Disclosure of Invention
In view of the above, the present application discloses a large-area fluorescence imaging detection device.
It is a primary object of the present application to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection device with a reflecting mirror, which reduces the volume of the fluorescence detection device, shortens the distance between the optical imaging lens and the objective table, and improves the fluorescence receiving efficiency.
It is a primary object of the present invention to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection device with a limited aperture plate, which limits excitation light to excite only the fluorescence imaging region, and avoids fluorescence quenching of other regions due to long-term exposure to excitation light.
One main object of the present application is to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection device with a temperature-controlled circulating stage, which implements real-time fluorescence imaging detection of a single-layer tiled micro-droplet array while performing real-time temperature-controlled circulating operation.
It is therefore one of the primary objectives of the present application to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection device with simple optical path hardware, which can integrate a multi-color composite light source or multiple single-color light sources, an excitation light filter assembly and a fluorescence filter assembly to realize multiple digital PCR detection (more than 4 weight).
It is a primary object of the present application to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection device with a motion device, where the motion device can drive the objective table or the optical path system to move in at least one motion degree direction, so as to implement single-layer tiled micro-droplet array large-area fluorescence imaging detection.
In order to achieve the above purpose, the present application adopts the following technical scheme:
1. a large-area fluorescence imaging detection device is characterized by comprising a fluorescence excitation light mechanism, an excitation light reflection mechanism and a fluorescence projection mechanism, wherein,
the fluorescence excitation mechanism comprises an excitation light source assembly, a collimation lens group, an excitation light filter assembly and a dichroic mirror which are sequentially arranged, and light rays emitted by the excitation light source assembly sequentially pass through the collimation lens group, the excitation light filter assembly and the dichroic mirror to form a fluorescence excitation light path;
the excitation light reflection mechanism comprises a light limiting orifice plate and an objective table which are sequentially arranged, and light rays emitted by the dichroic mirror pass through the light limiting orifice plate and the objective table to form an excitation light reflection light path;
the fluorescent projection mechanism comprises a fluorescent filter assembly, an optical imaging lens and an image sensor which are sequentially arranged; the light reflected by the object stage passes through the light limiting hole plate, the dichroic mirror, the fluorescent filter component, the optical imaging lens and the image sensor to form a fluorescent projection light path;
the light entering the dichroic mirror forms an angle of 45+/-10 degrees with the dichroic mirror, and the light reflected by the dichroic mirror forms an angle of 0+/-10 degrees with the normal of the object stage;
the excitation light reflection light path is coaxial with the fluorescence projection light path.
2. The large area fluorescence imaging detection device of item 1, further comprising a mirror positioned between the excitation light filter assembly and a dichroic mirror; light rays emitted by the excitation light filter assembly pass through the reflecting mirror and are emitted to the dichroic mirror.
3. The large-area fluorescence imaging detection device of item 2, wherein the light rays emitted by the excitation light source component form an angle of 45 ° ± 10 ° with the mirror surface of the reflector;
the reflecting mirror is arranged in parallel with the dichroic mirror;
the reflecting mirror is a plane reflecting mirror.
4. The large area fluorescence imaging detection apparatus of any of claims 1 or 2, wherein the excitation light filter assembly comprises a first motor, a first turntable, and an excitation light filter, the first motor is coupled to the first turntable, and the excitation light filter is disposed on the first turntable.
5. The large-area fluorescence imaging detection device according to item 4, wherein one or more first mounting holes are uniformly provided on the first turntable with the center of the first turntable as a center of symmetry, and the excitation light filters are provided in the first mounting holes;
the OD of the outside of the excitation light filter is inhibited to be more than or equal to 5.
6. The large-area fluorescence imaging detection apparatus according to item 1 or 2, wherein the excitation light source assembly includes a second motor, a second turntable, and an excitation light source, the second motor is connected to the second turntable, and the excitation light filter is disposed on the second turntable.
7. The large-area fluorescence imaging detection device according to item 6, wherein the center of the second turntable is taken as a symmetry center, two or more second mounting holes are uniformly provided on the second turntable, and the excitation light source is provided in the second mounting holes;
the excitation light source is one or more than two of a single-color light source, a wide-spectrum light source or a multi-color composite light source.
8. The large-area fluorescence imaging detection device of item 1 or 2, further comprising a dichroic mirror assembly comprising a third motor, a third turntable, and the dichroic mirror, the third motor being connected to the third turntable, the dichroic mirror being disposed on the third turntable.
9. The large-area fluorescence imaging detection device according to item 8, wherein two or more third mounting holes are uniformly provided on the third turntable with the center of the third turntable as a center of symmetry, and the dichroic mirror is provided in the third mounting holes;
the dichroic mirror is a single-pass single-inverse dichroic mirror or a multi-pass multi-inverse dichroic mirror; the transmittance or reflectivity in the channel of the dichroic mirror is more than 0.9, and the out-of-channel inhibition OD is more than or equal to 3.
10. The large area fluorescence imaging detection device of any of claims 1 or 2, wherein the fluorescence filter assembly comprises a fourth motor, a fourth turntable, and the fluorescence filter, the fourth motor is connected to the fourth turntable, and the fluorescence filter is disposed on the fourth turntable.
11. The large-area fluorescence imaging detection device according to item 10, wherein the center of the fourth turntable is taken as a symmetry center, and two or more fourth mounting holes are uniformly formed in the fourth turntable, and the fluorescence filter is disposed in the fourth mounting holes;
the transmittance or reflectivity in the channel of the fluorescent filter is more than 0.9, and the out-of-channel inhibition OD is more than or equal to 5.
12. The large-area fluorescence imaging detection apparatus according to item 1 or 2, further comprising a movement mechanism including a first movement mechanism and a second movement mechanism, the first movement mechanism being connected to the image sensor, the first movement mechanism being configured to control movement of the image sensor relative to the optical imaging lens; the second movement mechanism is connected with the objective table, so the second movement mechanism is used for controlling the objective table to move relative to the light limiting hole plate.
13. The large area fluorescence imaging detection apparatus of item 1, wherein the image sensor is one of a complementary metal oxide semiconductor, a charge coupled device, or an array photosensor;
the pixel size of the image sensor is smaller than 12.4 micrometers, and the dynamic range is larger than 4700dB.
14. The large area fluorescence imaging detection apparatus of item 1 or 2, wherein the collimating lens group comprises a spherical lens and/or an aspherical lens.
15. The large-area fluorescence imaging detection device according to item 1 or 2, wherein the light-limiting aperture plate is provided with a plurality of light-limiting apertures, the light-limiting apertures are in the shape of one of a circle, a rectangle, a triangle, a diamond or other polygons, and the area of the light-limiting apertures is not less than 16mm 2 。
16. The large-area fluorescence imaging detection apparatus according to item 1 or 2, wherein a temperature control mechanism is provided on the stage, the temperature control mechanism being used for accurate temperature setting of the reaction of the sample to be detected and cyclic temperature control reaction control.
17. The large-area fluorescence imaging detection device according to item 1 or 2, wherein the optical imaging lens is automatically or manually continuously adjusted steplessly within a magnification of 0.1 to 5 times.
Compared with the prior art, the large-area fluorescence imaging detection device disclosed by the application has the beneficial effects that: by arranging the reflective mirror, the volume of the fluorescence detection device is reduced, the distance between the optical imaging lens and the objective table is shortened, and the fluorescence receiving efficiency is improved; by arranging the light limiting pore plate, the excitation light is limited to only excite the fluorescence imaging area, so that fluorescence quenching caused by long-time excitation light exposure of other areas is avoided; the real-time fluorescence detection is carried out on the single-layer tiled micro-droplet array while the real-time temperature control circulation operation is realized through the objective table provided with the temperature control circulation; the simple light path hardware system can integrate a single multicolor composite light source or a plurality of monochromatic light sources, an excitation light filter assembly and a fluorescence filter assembly to realize multiple digital PCR detection (more than 4 weight); through the device motion device, the motion device can drive the objective table or the light path system moves in at least one motion degree direction, and large-area fluorescence imaging detection of the single-layer tiled micro-droplet array is realized.
Detailed Description
Exemplary embodiments of the present application are described below in conjunction with the accompanying drawings, which include various details of the embodiments of the present application to facilitate understanding, and should be considered as merely exemplary. Accordingly, one of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the present application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.
In the application, as shown in fig. 1, the large-area fluorescence imaging detection device disclosed in the application comprises a fluorescence excitation mechanism, an excitation reflection mechanism and a fluorescence projection mechanism, wherein the fluorescence excitation mechanism comprises an excitation light source 15, a collimating lens group 2, an excitation light filter 14, a reflecting mirror 4 and a dichroic mirror 5 which are sequentially arranged; light rays emitted by the excitation light source 15 sequentially pass through the collimating lens group 2, the excitation light filter 14, the reflecting mirror 4 and the dichroic mirror 5 to form a fluorescence excitation light path;
the excitation light reflection mechanism comprises a light limiting hole plate 6 and a stage 7 which are sequentially arranged, and the light emitted by the dichroic mirror 5 passes through the light limiting hole plate 6 and the stage 7 to form an excitation light reflection light path;
the fluorescent projection mechanism comprises a fluorescent filter 8, an optical imaging lens 9 and an image sensor 10 which are sequentially arranged; the light reflected by the object stage 7 passes through the light limiting hole plate 6, the dichroic mirror 5, the fluorescent filter 8, the optical imaging lens 9 and the image sensor 10 to form a fluorescent projection light path; the light entering the dichroic mirror 5 forms 45 degrees plus or minus 10 degrees with the mirror surface of the dichroic mirror 5, and the light reflected by the dichroic mirror 5 forms 0 degrees plus or minus 10 degrees with the normal line of the object stage 7; the excitation light reflection light path is coaxial with the fluorescence projection light path.
The direct-downward excitation light source 15 is provided with the collimating lens group 2, the direct-downward excitation light filter 14 is fixedly arranged under the collimating lens group 2, the direct-downward excitation light filter 14 is fixedly provided with the reflecting mirror 4, the reflecting mirror 4 is parallel to the dichroic mirror 5, and the reflecting mirror 4 and the dichroic mirror 5 are positioned on the same horizontal height. A limiting hole is fixedly formed under the dichroic mirror 5, and an objective table 7 is arranged under the limiting hole. A fluorescence filter 8 is arranged right above the dichroic mirror 5, an optical imaging lens 9 is arranged right above the fluorescence filter 8, and an image sensor 10 is arranged right above the optical imaging lens 9.
The collimating lens group 2 is disposed between the excitation light source 15 and the excitation light filter 14, and is configured to collimate the excitation light emitted by the excitation light source 15 to form a parallel light beam. As shown in fig. 4, the collimator lens group 2 includes a spherical lens 16 and an aspherical lens 17, and preferably, the working distance of the collimator lens group 2 is not more than 5cm to increase the amount of light entering the fluorescence excitation light path.
The excitation light source 15 may be one of a monochromatic light source, a broad spectrum light source, or a polychromatic composite light source.
The reflecting mirror 4 is a plane reflecting mirror 4, is fixedly arranged between the excitation light filter 14 and the dichroic mirror 5, is arranged at an angle of 45+/-10 degrees relative to the light emitted by the excitation light source 15, and is used for reflecting the light emitted by the excitation light source 15 to the dichroic mirror 5. By arranging the reflective mirror, the volume of the fluorescence detection device is reduced, the distance between the optical imaging lens 9 and the objective table 7 is shortened, and the fluorescence receiving efficiency is improved.
The OD of the excitation light filter 14 is restrained to be more than or equal to 5 in an out-band mode.
The dichroic mirror 5 is a single-pass single-reverse dichroic mirror or a multi-pass multi-reverse dichroic mirror; the transmittance or reflectivity in the channel of the dichroic mirror 5 is more than 0.9, and the out-of-channel inhibition OD is more than or equal to 3.
The transmittance or reflectivity in the channel of the fluorescent filter 8 is more than 0.9, and the out-of-channel inhibition OD is more than or equal to 5.
The image sensor 10 has a pixel size of less than 12.4 microns and a dynamic range of greater than 4700dB. The dynamic range is the ratio of the maximum output signal level that an image sensor individual pixel can produce (full well capacity) divided by the signal level that the pixel would produce (noise floor) even if no light was incident.
The light limiting hole plate 6 is provided with a plurality of light limiting holes, the shape of each light limiting hole is one of a circle, a rectangle, a triangle, a diamond or other polygons, and the area of each light limiting hole is not less than 16mm 2 . The shape of the light limiting hole can be determined according to practical application and requirements, and the area of the light limiting hole is not smaller than 16mm 2 . The light limiting aperture plate 6 is used for limiting excitation light to only irradiate the fluorescence imaging area, so that fluorescence quenching caused by long-time exposure of the excitation light in other areas is avoided.
In this application, as shown in fig. 10, in the large-area fluorescence imaging detection device disclosed in this application, the excitation light source 15, the collimating lens group 2, the excitation light filter 14 and the dichroic mirror 5 are located on the same horizontal level, and the light emitted by the excitation light source 15 is emitted to the dichroic mirror through the collimating lens group 2 and the excitation light filter 14, and the light emitted by the excitation light filter 14 is reflected to 45 ° ± 10 ° on the mirror surface of the dichroic mirror.
In this application, as shown in fig. 2 and fig. 5, a large-area fluorescence imaging detection device disclosed in this application, the excitation light filter assembly 3 includes a first motor 12, a first turntable 13, and an excitation light filter 14, the first motor 12 is connected with the first turntable 13, and the excitation light filter 14 is disposed on the first turntable 13. One or more first mounting holes 28 are uniformly formed in the first turntable 13 with the center of the first turntable 13 as a symmetry center, and the excitation light filters 14 are arranged in the first mounting holes 28; the OD of the excitation light filter 14 is restrained to be more than or equal to 5 in an out-band mode.
The number of the first mounting holes 28 on the first turntable 13 may be 1, 2, 3, 4, 5, 6, 7, etc., and the number and the size of the first mounting holes 28 may be determined according to actual needs. The excitation light filters 14 on the plurality of first mounting holes 28 may be the excitation light filters 14 having the same out-of-band rejection, or may be excitation light filters 14 having different out-of-band rejection. The relative mounting positions and the number of the excitation light filters 14 of a plurality of different kinds may be determined according to actual needs.
The plane of the excitation light filter 14 is perpendicular to the light emitted by the excitation light source 15.
The first motor 12 may be one of a stepper servo motor, a dc servo motor, or an ac servo motor. The first motor 12 drives the first turntable 13 to rotate through a first transmission mechanism, so that the required excitation light filter 14 is rotated to a designated position, the excitation light filters 14 are respectively configured into the fluorescence excitation light paths in a switchable mode, the outside of the excitation light filters 14 inhibit OD (optical density) from being more than or equal to 5, and monochromatic light output of different wavelengths of the excitation light source assembly 1 is realized.
In this application, as shown in fig. 3, which is a large-area fluorescence imaging detection device disclosed in this application, the excitation light source assembly 1 includes a second motor 18, a second turntable 19, and an excitation light source 15, the second motor 18 is connected to the second turntable 19, and the excitation light filter is disposed on the second turntable 19. The center of the second turntable 19 is taken as a symmetry center, more than two second mounting holes are uniformly formed in the second turntable 19, and the excitation light source 15 is arranged in each second mounting hole;
the excitation light source 15 is one or more of a monochromatic light source, a broad spectrum light source or a polychromatic composite light source.
The number of the second mounting holes on the second turntable 19 may be 1, 2, 3, 4, 5, 6, 7, etc., and the number and the size of the second mounting holes may be determined according to actual needs. The excitation light sources 15 on the second mounting holes may be monochromatic light sources, broad spectrum light sources or polychromatic composite light sources, or may be a combination of monochromatic light sources, broad spectrum light sources and polychromatic composite light sources. When the excitation light source 15 is a single-color light source, a broad-spectrum light source, or a multi-color composite light source, the relative installation positions and the number of the excitation light sources 15 can be determined according to actual needs.
The second motor 18 drives the second turntable 19 to rotate through a second transmission mechanism, so that the required excitation light source 15 is rotated to a designated position, and the excitation light source 15 is respectively configured into the fluorescence excitation light path in a switchable manner, so that monochromatic excitation light output of different wavelengths of the multicolor and multiple composite light sources is realized.
The second motor 18 may be one of a stepper motor, a dc servo motor, or an ac servo motor. When the excitation light source 15 adopts a multicolor composite light source, the multicolor composite light source and the excitation light filter assembly 3 generate monochromatic excitation light, so that monochromatic excitation light with multiple wavelengths can be provided at lower cost, and the multiple digital PCR function is realized.
When the excitation light source 15 is a plurality of monochromatic light sources, the implementation manner of realizing the excitation light excitation single-layer tiled micro-droplet array with different wavelengths is as follows: the second motor 18 drives the second turntable 19 to enable the monochromatic light sources on the second turntable 19 to be respectively configured into the fluorescence excitation light paths in a switchable mode, and monochromatic excitation light with different wavelengths is emitted; the collimating lens group 2 collimates the monochromatic light emitted by the monochromatic light source to form a parallel light beam, the excitation light filter assembly 3 includes a first motor 12, a first turntable 13 with a plurality of first mounting holes 28 distributed along the circumferential direction, and a plurality of excitation light filters 14, the first motor 12 drives the first turntable 13 to make the excitation light filters 14 thereon be respectively configured in the fluorescence excitation light paths in a switchable manner, so as to remove the unwanted excitation light wavelength.
In this application, as shown in fig. 6, a large-area fluorescence imaging detection device disclosed in this application, the device further includes a dichroic mirror 5 assembly, where the dichroic mirror 5 assembly includes a third motor 20, a third turntable 21, and the dichroic mirror 5, the third motor 20 is connected with the third turntable 21, and the dichroic mirror 5 is disposed on the third turntable 21. The center of the third turntable 21 is taken as a symmetry center, more than two third mounting holes are uniformly formed in the third turntable 21, and the dichroic mirror 5 is arranged in the third mounting holes;
the dichroic mirror 5 is a single-pass single-reverse dichroic mirror or a multi-pass multi-reverse dichroic mirror; the transmittance or reflectivity in the channel of the dichroic mirror 5 is more than 0.9, and the out-of-channel inhibition OD is more than or equal to 3. Out-of-band rejection refers to the ability of the module to reject optical signals that deviate from the effective occupancy band range.
The number of the third mounting holes on the third turntable 21 may be 1, 2, 3, 4, 5, 6, 7, etc., and the number and the size of the third mounting holes may be determined according to actual needs. The dichroic mirrors 5 on the plurality of third mounting holes may be single-pass single-inverse dichroic mirrors or multi-pass multi-inverse dichroic mirrors, or may be a combination of single-pass single-inverse dichroic mirrors or multi-pass multi-inverse dichroic mirrors. When the dichroic mirror 5 is a single-pass single-reverse dichroic mirror or a multi-pass multi-reverse dichroic mirror, the relative mounting positions and the number between the two dichroic lenses can be determined according to actual needs.
The third motor 20 drives the third turntable 21 to rotate through a third transmission mechanism, so that the required two-way lens is rotated to a designated position, and is respectively configured into the fluorescence excitation light paths in a switchable manner. When the single-pass single-reverse dichroic mirror is disposed at an angle of 45 deg. + -10 deg. with respect to the fluorescence excitation light path and the fluorescence transmission light path, the single-pass single-reverse dichroic mirror reflects the excitation light of a short wavelength, enters the stage 7 at a vertical angle, and simultaneously enters the image sensor 10 through the fluorescence of a long wavelength.
When the dichroic mirror 5 is a multi-pass multi-reverse dichroic mirror, it is fixedly disposed in the fluorescence excitation light path. When the multipass dichroic mirror is disposed at an angle of 45 deg. + -10 deg. with respect to the fluorescence excitation light path and the fluorescence transmission light path, the multipass dichroic mirror reflects excitation light of a plurality of wavelength bands, enters the stage 7 at a vertical angle, and simultaneously enters the image sensor 10 through fluorescence of a plurality of wavelength bands.
The third motor 20 may be one of a stepping servo motor, a direct current servo motor, or an alternating current servo motor.
In this application, as shown in fig. 7, in the large-area fluorescence imaging detection device disclosed in this application, the fluorescence filter assembly 11 includes a fourth motor 24, a fourth turntable 25, and the fluorescence filter 8, the fourth motor 24 is connected with the fourth turntable 25, and the fluorescence filter 8 is disposed on the fourth turntable 25. The center of the fourth turntable 25 is taken as a symmetry center, more than two fourth mounting holes are uniformly formed in the fourth turntable 25, and the fluorescent filter 8 is arranged in the fourth mounting holes;
the number of the fourth mounting holes on the fourth turntable 25 may be 1, 2, 3, 4, 5, 6, 7, etc., and the number and the size of the fourth mounting holes may be determined according to actual needs.
The fourth motor 24 drives the fourth turntable 25 to rotate through a fourth transmission mechanism, so that the required fluorescent filter 8 is rotated to a designated position, switchable modes are respectively configured in the fluorescent transmission light paths, the transmittance or the reflectivity in the channel of the fluorescent filter 8 is more than 0.9, and the OD of the channel out-of-band inhibition is more than or equal to 5, so that the unnecessary fluorescent wavelength is removed.
The optical imaging lens 9 is disposed between the fluorescence filter assembly 11 and the image sensor 10, and the magnification thereof can be continuously and steplessly adjusted within 0.1 x-5 x, so as to collect fluorescence emitted by the excited single-layer tiled micro-droplet array.
In the present application, as shown in fig. 8 and 9, a large-area fluorescence imaging detection device disclosed in the present application further includes a motion mechanism, where the motion mechanism includes a first motion mechanism 22 and a second motion mechanism 26, the first motion mechanism 22 is connected to the image sensor 10, and the first motion mechanism 22 is used to control the image sensor 10 to move relative to the optical imaging lens 9; the second movement mechanism 22 is connected to the stage 7, so the second movement mechanism 22 is used to control the movement of the stage 7 relative to the light-restricting orifice plate 6.
The first motion mechanism 22 includes a fifth motor and a fifth transmission mechanism 23, the fifth transmission mechanism 23 is connected to the image sensor 10, and the fifth motor can drive the image sensor 10 to move in one or two dimensions through the fifth transmission mechanism 23, so as to realize the motion of the image sensor 10 in one or two motion degrees of freedom. Therefore, the one-dimensional or two-dimensional movement of the image sensor 10 in the horizontal X direction or the Y direction can be precisely controlled, and one-dimensional or two-dimensional scanning detection of the micro-droplet arrays tiled at different hole bottoms of the open-pore container by the fluorescence imaging detection device can be realized, so that the detection range is increased, and the large-area high-flux fluorescence imaging detection is realized.
The second motion mechanism 26 includes a sixth motor and a sixth transmission mechanism 27, the sixth transmission mechanism 27 is connected to the objective table 7, and the sixth motor can drive the objective table 7 to move in one-dimensional or two-dimensional directions through the sixth transmission mechanism 27, so as to realize movement of the objective table 7 in one or two degrees of freedom of movement. Therefore, the one-dimensional or two-dimensional movement of the objective table 7 in the horizontal X direction or the Y direction can be precisely controlled, one-dimensional or two-dimensional scanning detection of the micro-droplet arrays paved at different hole bottoms of the hole opening container by the fluorescence imaging detection device can be realized, so that the detection range is increased, and the large-area high-flux fluorescence imaging detection is realized.
The fifth motor and the sixth motor can be one of a stepping servo motor, a direct current servo motor or an alternating current servo motor.
In this application, the stage 7 is provided with a temperature control mechanism for accurate temperature setting of the reaction of the sample to be measured and cyclic temperature control reaction control. Multiple digital PCR can be realized by adding multiple fluorescent dyes or probes to a PCR reaction system and matching different PCR reaction premix solutions and primers. Wherein the PCR is the same in number as the added fluorescent dye or probe, for example, four fluorescent dyes or probes are required for a four-digit PCR.
In the present application, the image sensor 10 is one of a Complementary Metal Oxide Semiconductor (CMOS), a Charge Coupled Device (CCD) or an array photosensor, and the pixel size of the image sensor 10 is smaller than 12.4 microns, and the dynamic range is larger than 4700dB, for converting the optical signal into the image signal for subsequent analysis.
Examples
The experimental methods used in the following examples are conventional methods, if no special requirements are imposed.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
Using the large-area fluorescence imaging detection device shown in fig. 8, starting an excitation light source (the excitation light source is a self-grinding high-power multicolor composite LED light source), placing an open container with a single-layer tiled micro-droplet (the micro-droplet is an aqueous solution containing 0.4mg/mL rhodamine B) array on a stage (Zhuo Lihan light), and adjusting the movement mechanism to enable a sample pool of the open container to be positioned in the center of a visual field; the light emitted by the excitation light source enters an excitation light filter (the excitation light filter is a filter with the diameter of 520nm-540 nm) after being collimated by a collimating lens group (the diameter of 25mm and the focal length of 50 mm), the excitation light enters a single-pass single-inverse dichroic mirror (the total reflection of the wavelength below 550nm and the total transmission of the wavelength above 550 nm) after being filtered by the excitation light filter group, the excitation light with the short wavelength is reflected by the single-pass single-inverse dichroic mirror, the light spot size and the shape of the excitation light are limited by a light limiting aperture plate (the aperture of 15 mm), the single-layer tiled micro-droplet array in an opening container is excited, the micro-droplet is excited and emitted by fluorescence, the fluorescence is transmitted through the light limiting aperture plate and the single-pass single-inverse dichroic filter, the unwanted band is filtered by the fluorescence filter (the fluorescence is 554nm-576nm filter), the fluorescence is collected by an optical imaging lens, the image sensor is reached, the image signal is converted, the experimental result of fluorescence imaging detection of the micro-droplet array is shown in fig. 11, the imaging area of the micro-droplet array is 64mm 2 。
Example 2
Fluorescence imaging detection was performed on a single layer tiled array of microdroplets, which were aqueous solutions containing 200mM green fluorescein, excitation light was a polychromatic composite light source,the excitation light filter is a 440nm-460nm filter, and the fluorescence filter is a 500nm-550nm filter. The experimental results of fluorescence imaging detection of the micro-droplet array are shown in FIG. 12, in which the imaging area of the micro-droplet array is 64mm 2 。
Example 3
Fluorescence imaging detection of a single layer tiled array of micro-droplets comprising: 10 Xreaction buffer 2. Mu.L, 10mM forward primer (5'-GGACTC TGGACA TGCAAGAT-3') 1.5. Mu.L, 10mM reverse primer (5'-ATACCC TTC TTAACACCT GG-3') 1.5. Mu.L, 10mM Taqman probe (5 '-FAM-GTATCAATTTAGAGAAAA CGC TCT GAA GGG-BHQ 1-3') 0.5. Mu.L, 4mM dNTPMmix 1. Mu.L, 100mM MgCl 20.4. Mu.L, 2U/. Mu. L TransScript DNA polymerase 0.4. Mu.L, saccharomyces cerevisiae Saccharomyces cerevisiae HansenATCC 204508/S288C genomic DNA (10-fold dilution) 1. Mu.L, deionized water 11.7. Mu.L. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 20s and annealing at 55℃for 40s for 40 cycles. The excitation light is a multicolor composite light source, the excitation light filter is a 440nm-460nm filter, and the fluorescence filter is a 500nm-550nm filter. The experimental results of fluorescence imaging detection of the micro-droplet array are shown in FIG. 13, in which the imaging area of the micro-droplet array is 64mm 2 。
Although embodiments of the present application have been described above with reference to the accompanying drawings, the present application is not limited to the specific embodiments and fields of application described above, which are merely illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may make numerous forms, and equivalents thereof, without departing from the scope of the invention as defined by the claims.