorescence imaging device which is used for detecting the fluorescence intensity of a fluorescence sample and comprises a laser, a first optical filter, a semi-permeable mirror, a dichroic mirror, a reflecting mirror, a two-dimensional vibrating mirror, an F-theta lens, a micro-lens array, a converging lens, a second optical filter, a first signal detection device and a second signal detection device;
the laser, the first optical filter, the semi-transparent mirror and the first signal detection device are sequentially arranged along a first optical axis;
the semi-transparent mirror and the dichroic mirror are sequentially arranged along a second optical axis perpendicular to the first optical axis; the dichroic mirror is arranged on the reflecting surface of the semi-transparent mirror;
the reflecting mirror, the dichroic mirror, the second optical filter, the converging lens and the second signal detection device are sequentially arranged along a third optical axis perpendicular to the second optical axis; wherein a reflective surface of the mirror faces a reflective surface of the dichroic mirror;
the reflector and the two-dimensional galvanometer are sequentially arranged along a fourth optical axis perpendicular to the third optical axis;
the two-dimensional galvanometer, the F-theta lens and the micro lens array are sequentially arranged along a fifth optical axis which is vertical to the fourth optical axis;
the first signal detection device is used for receiving second excitation light which is emitted by the laser and transmitted through the semi-transparent mirror, and determining the intensity of the second excitation light;
the second signal detection device is used for receiving second fluorescent light which is emitted by the fluorescent sample and converged by the converging lens and determining the fluorescent intensity of the second fluorescent light; and a correction value of the fluorescence intensity of the second fluorescence is determined based on the intensity of the second excitation light and the fluorescence intensity of the second fluorescence.
Optionally, the first, second, third, fourth and fifth optical axes lie in the same plane.
Optionally, the semi-transparent mirror, the dichroic mirror and the reflecting mirror are arranged in parallel.
Optionally, the first signal detection device comprises a photosensor; the photoelectric sensor is used for receiving the second exciting light, performing photoelectric conversion on the second exciting light and determining an electric signal corresponding to the intensity of the second exciting light;
the second signal detection device comprises a photomultiplier and a signal processing module;
the photomultiplier is used for receiving the second fluorescence and performing photoelectric conversion on a fluorescence signal of the second fluorescence to obtain an electric signal corresponding to the fluorescence intensity of the second fluorescence;
the information processing module is used for determining the correction value of the fluorescence intensity of the second fluorescence according to the electric signal corresponding to the intensity of the second excitation light and the electric signal corresponding to the fluorescence intensity of the second fluorescence.
Optionally, another scanning fluorescence imaging device provided by the present invention further includes an optical diaphragm, where the optical diaphragm is disposed between the converging lens and the second signal detection device, and is used to improve a signal-to-noise ratio of a fluorescence signal of the second fluorescence converged by the converging lens.
The invention also provides a portable QPCR device, comprising a housing, and a scanning fluorescence imaging device according to the first or second aspect of the invention, a temperature-controlled platform and at least one cooling fan located in the housing;
the shell is divided into a first grid chamber and a second grid chamber which are distributed in the horizontal direction through a partition plate; the heat radiation fan, the temperature control platform and the two-dimensional galvanometer, the F-theta lens and the micro-lens array in the scanning fluorescence imaging device are positioned in a first grid chamber; other components in the scanning fluorescence imaging device are positioned in the second cell;
the temperature control platform is arranged below a micro-lens array in the scanning fluorescence imaging device and sequentially comprises a first silica gel heating sheet, a first soaking platform, a second soaking platform and a second silica gel heating sheet from bottom to top; the first soaking platform is fixed on the upper surface of the first silica gel heating sheet, and the second soaking platform is fixed on the lower surface of the second silica gel heating sheet; the second silica gel heating sheet and the second soaking platform are provided with corresponding through porous structures;
temperature sensors are respectively arranged on the lower surface of the first silica gel heating sheet, the upper surface of the first soaking platform and the upper surface of the second silica gel heating sheet; the heat radiation fan is arranged below the temperature control platform;
when the QPCR device is used to perform a PCR reaction, the sample plate is placed between the first and second soak platforms; the positions of the sample holes in the sample plate correspond to the positions of the second silica gel heating sheet and the holes of the second soaking platform one by one;
a sliding door is arranged on the shell corresponding to the first grid room and connected with the temperature control platform; when the sliding door is pulled open, the temperature control platform can be pulled out of the first grid chamber.
Optionally, the laser of the scanning fluorescence imaging device in the portable QPCR device provided by the present invention is a 470nm laser.
Optionally, the first filter of the scanning fluorescence imaging device in the portable QPCR device provided by the present invention is selected from a narrowband filter with a center wavelength of 470 nm; the second filter is selected from a narrow-band filter with the central wavelength of 532 nm; the dichroic mirror is selected from long-pass dichroic mirrors with cut-off wavelength between 470nm-532 nm.
Optionally, the first soaking platform and the second soaking platform in the portable QPCR device provided by the present invention each comprise an aluminum plate.
Optionally, a silica gel pad is disposed between the aluminum plate of the first soaking platform and the sample plate in the portable QPCR apparatus provided by the present invention.
Optionally, the sample plate in the portable QPCR device provided by the invention is a 384 well plate.
Optionally, the first silica gel heating plate and the first soaking platform in the portable QPCR device provided by the present invention are fixed in a first fixing frame; the second silica gel heating sheet and the second soaking platform are fixed in the second fixing frame; the first fixed frame and the second fixed frame are detachably connected to realize the relative fixation of the first fixed frame and the second fixed frame in the horizontal direction;
when performing a PCR reaction using the QPCR device, the sample plate is placed within the first fixed frame.
Optionally, the materials of the first fixing frame and the second fixing frame in the portable QPCR device provided by the present invention are selected from high performance nylon; the thermal deformation temperature of the high-performance nylon is more than 145 ℃; the thermal conductivity is less than 0.2W/(m.K).
Optionally, the microlens array in the portable QPCR device provided by the present invention is fixed above the second fixing frame by a third fixing frame.
Optionally, in the portable QPCR apparatus provided by the present invention, rails are respectively disposed on inner walls of two sides of the housing corresponding to the first cells; bulges are arranged on two sides of the first fixing frame of the temperature control platform corresponding to the rails; the protrusion is embedded in the track, so that the temperature control platform can move along the track.
Optionally, the temperature control platform in the portable QPCR device provided by the invention divides the first compartment into two optically isolated spaces, upper and lower.
Optionally, an air inlet fan is disposed on an upper portion of a sidewall of the housing corresponding to the second cell in the portable QPCR device provided by the present invention, and an exhaust fan is disposed on a lower portion of the sidewall; and the bottom of the partition plate is provided with a vent hole.
Optionally, the portable QPCR device provided by the present invention further includes an ac-dc conversion module and a power board card; the alternating current-direct current conversion module is used for converting alternating current into direct current; the power supply board card is used for converting direct current converted by the alternating current-direct current conversion module into direct current with different voltages so as to match voltage requirements of different parts in the device.
Optionally, the portable QPCR device provided by the present invention further comprises a temperature control module, wherein the temperature control module is configured to control a heating state of the silica gel heating sheet and start or stop of the at least one cooling fan according to temperature data fed back by the temperature sensor.
According to the scanning fluorescence imaging device and the portable QPCR device using the same, provided by the embodiment of the invention, the average power consumption of the QPCR device is lower than 250W, the size of the device is only 24cm multiplied by 16cm multiplied by 32cm, the weight of the device is less than 10kg, the time for completing scanning imaging of 384 samples is only 1s, and the miniaturization and high-throughput detection of a real-time quantitative PCR device can be realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is an optical schematic of a scanning fluorescence imaging device.
FIG. 2 is an optical schematic diagram of a second scanning fluorescence imaging device.
FIG. 3 is an assembly view of a second scanning fluorescence imaging device.
Fig. 4 is a schematic structural diagram of a temperature control platform of a portable QPCR device.
Fig. 5 is an exploded view of a temperature-controlled platform of a portable QPCR device.
Figure 6 is a schematic diagram of a first internal configuration of a portable QPCR device.
Figure 7 is a second internal block diagram of a portable QPCR device.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a scanning fluorescence imaging device, which is used for detecting the fluorescence intensity of a fluorescence sample, and as shown in fig. 1, the scanning fluorescence imaging device comprises a laser 1, a first optical filter 2, a semi-transparent mirror 3, a dichroic mirror 9, a two-dimensional galvanometer 14, an F-theta lens 13, a micro-lens array 12, a second optical filter 8, a convergent lens 7, a first signal detection device 4 and a second signal detection device 5;
the laser 1, the first optical filter 2, the semi-transparent mirror 3 and the dichroic mirror 9 are sequentially arranged along a first optical axis;
the two-dimensional galvanometer 14, the dichroic mirror 9, the second optical filter 8, the converging lens 7 and the second signal detection device 5 are sequentially arranged along a second optical axis perpendicular to the first optical axis; wherein, the incident direction of the two-dimensional galvanometer 14 forms an included angle of 45 degrees with the reflecting surface of the dichroic mirror 9;
the two-dimensional galvanometer 14, the F-theta lens 13 and the micro-lens array are sequentially arranged along a third optical axis 12 which is vertical to the second optical axis;
the first signal detection device 4 is used for receiving the first excitation light emitted by the laser 1 and reflected by the semi-transparent mirror 3 and determining the intensity of the first excitation light;
the second signal detection device 5 is used for receiving the first fluorescence emitted by the fluorescence sample and converged by the converging lens 7 and determining the fluorescence intensity of the first fluorescence; and a correction value of the fluorescence intensity of the first fluorescence is determined based on the intensity of the first excitation light and the fluorescence intensity of the first fluorescence.
In the present invention, the laser 1, the first optical filter 2, the second optical filter 8 and the dichroic mirror 9 can be selected according to the fluorescent sample for detection, for example, the laser 1 and the first optical filter 2 can be selected according to the excitation wavelength of the fluorescent sample (i.e. the wavelength of the excitation light of the fluorescent sample), wherein the optical filters are used for filtering out possible stray light emitted by the laser; selecting a second filter 8 according to the detection wavelength of the fluorescent sample to be detected (i.e., the wavelength for detecting the fluorescent sample); the dichroic mirror 9 is used to reflect a certain range of wavelengths and transmit another range of wavelengths, so that the dichroic mirror 9 is selected according to the excitation wavelength and the detection wavelength of the fluorescent sample; in some embodiments of the first aspect of the present invention, when SYBR Green is used as the QPCR fluorochrome, for example, the excitation wavelength is typically 470nm and the detection wavelength is 532nm, so a 470nm laser is used accordingly; the first filter 2 is selected from a narrow-band filter with the central wavelength of 470 nm; the second filter 8 is selected from a narrow-band filter with the central wavelength of 532 nm; the dichroic mirror 9 is selected from long-pass dichroic mirrors with a cut-off wavelength between 470nm-532 nm. The laser 1, the first filter 2, the second filter 8 and the dichroic mirror 9 are selected by a common technical means in the art, and the invention is not limited herein.
The two-dimensional galvanometer 14 and the matched F-theta13 lens are selected according to the area of the sample plate 11 to be scanned; in some embodiments of the invention, when a 384 well plate is used as the sample plate, the scan format is required to be greater than 120mm x 80mm, and at least the scan format diameter is required to be greater than its diagonal (i.e., 145 mm); the focal length F of the F-theta lens is 160mm, the working distance is 182.5mm, and the scanning range is 156.4mm in diameter, so that the requirement of scanning format is met. Further, in the present invention, in order to collect as many fluorescence signals emitted from the fluorescent sample as possible, it is also necessary that the two-dimensional galvanometer 14 has as large a reflecting surface as possible; thus, illustratively, in some embodiments of the invention, the two-dimensional galvanometer 14 employs a 10mm beam system. In addition, the reflecting surface of the two-dimensional galvanometer 14 is generally coated according to the fluorescent wave band requirement of the sample to be detected; in some embodiments of the present invention, when SYBR Green is used as the fluorescent dye of QPCR, the mirror reflection coating band should include 470nm-532nm, and in some embodiments of the present invention, the reflection coating band is illustratively 450nm-700 nm.
After the excitation light for exciting the fluorescent sample is reflected by the two-dimensional galvanometer 14, the light beam of the excitation light forms a certain angle with the vertical direction, so that deviation is possibly generated and the excitation light cannot accurately irradiate the sample; therefore, the micro-lens array 12 is needed to correct the light beam of the excitation light reflected by the two-dimensional galvanometer, and the nonlinear offset of the light beam on the target plane is converted into linear offset; the microlens array 12 is selected by calculating the equivalent focal length through the reverse convergence of the actual light beams. Illustratively, in some embodiments of the invention, the scanning diameter of the F-theta lens 13 is 145mm, from which the equivalent focal length of the microlens array 12 is calculated to be 313.64mm, so a Fresnel lens with a focal length of 313.64mm is selected. The method of calculating the equivalent focal length of the microlens array according to the scanning diameter of the F-theta lens is a conventional technical means in the field, and the present invention is not described herein.
In the invention, the converging lens 7 is used for focusing the fluorescent signal transmitted from the second optical filter 8, so that the second signal detection device 5 can correctly collect the fluorescent signal; in order to reduce the size of the scanning fluorescence imaging device, a converging lens with a short focal length is adopted as far as possible, and in some embodiments of the invention, a plano-convex lens is preferably adopted.
The inventor finds in research that the scanning fluorescence imaging device of the invention realizes point signal detection by adopting a two-dimensional galvanometer scanning mode, not only can greatly reduce the volume of the fluorescence imaging device, but also only needs 1s for completing 384-hole scanning, greatly improves the detection efficiency and realizes high-flux detection.
In some embodiments of the first aspect of the present invention, the first signal detection means 4 comprises a photosensor; in particular a photodiode; the photoelectric sensor is used for receiving the first exciting light, performing photoelectric conversion on the first exciting light and determining an electric signal corresponding to the intensity of the first exciting light;
the second signal detection device 5 comprises a photomultiplier and a signal processing module;
the photomultiplier is used for receiving the first fluorescence and performing photoelectric conversion on a fluorescence signal of the first fluorescence to obtain an electric signal corresponding to the fluorescence intensity of the first fluorescence;
the information processing module is used for determining a correction value of the fluorescence intensity of the first fluorescence according to the electric signal corresponding to the intensity of the first excitation light and the electric signal corresponding to the fluorescence intensity of the first fluorescence.
The photoelectric sensor and the photomultiplier are respectively selected according to the power of the exciting light or the fluorescence received by the photoelectric sensor and the photomultiplier; illustratively, in some embodiments of the invention, the power per unit area of the laser 1 is in watts, a photosensor with sensitivity in W/a is used; the unit area power of the fluorescence power is in milliwatt level, and a photomultiplier with the sensitivity unit of mW/A level is adopted. This is a commonly used technique in the art, and the present invention is not limited thereto.
In some embodiments of the first aspect of the present invention, the information processing module specifically includes an analog-to-digital conversion module and a data processing module, the photoelectric sensor and the photomultiplier respectively convert the light intensity signals of the first excitation light and the first fluorescence into analog electrical signals, the analog-to-digital conversion module further converts the analog electrical signals into digital electrical signals, and the data processing module collects, integrates, and operates the digital electrical signals according to a preset program, so as to obtain a corrected value of the fluorescence intensity of the first fluorescence.
In some embodiments of the first aspect of the present invention, the optical stop 6 is further included, and the optical stop 6 is disposed between the converging lens 7 and the second signal detection device 5, and is used for improving the signal-to-noise ratio of the fluorescence signal of the first fluorescence converged by the converging lens 7.
In some embodiments of the first aspect of the present invention, the diaphragm 6 is an aperture diaphragm, and the aperture of the diaphragm can be selected by those skilled in the art according to actual needs, and the present invention is not limited herein; illustratively, in some embodiments of the first aspect of the present invention, the aperture stop has an aperture diameter of 100 μm.
The second aspect of the present invention provides a method for performing scanning fluorescence imaging by using the scanning fluorescence imaging apparatus according to the first aspect of the present invention, specifically:
a sample plate 11 is placed under a microlens array 12 in the scanning fluorescence imaging device;
exciting light emitted by a laser 1 is irradiated onto a semi-transparent mirror 3 through a first optical filter 2, and is divided into two beams of light with equal intensity by the semi-transparent mirror 3, wherein one beam of light is reflected by the semi-transparent mirror 3 and is irradiated to a first signal detection device 4; the other beam is transmitted by the semi-transparent mirror 3, irradiated to the dichroic mirror 9, reflected by the dichroic mirror 9 to the two-dimensional vibrating mirror 14, and vibrated by the two-dimensional vibrating mirror 14, so that the exciting light from the dichroic mirror 9 converts the nonlinear offset of the beam on the target plane into linear offset through the F-theta lens 13, and is subjected to optical distortion correction through the micro-lens array 12 and then irradiated to different positions of the sample plate 11;
the fluorescence sample on the sample plate 11 is excited by the excitation light to generate fluorescence, and the fluorescence reaches the dichroic mirror 9 along a path opposite to the excitation light from the dichroic mirror 9, is transmitted to the second optical filter 8 through the dichroic mirror 9, and reaches the second signal detection device 5 through the converging lens 7;
the first signal detection device 4 is used for receiving the first excitation light emitted by the laser 1 and reflected by the semi-transparent mirror 3 and determining the intensity of the first excitation light;
the second signal detection device 5 is used for receiving the first fluorescence emitted by the fluorescence sample and converged by the converging lens 7 and determining the fluorescence intensity of the first fluorescence; and a correction value of the fluorescence intensity of the first fluorescence is determined based on the intensity of the first excitation light and the fluorescence intensity of the first fluorescence.
In some embodiments of the second aspect of the present invention, the first signal detection means 4 comprises a photosensor; the photoelectric sensor is used for receiving the first exciting light, performing photoelectric conversion on the first exciting light and determining an electric signal corresponding to the intensity of the first exciting light;
the second signal detection device 5 comprises a photomultiplier and a signal processing module;
the photomultiplier is used for receiving the first fluorescence and performing photoelectric conversion on a fluorescence signal of the first fluorescence to obtain an electric signal corresponding to the fluorescence intensity of the first fluorescence;
the information processing module is used for determining a correction value of the fluorescence intensity of the first fluorescence according to the electric signal corresponding to the intensity of the first excitation light and the electric signal corresponding to the fluorescence intensity of the first fluorescence.
In order to change the volume and shape of the scanning fluorescence imaging device of the present invention, one or more reflectors may be disposed on different optical axes in the scanning fluorescence imaging device of the first aspect of the present invention to change the direction of the optical path, and further change the position and installation direction of each optical element, thereby achieving the purpose of changing the volume and shape of the scanning fluorescence imaging device.
Specifically, the third aspect of the present invention provides a scanning fluorescence imaging device, as shown in fig. 2 and 3, including a laser 1, a first optical filter 2, a semi-transparent mirror 3, a dichroic mirror 9, a reflecting mirror 10, a two-dimensional galvanometer 14, an F-theta lens 13, a micro-lens array 12, a second optical filter 8, a converging lens 7, a first signal detection device 4, and a second signal detection device 5;
the laser 1, the first optical filter 2, the semi-transparent mirror 3 and the first signal detection device 4 are sequentially arranged along a first optical axis;
the semi-transparent mirror 3 and the dichroic mirror 9 are sequentially arranged along a second optical axis perpendicular to the first optical axis; the dichroic mirror 9 is arranged on the reflecting surface of the semi-transparent mirror 3;
the reflecting mirror 10, the dichroic mirror 9, the second optical filter 8, the converging lens 7 and the second signal detection device 5 are sequentially arranged along a third optical axis perpendicular to the second optical axis; wherein the reflecting surface of the mirror 10 faces the reflecting surface of the dichroic mirror 9;
the reflector 10 and the two-dimensional galvanometer 14 are sequentially arranged along a fourth optical axis perpendicular to the third optical axis;
the two-dimensional galvanometer 14, the F-theta lens 13 and the micro-lens array 12 are sequentially arranged along a fifth optical axis perpendicular to the fourth optical axis;
the first signal detection device 4 is used for receiving the second excitation light emitted by the laser 1 and transmitted by the semi-transparent mirror 3 and determining the intensity of the second excitation light;
the second signal detection device 5 is used for receiving second fluorescence emitted by the fluorescence sample and converged by the converging lens 7 and determining the fluorescence intensity of the second fluorescence; and a correction value of the fluorescence intensity of the second fluorescence is determined based on the intensity of the second excitation light and the fluorescence intensity of the second fluorescence.
The same selection of the respective optical elements in the third aspect of the present invention as in the first aspect of the present invention can be referred to the expression of the first aspect of the present invention.
In some embodiments of the third aspect of the present invention, the first signal detection means 4 comprises a photosensor; the photoelectric sensor is used for receiving the second exciting light, performing photoelectric conversion on the second exciting light and determining an electric signal corresponding to the intensity of the second exciting light;
the second signal detection device 5 comprises a photomultiplier and a signal processing module;
the photomultiplier is used for receiving the second fluorescence and performing photoelectric conversion on a fluorescence signal of the second fluorescence to obtain an electric signal corresponding to the fluorescence intensity of the second fluorescence;
the information processing module is used for determining the correction value of the fluorescence intensity of the second fluorescence according to the electric signal corresponding to the intensity of the second excitation light and the electric signal corresponding to the fluorescence intensity of the second fluorescence.
In some embodiments of the third aspect of the present invention, the information processing module specifically includes an analog-to-digital conversion module and a data processing module, the photoelectric sensor and the photomultiplier respectively convert the light intensity signals of the second excitation light and the second fluorescence into analog electrical signals, the analog-to-digital conversion module further converts the analog electrical signals into digital electrical signals, and the data processing module collects, integrates, and operates the digital electrical signals according to a preset program, so as to obtain a corrected value of the fluorescence intensity of the second fluorescence.
In some embodiments of the third aspect of the present invention, the optical stop 6 is further included, and the optical stop 6 is disposed between the converging lens 7 and the second signal detection device 5, and is used for improving the signal-to-noise ratio of the fluorescence signal of the second fluorescence converged by the converging lens 7.
In order to achieve a smaller device volume, in some embodiments of the third aspect of the present invention, the first, second, third, fourth and fifth optical axes lie in the same plane; in further embodiments of the third aspect of the present invention, the semi-transparent mirror 3, the dichroic mirror 9 and the reflecting mirror 10 are arranged in parallel.
The fourth aspect of the present invention provides a method for performing scanning fluorescence imaging by using the scanning fluorescence imaging apparatus according to the third aspect of the present invention, specifically:
a sample plate 11 is placed below a microlens array 12 in the scanning fluorescence imaging device;
exciting light emitted by a laser 1 is irradiated onto a semi-transparent mirror 3 through a first optical filter 2, and is divided into two beams of light with equal intensity by the semi-transparent mirror 3, wherein one beam of light is transmitted through the semi-transparent mirror 3 and is irradiated to a first signal detection device 4; the other beam is reflected by the semi-transparent mirror 3, irradiated to the dichroic mirror 9, reflected by the dichroic mirror 9 to the reflecting mirror 10, reflected by the reflecting mirror 10 to the two-dimensional vibrating mirror 14, and subjected to vibration of the two-dimensional vibrating mirror 14, so that the excitation light from the dichroic mirror 9 converts nonlinear offset of the light beam on a target plane into linear offset through the F-theta lens 13, and is irradiated to different positions of the sample plate 11 after optical distortion correction is performed through the micro-lens array 12;
the fluorescence sample on the sample plate 11 is excited by the exciting light to generate fluorescence, and the fluorescence reaches the dichroic mirror 9 along a path opposite to the exciting light from the dichroic mirror 9, is transmitted to the second optical filter 8 through the dichroic mirror 9, and reaches the second signal detection device 5 through the converging lens 7;
the first signal detection device 4 is used for receiving the second excitation light emitted by the laser 1 and transmitted by the semi-transparent mirror 3 and determining the intensity of the second excitation light;
the second signal detection device 5 is used for receiving second fluorescence emitted by the fluorescence sample and converged by the converging lens 7 and determining the fluorescence intensity of the second fluorescence; and a correction value of the fluorescence intensity of the second fluorescence is determined based on the intensity of the second excitation light and the fluorescence intensity of the second fluorescence.
In some embodiments of the fourth aspect of the present invention, the first signal detection means 4 comprises a photosensor; the photoelectric sensor is used for receiving the second exciting light, performing photoelectric conversion on the second exciting light and determining an electric signal corresponding to the intensity of the second exciting light;
the second signal detection device 5 comprises a photomultiplier and a signal processing module;
the photomultiplier is used for receiving the second fluorescence and performing photoelectric conversion on a fluorescence signal of the second fluorescence to obtain an electric signal corresponding to the fluorescence intensity of the second fluorescence;
the information processing module is used for determining the correction value of the fluorescence intensity of the second fluorescence according to the electric signal corresponding to the intensity of the second excitation light and the electric signal corresponding to the fluorescence intensity of the second fluorescence.
In order to reduce the volume and power consumption of optical equipment in the scanning fluorescence imaging device, the invention abandons a large-volume high-precision laser optical drive module and a constant temperature module; therefore, temperature drift occurs during the long-time operation of the laser 1, that is, the excitation light intensity is unstable due to the over-high temperature of the laser 1, so that the excitation light intensity corresponding to the fluorescence intensity of the fluorescent sample is different at different time points.
Therefore, in a fifth aspect of the present invention, an excitation light source intensity correction method applied to the scanning fluorescence imaging apparatus according to the first and/or third aspect of the present invention is provided, wherein the fluorescence intensity signal received by the second signal detection device 5 is corrected according to the excitation light intensity signal received by the first signal detection device 4:
correction value of fluorescence intensity If' satisfies:
wherein, IfThe fluorescence intensity detected by the second signal detection means; i isαThe intensity of the excitation light detected by the first signal detection means;a fluorescence quantum yield constant; i isαmidIs the global median of the excitation light intensity;
wherein,
Iαmaxthe maximum value of the intensity of the exciting light detected by the first signal detection device; i isαminIs the minimum value of the intensity of the exciting light detected by the first signal detection device.
In the method, IfAnd IαThe fluorescence intensity and the excitation light intensity detected by the second signal detection device and the first signal detection device are the same time point; i isamaxAnd IαminRespectively, the maximum and minimum values of all the excitation light intensities detected by the first signal detection device during the whole fluorescence imaging process.
In some embodiments of the fifth aspect of the present invention, the first signal detection means comprises a photosensor for detecting the excitation light intensity measurement IαMaximum value of excitation light intensity IαmaxAnd minimum value of excitation light intensity Iαmin。
In some embodiments of the fifth aspect of the present invention, the second signal detection device comprises a photomultiplier tube and an information processing module;
the photomultiplier is used for detecting the fluorescence intensity If；
The information processing module is used for measuring I according to the intensity of the exciting light detected by the photoelectric sensorαMaximum value of excitation light intensity IαmaxMinimum value of excitation light intensity IαminAnd the intensity of fluorescence I detected by the photomultiplierfCalculating to obtain a corrected value I of the fluorescence intensityf’。
A sixth aspect of the present invention provides a portable QPCR device, as shown in figures 4-7, comprising a housing 28, and located within the housing 28, a scanning fluorescence imaging device according to the first or third aspect of the present invention, a temperature-controlled platform and at least one cooling fan 15;
the inside of the shell 28 is divided into a first cell and a second cell which are distributed in the horizontal direction through a partition plate 25; the heat radiation fan 15, the temperature control platform, the two-dimensional galvanometer 14 in the scanning fluorescence imaging device, the F-theta lens 13 and the micro-lens array 12 are positioned in a first grid chamber; other components in the scanning fluorescence imaging device are positioned in the second cell;
the temperature control platform is arranged below the micro-lens array 12 in the scanning fluorescence imaging device and sequentially comprises a first silica gel heating sheet 16, a first soaking platform 17, a second soaking platform 18 and a second silica gel heating sheet 24 from bottom to top; the first soaking platform 17 is fixed on the upper surface of the first silica gel heating sheet 16, and the second soaking platform 18 is fixed on the lower surface of the second silica gel heating sheet 24; the second silica gel heating sheet 24 and the second soaking platform 18 are provided with corresponding through porous structures;
temperature sensors 19 are respectively arranged on the lower surface of the first silica gel heating sheet 16, the upper surface of the first soaking platform 17 and the upper surface of the second silica gel heating sheet 24; the heat dissipation fan 15 is arranged below the temperature control platform;
when performing PCR reactions using the QPCR device, the sample plate 11 is placed between the first soaking stage 17 and the second soaking stage 18; the positions of the sample holes on the sample plate 11 correspond to the positions of the holes of the second silica gel heating sheet 24 and the second soaking platform 18 one by one;
a sliding door 23 is arranged on the shell corresponding to the first grid room, and the sliding door 23 is connected with the temperature control platform; when the sliding door 23 is pulled open, the temperature control platform can be pulled out of the first grid chamber.
The QPCR device provided by the present invention needs to consider requirements of optical confinement, electromagnetic compatibility, heat dissipation design, etc. in a narrow space at the same time to ensure that a more accurate result is obtained in a biological experiment, therefore, the housing 28 realizes optical isolation of two cells by the partition 25, necessary electronic or optical connection is realized by arranging a connector on the partition, and the first cell creates a "dark room" environment for optical detection to avoid stray light from affecting a fluorescence detection result.
It should be noted that, when the two-dimensional galvanometer 14 is used, a two-dimensional galvanometer driver 27 needs to be arranged, which is common knowledge, and therefore the description of the present invention is not repeated, in some embodiments of the sixth aspect of the present invention, the two-dimensional galvanometer driver 27 may be arranged in the first cell, and a specific installation manner thereof is a common technical means in the art, and the present invention is not limited herein.
In addition, the laser 1 is also used with the laser driver 33, in some embodiments of the sixth aspect of the present invention, the laser driver 33 may be disposed in the second compartment, and the specific installation position thereof may be designed by those skilled in the art according to actual needs, and the present invention is not limited thereto.
The present invention employs a sliding door 23 that allows the temperature controlled platform to be pulled out of the apparatus for easy placement and replacement of the sample plate 11.
SYBR Green is the most commonly used fluorescent dye in QPCR, in some embodiments of the sixth aspect of the invention, when SYBR Green is used as the fluorescent dye in QPCR, the laser 1 of the scanning fluorescence imaging device is a 470nm laser; the first optical filter 2 is selected from a narrow-band optical filter with the central wavelength of 470 nm; the second filter 8 is selected from a narrow-band filter with the central wavelength of 532 nm; the dichroic mirror 9 is selected from long-pass dichroic mirrors with a cut-off wavelength between 470nm and 532 nm.
In order to reduce the power consumption and the volume of the temperature control platform, the invention adopts a silica gel heating sheet and a soaking platform as heating elements of the temperature control platform, and adopts a cooling fan to cool the temperature control platform; because the heating temperatures of different positions of the silica gel heating sheet are not uniform, a layer of soaking platform is added between the silica gel heating sheet and the sample plate to ensure that the heating temperatures of different positions on the sample plate are consistent; in some embodiments of the sixth aspect of the present invention, the first soaking stage 17 and the second soaking stage 18 each comprise an aluminum plate.
In the prior art, the sample plate 11 is usually rectangular, so the silica gel heating sheet and the soaking platform in the invention can be directly processed into rectangles, and the area of the soaking platform is larger than the bottom area of the sample plate, so as to conveniently heat the sample plate.
In certain embodiments of the sixth aspect of the present invention, the aluminum plate has a thickness of about 2 to 3 mm.
In some embodiments of the sixth aspect of the present invention, a silica gel pad is further disposed between the aluminum plate of the first soaking platform and the sample plate; on one hand, the silica gel pad can enable the contact between the aluminum plate and the sample plate to be tighter, so that the sample plate is heated more uniformly; on the other hand, the aluminum plate adopted in the invention has smaller thickness, so that the temperature change of the aluminum plate is quicker when the aluminum plate is heated, and the silica gel pad can reduce the temperature impact of the aluminum plate on the sample plate, so that the sample plate is heated more smoothly. In some embodiments of the sixth aspect of the present invention, the silicone pad has a thickness of about 0.5 to 1.5mm, preferably 1 mm.
In some embodiments of the sixth aspect of the present invention, the first silicone rubber heating sheet and the silicone rubber pad are respectively attached to both sides of the aluminum plate of the first soaking platform by using 3M double-faced adhesive tape.
In the invention, the second soaking platform 18 and the second silica gel heating sheet 24 are provided with through holes at corresponding positions, and the through holes have the function of ensuring that the light path is smooth, namely, the excitation light and the fluorescence can pass through the second silica gel heating sheet 24 and the second soaking platform 18. Illustratively, in some embodiments of the sixth aspect of the present invention, the sample plate 11 may be a 384-well plate, and the openings of the second silicone heating sheet 24 and the second soaking platform 18 correspond to the positions of the sample wells of the 384-well plate in a one-to-one manner.
In some embodiments of the sixth aspect of the present invention, the first silicone heating sheet 16 and the first soaking platform 17 are fixed in a first fixing frame 20; the second silica gel heating plate 24 and the second soaking platform 18 are fixed in the second fixing frame 21; the first fixed frame 20 and the second fixed frame 21 are detachably connected to realize the relative fixation of the two frames in the horizontal direction; illustratively, the first fixing frame 20 is a rectangular frame, four corners of the first fixing frame 20 are respectively provided with a cylindrical protrusion, four corresponding cylindrical grooves with the size corresponding to the cylindrical protrusions are arranged at corresponding positions of the second fixing frame 21, and the protrusions can be inserted into the grooves, so that the second fixing frame 21 and the first fixing frame 20 are relatively fixed in the horizontal direction; when a PCR reaction is performed using the QPCR device, the sample plate 11 is placed within the first fixed frame 20; when the sample plate 11 needs to be removed, the sample plate 11 can be taken out only by lifting the second fixing frame 21. Of course, the second fixing frame 21 can also be fixed to the first fixing frame 20 by other detachable connection methods, and those skilled in the art can specifically select the methods according to the needs, and the invention is not limited herein.
In some embodiments of the sixth aspect of the present invention, the material of the first and second fixed frames 20, 21 is selected from high performance nylon; the thermal deformation temperature of the high-performance nylon is more than 145 ℃ so as to ensure that the high-performance nylon is not deformed in the heating process; the thermal conductivity is less than 0.2W/(m.K) to ensure that the high-performance nylon has certain heat preservation performance, so that the temperature change is reduced in the heat preservation stage of PCR to reduce the power consumption of a heating device. Under the condition of meeting the heat preservation performance and the high-temperature deformation resistance, other materials can be adopted by the technical personnel in the field, and the invention is not limited herein.
In some embodiments of the sixth aspect of the present invention, the microlens array 12 is fixed above the second fixing frame 21 by a third fixing frame 22; in some embodiments of the sixth aspect of the present invention, the second fixing frame 21 and the third fixing frame 22 are fixedly connected by bolts, the microlens array 12 is fixed in the third fixing frame 22, and in the operating state of the device, the microlens array 12 and the sample plate 11 are relatively fixed in the horizontal position, so as to avoid the deviation of the optical path when the device is moved; in addition, the microlens array 12 is fixed above the second fixing frame 21, the distance between the microlens array 12 and the sample plate 11 in the vertical direction can be fixed, and the position of the microlens array 12 does not need to be adjusted again when the sample plate is placed or replaced.
In some embodiments of the sixth aspect of the present invention, when the silicone heating sheet and the soaking platform are fixed in the fixing frame, horizontal protrusions may be provided on both sides of the first fixing frame 20; the inner walls of the two sides of the shell corresponding to the first grid chamber are respectively provided with a track corresponding to the bulge; the protrusion is embedded in the track, so that the temperature control platform can move along the track.
In some embodiments of the sixth aspect of the present invention, the sliding door can be fixed on the device housing by a release screw, so as to facilitate the operation when the temperature control platform needs to be pulled out, and the device can prevent the temperature control platform from being pulled out by moving the device when working.
In some embodiments of the sixth aspect of the present invention, the temperature-controlled stage separates the first cell into two optically isolated spaces, one above the other.
In the sixth aspect of the present invention, in the cooling stage of the PCR reaction, the cooling fan 15 is used to cool the temperature control platform, and meanwhile, the fan is also used to cool the driving device, the power supply, and the like in the second compartment, which can reduce the power consumption and the volume of the device, in some embodiments of the sixth aspect of the present invention, the upper portion of the sidewall of the casing corresponding to the second compartment is provided with the air inlet fan 31, and the lower portion of the sidewall is provided with the exhaust fan 34; the bottom of the partition plate 25 is provided with a vent hole.
In some embodiments of the sixth aspect of the present invention, the vent hole at the bottom of the partition 25 is disposed below the temperature-controlled stage; the temperature control platform will two light isolation's space about first check room is separated into, consequently the setting in ventilation hole can not influence the "darkroom" environment of temperature control platform top to influence sample fluorescence detection result.
The cooling fan 15, the vent holes and the exhaust fan 34 form a first air channel which is mainly used for cooling the temperature control platform; the air inlet fan 31 and the exhaust fan 34 form a second air duct, which is mainly used for cooling the heat-releasing component in the second compartment.
In some embodiments of the sixth aspect of the present invention, the heat dissipation fan 15 may be fixed at the bottom of the QPCR device, or below the first fixing frame 20; when the heat dissipation fan 15 is fixed under the first fixing frame 20, the heat dissipation fan 15 can be pulled out of the device along with the temperature control platform. The skilled person can specifically select the installation manner of the heat dissipation fan 15 according to the actual needs, and the invention is not limited herein.
In the present invention, the number of the heat dissipation fans may be 2, 3, or 4, and the number and the installation positions of the heat dissipation fan 15, the air inlet fan 31, and the air outlet fan 34 may be designed by those skilled in the art according to actual needs, and the present invention is not limited herein.
In some embodiments of the sixth aspect of the present invention, the power supply further includes an ac-dc conversion module 32 and a power board 29; the ac-dc conversion module 32 is configured to convert ac power into dc power; the power board 29 is used for converting the dc power converted by the ac-dc conversion module 32 into dc power with different voltages, so as to match the voltage requirements of different components in the device.
In some embodiments of the sixth aspect of the present invention, the ac-dc conversion module 32 and the power board 29 are disposed in the second room; the ac-dc conversion module 32 and the power board 29 may be wrapped by a shielding case to prevent electromagnetic interference to other components.
In some embodiments of the sixth aspect of the present invention, the heat dissipation device further comprises a temperature control module, and the temperature control module is configured to control a heating state of the silicone heating sheet and start or stop of the at least one heat dissipation fan according to temperature data fed back by the temperature sensor.
In some embodiments of the sixth aspect of the present invention, the temperature sensor 19 may be a PT1000 platinum resistance temperature sensor.
In some embodiments of the sixth aspect of the present invention, since a thinner aluminum plate is used as the soaking platform, which results in a fast temperature change of the aluminum plate when the aluminum plate is heated and is difficult to control, a multi-point temperature measurement manner is used to construct a temperature control platform thermal distribution model, and algorithm parameters of a PID control algorithm (proportional-integral-derivative control algorithm, PID for short) are determined by the thermal distribution model to obtain PID control coefficients for controlling the heating state of the silica gel heating sheet, where the heating state includes operation, stop and operation power.
Illustratively, three temperature sensors respectively acquire the temperature t1 of the first silica gel heating sheet, the temperature t of the first soaking platform and the temperature t2 of the second silica gel heating sheet, and the algorithm parameters of the PID system, namely the error input e1 and the control output u1, are adjusted through the three temperature parameters. The target of the PID control is the first soaking stage, and the target temperature of the first soaking stage is set to be ts, so the input error e1 is ts-t. After the PID parameter calculation, the output parameter u1 is obtained. Since the temperature change of the first soaking platform is simultaneously acted by the first silica gel heating sheet and the second silica gel heating sheet, the first silica gel heating sheet and the second silica gel heating sheet can be approximately regarded as linear conduction according to a thermodynamic conduction formula, and if the conduction coefficient of the first silica gel heating sheet to the first soaking platform is k1 and the conduction coefficient of the second silica gel heating sheet to the first soaking platform is k2, the final system control parameter coefficient u is u1+ k1 x (t1-t) + k2 x (t 2-t). And the control parameter coefficient u is in the interval [0,1] after normalization treatment, namely the percentage of the operating power of the first silica gel heating plate.
Wherein, after the materials and structures of the silica gel heating plate and the soaking platform are determined, the conductivity coefficient k1 and the conductivity coefficient k2 are constants, and the determination method is a conventional technical means in the field, which is not described herein again.
In some embodiments of the sixth aspect of the present invention, the information processing module and the temperature control module are integrated in the control board card 30; the control board card 30 is disposed in the second compartment.
In some embodiments of the sixth aspect of the present invention, a display 26 is further disposed on the housing 28 for displaying data information of fluorescence intensity.
In some embodiments of the sixth aspect of the present invention, the power board 29 is used to supply power to the laser driver 33, the two-dimensional galvanometer driver 27, the silicone heating plate, the heat dissipation fan 15, the exhaust fan 34, the intake fan 31, the control board 30, and the display 26.
In some embodiments of the sixth aspect of the present invention, the control board card 30 is provided with a data transmission interface 35; the data interface 35 is used for controlling transmission between data in the board card 30 and an external device.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
All the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments may be referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (21)
1. A scanning fluorescence imaging device is used for detecting the fluorescence intensity of a fluorescence sample and is characterized by comprising a laser, a first optical filter, a semi-transparent mirror, a dichroic mirror, a two-dimensional vibrating mirror, an F-theta lens, a micro-lens array, a converging lens, a second optical filter, a first signal detection device and a second signal detection device;
the laser, the first optical filter, the semi-transparent mirror and the dichroic mirror are sequentially arranged along a first optical axis;
the two-dimensional galvanometer, the dichroic mirror, the second optical filter, the converging lens and the second signal detection device are sequentially arranged along a second optical axis perpendicular to the first optical axis; the incident direction of the two-dimensional galvanometer and the reflecting surface of the dichroic mirror form an included angle of 45 degrees;
the two-dimensional galvanometer, the F-theta lens and the micro-lens array are sequentially arranged along a third optical axis which is vertical to the second optical axis;
the first signal detection device is used for receiving first exciting light emitted by the laser and reflected by the semi-transparent mirror and determining the intensity of the first exciting light;
the second signal detection device is used for receiving the first fluorescence emitted by the fluorescence sample and converged by the converging lens and determining the fluorescence intensity of the first fluorescence; and a correction value of the fluorescence intensity of the first fluorescence is determined based on the intensity of the first excitation light and the fluorescence intensity of the first fluorescence.
2. The scanning fluorescence imaging device of claim 1, wherein said first signal detection means comprises a photosensor; the photoelectric sensor is used for receiving the first exciting light, performing photoelectric conversion on the first exciting light and determining an electric signal corresponding to the intensity of the first exciting light;
the second signal detection device comprises a photomultiplier and a signal processing module;
the photomultiplier is used for receiving the first fluorescence and performing photoelectric conversion on a fluorescence signal of the first fluorescence to obtain an electric signal corresponding to the fluorescence intensity of the first fluorescence;
the information processing module is used for determining a correction value of the fluorescence intensity of the first fluorescence according to the electric signal corresponding to the intensity of the first excitation light and the electric signal corresponding to the fluorescence intensity of the first fluorescence.
3. A scanning fluorescence imaging device is used for detecting the fluorescence intensity of a fluorescence sample and is characterized by comprising a laser, a first optical filter, a semi-transparent mirror, a dichroic mirror, a reflecting mirror, a two-dimensional vibrating mirror, an F-theta lens, a micro-lens array, a converging lens, a second optical filter, a first signal detection device and a second signal detection device;
the laser, the first optical filter, the semi-transparent mirror and the first signal detection device are sequentially arranged along a first optical axis;
the semi-transparent mirror and the dichroic mirror are sequentially arranged along a second optical axis perpendicular to the first optical axis; the dichroic mirror is arranged on the reflecting surface of the semi-transparent mirror;
the reflecting mirror, the dichroic mirror, the second optical filter, the converging lens and the second signal detection device are sequentially arranged along a third optical axis perpendicular to the second optical axis; wherein a reflective surface of the mirror faces a reflective surface of the dichroic mirror;
the reflector and the two-dimensional galvanometer are sequentially arranged along a fourth optical axis perpendicular to the third optical axis;
the two-dimensional galvanometer, the F-theta lens and the micro lens array are sequentially arranged along a fifth optical axis which is vertical to the fourth optical axis;
the first signal detection device is used for receiving second excitation light which is emitted by the laser and transmitted through the semi-transparent mirror, and determining the intensity of the second excitation light;
the second signal detection device is used for receiving second fluorescent light which is emitted by the fluorescent sample and converged by the converging lens and determining the fluorescent intensity of the second fluorescent light; and a correction value of the fluorescence intensity of the second fluorescence is determined based on the intensity of the second excitation light and the fluorescence intensity of the second fluorescence.
4. The scanning fluorescence imaging device of claim 3, wherein the first optical axis, the second optical axis, the third optical axis, the fourth optical axis, and the fifth optical axis lie in a same plane.
5. The scanning fluorescence imaging device of claim 3, wherein the semi-transparent mirror, the dichroic mirror, and the reflecting mirror are arranged in parallel.
6. The scanning fluorescence imaging device of claim 3, wherein said first signal detection means comprises a photosensor; the photoelectric sensor is used for receiving the second exciting light, performing photoelectric conversion on the second exciting light and determining an electric signal corresponding to the intensity of the second exciting light;
the second signal detection device comprises a photomultiplier and a signal processing module;
the photomultiplier is used for receiving the second fluorescence and performing photoelectric conversion on a fluorescence signal of the second fluorescence to obtain an electric signal corresponding to the fluorescence intensity of the second fluorescence;
the information processing module is used for determining the correction value of the fluorescence intensity of the second fluorescence according to the electric signal corresponding to the intensity of the second excitation light and the electric signal corresponding to the fluorescence intensity of the second fluorescence.
7. The scanning fluorescence imaging device of any of claims 1-6, further comprising an optical stop disposed between the converging lens and the second signal detection device for increasing the signal-to-noise ratio of the fluorescence signal converged by the converging lens.
8. A portable QPCR device comprising a housing, and within the housing a scanning fluorescence imaging device according to any of claims 1 to 7, a temperature controlled platform and at least one heat sink fan;
the shell is divided into a first grid chamber and a second grid chamber which are distributed in the horizontal direction through a partition plate; the heat radiation fan, the temperature control platform and the two-dimensional galvanometer, the F-theta lens and the micro-lens array in the scanning fluorescence imaging device are positioned in a first grid chamber; other components in the scanning fluorescence imaging device are positioned in the second cell;
the temperature control platform is arranged below a micro-lens array in the scanning fluorescence imaging device and sequentially comprises a first silica gel heating sheet, a first soaking platform, a second soaking platform and a second silica gel heating sheet from bottom to top; the first soaking platform is fixed on the upper surface of the first silica gel heating sheet, and the second soaking platform is fixed on the lower surface of the second silica gel heating sheet; the second silica gel heating sheet and the second soaking platform are provided with corresponding through porous structures;
temperature sensors are respectively arranged on the lower surface of the first silica gel heating sheet, the upper surface of the first soaking platform and the upper surface of the second silica gel heating sheet; the heat radiation fan is arranged below the temperature control platform;
when the QPCR device is used to perform a PCR reaction, the sample plate is placed between the first and second soak platforms; the positions of the sample holes in the sample plate correspond to the positions of the second silica gel heating sheet and the holes of the second soaking platform one by one;
a sliding door is arranged on the shell corresponding to the first grid room and connected with the temperature control platform; when the sliding door is pulled open, the temperature control platform can be pulled out of the first grid chamber.
9. The QPCR device according to claim 8, wherein the laser of the scanning fluorescence imaging device is a 470nm laser.
10. The QPCR device according to claim 8, wherein the first filter of the scanning fluorescence imaging device is selected from a narrowband filter with a central wavelength of 470 nm; the second filter is selected from a narrow-band filter with the central wavelength of 532 nm; the dichroic mirror is selected from long-pass dichroic mirrors with cut-off wavelength between 470nm-532 nm.
11. The QPCR device of claim 8, wherein the first and second soaking platforms each comprise an aluminium plate.
12. The QPCR device according to claim 11, wherein a silicone pad is provided between the aluminium plate of the first soaking platform and the sample plate.
13. The QPCR device according to claim 8, wherein the sample plate is a 384 well plate.
14. The QPCR device according to any of claims 8 to 13, wherein the first silicone heating strip and the first soaking platform are fixed within a first fixed frame; the second silica gel heating sheet and the second soaking platform are fixed in the second fixing frame; the first fixed frame and the second fixed frame are detachably connected to realize the relative fixation of the first fixed frame and the second fixed frame in the horizontal direction;
when performing a PCR reaction using the QPCR device, the sample plate is placed within the first fixed frame.
15. The QPCR device according to claim 14, wherein the material of the first and second fixing frames is selected from high performance nylon; the thermal deformation temperature of the high-performance nylon is more than 145 ℃; the thermal conductivity is less than 0.2W/(m.K).
16. The QPCR device according to claim 14, wherein the microlens array is fixed above the second fixing frame by a third fixing frame.
17. A QPCR device according to claim 14, wherein a track is provided on each of the two side inner walls of the housing corresponding to the first compartment; bulges are arranged on two sides of the first fixing frame of the temperature control platform corresponding to the rails; the protrusion is embedded in the track, so that the temperature control platform can move along the track.
18. The QPCR device according to any one of claims 8 to 13, wherein the temperature-controlled platform divides the first cell into two optically isolated spaces, one above the other.
19. A QPCR device according to any of claims 8 to 13, wherein the housing corresponding to the second compartment is provided with an inlet fan at an upper part of the side wall and an outlet fan at a lower part of the side wall; and the bottom of the partition plate is provided with a vent hole.
20. The QPCR device according to any one of claims 8 to 13, further comprising an ac-dc conversion module and a power board card; the alternating current-direct current conversion module is used for converting alternating current into direct current; the power supply board card is used for converting direct current converted by the alternating current-direct current conversion module into direct current with different voltages so as to match voltage requirements of different parts in the device.
21. The QPCR device according to any one of claims 8 to 13, further comprising a temperature control module for controlling the heating state of the silicone heating sheet and the start or stop of the at least one cooling fan by temperature data fed back by the temperature sensor.
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