A wide-field fluorescence microcavity array imaging system and method for digital PCR

Abstract

This invention provides a large field-of-view fluorescence microcavity array imaging system and method for digital PCR. The system includes at least: a laser; a microarray lens group for homogenizing the laser beam to obtain a uniform excitation beam; a dichroic mirror for reflecting the uniform excitation beam emitted from the microarray lens group onto the microcavity array on the sample plane and transmitting the fluorescence beam emitted from the microcavity array on the sample plane into a subsequent optical path; a large field-of-view aberration-correcting lens group for imaging the microcavity array within the field of view; and a CMOS camera for receiving fluorescence signals and converting them into images. The microlens array group and large field-of-view fluorescence imaging technology used in this invention achieve a faster detection rate and eliminate the need for complex and time-consuming image stitching post-processing. Compared to traditional small field-of-view fluorescence imaging and image stitching techniques that may produce abnormal signals, this invention offers higher detection accuracy.

Description

Technical Field

[0001] This invention relates to the field of high-end equipment manufacturing technology, and in particular to the field of high-performance medical devices. Background Technology

[0002] Digital PCR is a nucleic acid quantitative detection technology that has been developed in recent years. Compared with traditional real-time PCR, it does not rely on the cycle Ct value of the nucleic acid amplification curve for result determination, is not affected by amplification efficiency, and can directly read the number of nucleic acid fragments in the sample. Therefore, it can perform absolute quantitative detection of nucleic acid molecules in the initial sample. The basic principle of digital PCR is to divide a sample into tens to tens of thousands of amplification units, each of which will amplify the target molecule. Then, the fluorescence signal of each unit will be detected and calculated.

[0003] Currently, most fluorescence detection technologies are based on small field-of-view multiple imaging and subsequent image stitching. This method involves dividing the target area into several parts, imaging each part in a specific order, and then using computer software to stitch them together into a complete fluorescence image. This method is time-consuming, and image stitching can lead to abnormalities in the microcavities at the edges of each sub-image, thus affecting the final detection results. Flow cytometry, on the other hand, places extremely high demands on the detection components. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a large field-of-view fluorescence microcavity array imaging system and method for digital PCR, which solves the technical problems of long detection time and low detection accuracy in the prior art of digital PCR.

[0005] To achieve the above and other related objectives, the present invention provides a large field-of-view fluorescence microcavity array imaging system for digital PCR, comprising at least: a laser, serving as a light source to generate laser light; a microarray lens group to homogenize the laser beam to obtain a uniform excitation beam; a dichroic mirror to reflect the uniform excitation beam emitted by the microarray lens group onto the microcavity array on the sample plane, and to transmit the fluorescence beam emitted by the microcavity array on the sample plane into a subsequent optical path; a large field-of-view aberration-correcting lens group to image the microcavity array within the field of view; and a CMOS camera for receiving fluorescence signals and converting them into images.

[0006] In one embodiment of the present invention, the microarray lens group includes: a collimating lens for collimating the laser beam emitted by the laser into parallel light; a microlens array for focusing and diverging the parallel beam emitted by the collimating lens; and a positive lens for deflecting the beam emitted by the microlens array onto the dichroic mirror.

[0007] In one embodiment of the present invention, the microlens array includes: a first microlens array for deflecting the parallel beam emitted by the collimating lens; and a second microlens array for appropriately reducing the angle of the beam emitted by the first microlens array before projecting it onto the positive lens.

[0008] In one embodiment of the present invention, it further includes: an excitation filter for filtering light in the non-excitation band; and an emission filter for filtering interfering light in the fluorescence emitted by the microcavity array of the sample plane transmitted through the dichroic mirror.

[0009] In one embodiment of the present invention, it further includes: a large field-of-view aberration-correcting lens group, used to eliminate aberrations generated by large field-of-view imaging.

[0010] In one embodiment of the present invention, the large field-of-view aberration-correcting lens group includes two sets of positive lenses and a negative lens.

[0011] The present invention also provides a large field-of-view fluorescence microcavity array imaging method for digital PCR, wherein a laser beam is emitted by a laser; the laser beam is homogenized to obtain a uniform excitation beam; the uniform excitation beam emitted through the microarray lens group is reflected onto the microcavity array of the sample plane, and the fluorescence beam emitted by the microcavity array of the sample plane is transmitted into the subsequent optical path; and the microcavity array of the field of view is imaged.

[0012] In one embodiment of the present invention, the homogenization process of the laser beam emitted by the laser to obtain a uniform excitation beam includes: collimating the laser beam emitted by the laser into parallel light; focusing and diverging the parallel beam; and deflecting the focused and diverged parallel beam to obtain a uniform beam.

[0013] In one embodiment of the present invention, the method further includes: filtering light in the non-excitation band; and filtering interfering light in the fluorescence emitted by the microcavity array of the sample plane transmitted through the dichroic mirror.

[0014] In one embodiment of the present invention, the aberrations generated by large field-of-view imaging are eliminated by a large field-of-view aberration-correcting lens group.

[0015] As described above, the large field-of-view fluorescence microcavity array imaging system and method for digital PCR of the present invention have the following beneficial effects:

[0016] 1. The microlens array and large field-of-view fluorescence imaging technology used in this invention can achieve a faster detection rate without the need for complex and time-consuming image stitching post-processing. Compared with the abnormal signals that may be generated by traditional small field-of-view fluorescence imaging and image stitching technology, this invention can ensure that the fluorescence signal in the test area is normal and the detection accuracy is higher.

[0017] 2. This invention has a higher degree of miniaturization and intelligence. It can be connected to a portable computer and use self-written software to automatically detect and analyze fluorescence images, which meets the urgent need for better nucleic acid testing equipment under the current global pandemic situation and has good application prospects. Attached Figure Description

[0018] Figure 1 The diagram shown is a schematic diagram of the large field-of-view fluorescence microcavity array imaging system for digital PCR in this invention.

[0019] Figure 2 The diagram shown is a specific structural schematic of a large field-of-view fluorescence microcavity array imaging system for digital PCR in this invention.

[0020] Figure 3 The diagram shows a schematic of the microarray lens group in the large field-of-view fluorescence microcavity array imaging system for digital PCR in this invention.

[0021] Figure 4 The diagram shows a microcavity array in the sample plane of the large field-of-view fluorescence microcavity array imaging system for digital PCR in this invention.

[0022] Figure 5 The diagram shows the structure of the aberration-correcting lens group in the large field-of-view fluorescence microcavity array imaging system for digital PCR in this invention.

[0023] Figure 6 The diagram shows a flowchart of the large field-of-view fluorescence microcavity array imaging method for digital PCR in this invention.

[0024] Component designation explanation

[0025] 100 Large Field-of-View Fluorescence Microcavity Array Imaging System for Digital PCR

[0026] 110 laser

[0027] 120 microarray lens group

[0028] 121 Collimating Lens

[0029] 122 First Microlens Array

[0030] 123 Second Microlens Array

[0031] 124 Positive Lens

[0032] 130 Dichroic Mirror

[0033] 140 reflector

[0034] 150 Excitation Filter

[0035] 160 Emission Filter

[0036] 170° wide field-of-view aberration-correcting lens group

[0037] Microcavity array with 200 sample planes

[0038] 300 CMOS camera Detailed Implementation

[0039] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0040] Please see Figures 1 to 6 It should be understood that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to facilitate understanding and reading. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to size, without affecting the effectiveness and purpose of the invention, should still fall within the scope of the technical content disclosed in this invention. Furthermore, the terms such as "upper," "lower," "left," "right," "middle," and "one" used in this specification are merely for clarity and not intended to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention's implementation.

[0041] The purpose of this embodiment is to provide a large field-of-view fluorescence microcavity array imaging system and method for digital PCR, which solves the technical problems of long detection time and low detection accuracy in the prior art of digital PCR.

[0042] The large field-of-view fluorescence microcavity array imaging system and method for digital PCR provided in this embodiment can achieve high-resolution imaging of microcavity arrays or microdroplets with a field of view of about 2cm*2cm, which greatly accelerates the detection rate and improves the detection accuracy.

[0043] The following will describe in detail the principle and implementation of a large field-of-view fluorescence microcavity array imaging system and method for digital PCR in this embodiment, so that those skilled in the art can understand the large field-of-view fluorescence microcavity array imaging system and method for digital PCR in this embodiment without creative effort.

[0044] Example 1

[0045] like Figure 1As shown, this embodiment provides a large field-of-view fluorescence microcavity array imaging system 100 for digital PCR, which includes at least: a laser 110, a microarray lens group 120, a dichroic mirror 130, and an imaging mirror.

[0046] In this embodiment, the laser 110 serves as a light source to generate laser light, that is, the laser 110 emits a laser beam.

[0047] In terms of the light source, the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR in this embodiment selects a laser as the excitation source in order to obtain a good excitation beam and sufficient illumination intensity. The laser source has good singleness and high energy density.

[0048] In this embodiment, the microarray lens group 120 homogenizes the beam emitted by the laser 110 to obtain a uniform excitation beam.

[0049] In other words, in this embodiment, a microlens array is used to achieve uniform illumination. After calibration by the microlens array, a highly uniform light field distribution can be obtained on the microcavity array 200 of the sample plane, thereby ensuring the consistency of fluorescence excitation conditions of all microcavities or microdroplets in the sample plane and ensuring detection accuracy.

[0050] Specifically, in this embodiment, as Figure 2 and Figure 3 As shown, the microarray lens group 120 includes: a collimating lens 121, a microlens array, and a positive lens 124.

[0051] In this embodiment, the collimating lens 121 collimates the laser beam emitted by the laser 110 into parallel light.

[0052] In this embodiment, the microlens array converges and diverges the parallel beam emitted by the collimating lens 121.

[0053] In this embodiment, the microlens array includes a first microlens array 122 and a second microlens array 123.

[0054] Specifically, the first microlens array 122 deflects the parallel beam emitted by the collimating lens 121, and the second microlens array 123 appropriately reduces the angle of the beam emitted by the first microlens array 122 before transmitting it to the positive lens 124.

[0055] That is, each microlens in the first microlens array 122 converges and diverges light in the same way, and the second microlens array 123 appropriately contracts the light generated by the first microlens array 122.

[0056] A larger field of view inevitably leads to more pronounced aberrations. Aberrations proportional to the field of view include coma, field curvature, astigmatism, and distortion. This embodiment uses aberration-correcting lenses to form the image, reducing aberrations to a level permissible in terms of imaging quality, thereby ensuring that the edges of the area under test can be imaged normally.

[0057] In this embodiment, the positive lens 124 deflects the light beam emitted by the microlens array onto the dichroic mirror 130.

[0058] In this embodiment, the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR further includes an excitation filter 150, which filters light in the non-excitation band.

[0059] Through the collimating lens 121, the microlens array, the positive lens 124, and the excitation filter 150, highly uniform light can be obtained on the microcavity array 200 of the sample plane.

[0060] In this embodiment, as Figure 4 As shown, the dichroic mirror 130 reflects the uniform excitation beam emitted from the microarray lens group 120 onto the microcavity array 200 of the sample plane, and transmits the fluorescence beam emitted from the microcavity array 200 of the sample plane into the subsequent optical path.

[0061] In this embodiment, the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR further includes an emission filter 160. The emission filter 160 filters out interfering light from the fluorescence emitted by the microcavity array 200 of the sample plane transmitted through the dichroic mirror 130.

[0062] In this embodiment, the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR further includes a large field-of-view aberration-correcting lens group 170, which images the microcavity array within the field of view and eliminates aberrations generated by large field-of-view imaging. Figure 5 This diagram illustrates the structure of the large field-of-view aberration-correcting lens group 170 in the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR in this embodiment. The large field-of-view aberration-correcting lens group eliminates, to a certain extent, various aberrations that are easily generated in large field-of-view imaging (especially at the edges of the field of view). In this embodiment, the large field-of-view aberration-correcting lens group 170 includes two sets of positive lenses 124 and a negative lens, which is the result of algorithm optimization. The large field-of-view aberration-correcting lens group 170 is used to eliminate aberrations that are easily generated at the edges of the field of view in conventional lens imaging, ensuring the imaging quality of the microcavity or microdroplet.

[0063] In this embodiment, the imaging mirror images the microcavity array within the field of view.

[0064] Specifically, the imaging mirror includes a reflector 140, which reflects the fluorescence of the microcavity array 200 on the sample plane transmitted by the dichroic mirror 130 to a CMOS camera 300 for receiving the fluorescence signal and converting it into an image. The CMOS camera 300 acquires the fluorescence image of the microcavity array 200 on the sample plane. The fluorescence image acquired by the CMOS camera 300 is then imported into preset analysis software for image processing, microcavity droplet identification, and statistical analysis to obtain accurate detection results.

[0065] In this embodiment, the operation of the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR is as follows:

[0066] Light is emitted by laser 110, homogenized by microlens array, and filtered out by emission filter 160 to remove non-excitation wavelengths. The light is then reflected by dichroic mirror 130 to reach microcavity array 200 on the sample plane. Fluorescence emitted from thousands of microcavities on the sample plane is transmitted through dichroic mirror 130, filtered out by emission filter 160 to remove interference signals, and finally reaches CMOS camera 300 through large field-of-view aberration-correcting lens group 170 and reflector 140, thereby obtaining a high-resolution large field-of-view fluorescence image.

[0067] Compared with existing traditional fluorescence imaging systems that require multiple image stitching, the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR described in this embodiment has the following advantages:

[0068] 1) Faster detection speed, eliminating the need for complex and time-consuming image stitching post-processing;

[0069] 2) Higher detection accuracy: Compared with the abnormal signals that may be generated by traditional image stitching technology, the microlens array group and large field of view fluorescence imaging technology we use can ensure that the fluorescence signal in the test area is normal.

[0070] 3) With a higher degree of miniaturization and intelligence, our system can be connected to a portable computer and use self-written software to automatically detect and analyze fluorescence images.

[0071] In summary, the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR described in this embodiment meets the urgent need for better nucleic acid testing equipment under the current global pandemic situation and has good application prospects.

[0072] Example 2

[0073] like Figure 6 As shown, this embodiment provides a large field-of-view fluorescence microcavity array imaging method for digital PCR, which includes:

[0074] Step S100: A laser beam is emitted by a laser device;

[0075] Step S200: The beam emitted by the laser is homogenized to obtain a uniform excitation beam.

[0076] Step S300: The uniform excitation beam emitted through the microarray lens group is reflected onto the microcavity array of the sample plane, and the fluorescence beam emitted by the microcavity array of the sample plane is transmitted into the subsequent optical path.

[0077] Step S400: Image the microcavity array within the field of view.

[0078] The following provides a detailed description of steps S100 to S400 of the large field-of-view fluorescence microcavity array imaging method for digital PCR in this embodiment.

[0079] Step S100: A laser beam is emitted by a laser.

[0080] In terms of the light source, the large field-of-view fluorescence microcavity array imaging method for digital PCR in this embodiment selects laser as the excitation source in order to obtain a good excitation beam and sufficient illumination intensity. Laser sources have good singleness and high energy density.

[0081] Step S200: The beam emitted by the laser is homogenized to obtain a uniform excitation beam.

[0082] In this embodiment, the laser beam emitted by the laser 110 is homogenized by the microarray lens group 120 to obtain a uniform excitation beam.

[0083] In other words, in this embodiment, a microlens array is used to achieve uniform illumination. After calibration by the microlens array, a highly uniform light field distribution can be obtained on the microcavity array 200 of the sample plane, thereby ensuring the consistency of fluorescence excitation conditions of all microcavities or microdroplets in the sample plane and ensuring detection accuracy.

[0084] Specifically, in this embodiment, the homogenization process of the laser beam emitted by the laser to obtain a uniform excitation beam includes:

[0085] 1) Collimate the laser beam emitted by the laser 110 into parallel light.

[0086] The laser beam emitted by the laser 110 is collimated into parallel light by a collimating lens 121.

[0087] 2) Focus and diverge the parallel beam.

[0088] The collimating lens 121 is focused and diffused by a microlens array.

[0089] In this embodiment, the microlens array includes a first microlens array 122 and a second microlens array 123.

[0090] Specifically, the first microlens array 122 deflects the parallel beam emitted by the collimating lens 121, and the second microlens array 123 appropriately reduces the angle of the beam emitted by the first microlens array 122 before transmitting it to the positive lens 124.

[0091] That is, each microlens in the first microlens array 122 converges and diverges light in the same way, and the second microlens array 123 appropriately contracts the light generated by the first microlens array 122.

[0092] A larger field of view inevitably leads to more pronounced aberrations. Aberrations proportional to the field of view include coma, field curvature, astigmatism, and distortion. This embodiment uses multi-lens combination imaging to reduce aberrations to a level permissible in terms of image quality, thereby ensuring that the edges of the area under test can be imaged normally.

[0093] 3) Deflect the converged and diverged parallel beams to obtain a uniform beam.

[0094] The beam emitted by the microlens array is deflected by the positive lens 124 to obtain a uniform beam.

[0095] In this embodiment, the method further includes filtering light in the non-excitation band. Specifically, an excitation filter 150 is used to filter light in the non-excitation band.

[0096] Through the collimating lens 121, the microlens array, the positive lens 124, and the excitation filter 150, highly uniform light can be obtained on the microcavity array 200 of the sample plane.

[0097] In step S300, the uniform excitation beam emitted through the microarray lens group is reflected onto the microcavity array of the sample plane, and the fluorescence beam emitted by the microcavity array of the sample plane is transmitted into the subsequent optical path.

[0098] In this embodiment, a dichroic mirror 130 reflects the uniform light beam emitted by the microarray lens group 120 onto the microcavity array 200 of the sample plane, and transmits the fluorescence emitted by the microcavity array 200 of the sample plane.

[0099] In this embodiment, it further includes filtering interfering light from the fluorescence emitted by the microcavity array 200 of the sample plane.

[0100] Specifically, an emission filter 160 filters out interfering light from the fluorescence emitted by the microcavity array 200 of the sample plane transmitted through the dichroic mirror 130.

[0101] In this embodiment, aberrations generated by large field-of-view imaging are also eliminated by a large field-of-view aberration-eliminating lens group 170. Figure 5 This diagram illustrates the structure of the large field-of-view aberration-correcting lens group 170 in the large field-of-view fluorescence microcavity array imaging system 100 for digital PCR in this embodiment. The large field-of-view aberration-correcting lens group 170 is used to eliminate aberrations that are easily generated at the edges of the imaging field of conventional lenses, ensuring the imaging quality of microcavities or microdroplets.

[0102] Step S400: Image the microcavity array within the field of view.

[0103] Specifically, in this embodiment, a reflector 140 reflects the fluorescence of the microcavity array 200 of the sample plane transmitted by the dichroic mirror 130 to a CMOS camera 300, which then acquires a fluorescence image of the microcavity array 200 of the sample plane. The fluorescence image acquired by the CMOS camera 300 is then imported into preset analysis software for image processing, microcavity or droplet identification, and statistical analysis, thereby obtaining accurate detection results.

[0104] In this embodiment, the specific procedure of the large field-of-view fluorescence microcavity array imaging method for digital PCR is as follows:

[0105] Laser 110 generates an excitation beam as a light source. The microlens array homogenizes the beam, and excitation filter 150 filters out light in the non-excitation band. The light is then reflected by dichroic mirror 130 to reach the microcavity array 200 on the sample plane. The fluorescence emitted from thousands of microcavities on the sample plane is transmitted through dichroic mirror 130, and then filtered out by emission filter 160 to remove interference signals. Finally, it reaches CMOS camera 300 through large field-of-view aberration-correcting lens group 170 and reflector 140, thereby obtaining a high-resolution large field-of-view fluorescence image.

[0106] In summary, the microlens array and large field-of-view fluorescence imaging technology employed in this invention achieve faster detection rates without the need for complex and time-consuming image stitching post-processing. Compared to the potential for abnormal signals generated by traditional small field-of-view fluorescence imaging and image stitching techniques, this invention ensures normal fluorescence signals in the test area, resulting in higher detection accuracy. Furthermore, this invention boasts a higher degree of miniaturization and intelligence, allowing connection to a portable computer and the use of self-developed software for automatic fluorescence image detection and analysis. This meets the urgent need for better nucleic acid testing equipment in the current global pandemic situation and has promising application prospects. Therefore, this invention effectively overcomes the shortcomings of existing technologies and possesses significant industrial application value.

[0107] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A large field-of-view fluorescence microcavity array imaging system for digital PCR, characterized in that: At least including: A laser, as the light source, generates laser light; A micro-array lens group homogenizes the laser beam emitted by the laser to obtain a uniform excitation beam. The micro-array lens group includes: a collimating lens to collimate the laser beam emitted by the laser into parallel light; a microlens array to converge and diverge the parallel beam emitted by the collimating lens; and a positive lens to deflect the beam emitted by the microlens array to a dichroic mirror. The microlens array includes: a first microlens array to deflect the parallel beam emitted by the collimating lens; and a second microlens array to appropriately reduce the angle of the beam emitted by the first microlens array before projecting it onto the positive lens. A dichroic mirror reflects the uniform excitation beam emitted from the microarray lens group onto the microcavity array of the sample plane, and transmits the fluorescence beam emitted from the microcavity array of the sample plane into the subsequent optical path. A large field-of-view aberration-correcting lens group is used to image a microcavity array within a wide field of view and eliminate aberrations generated by large field-of-view imaging; the large field-of-view aberration-correcting lens group includes two sets of positive lenses and a negative lens; A CMOS camera is used to receive fluorescence signals and convert them into images.

2. The large field-of-view fluorescence microcavity array imaging system for digital PCR according to claim 1, characterized in that: Also includes: Excitation filters filter out light in non-excitation wavelengths; An emission filter filters out interfering light from the fluorescence emitted by the microcavity array of the sample plane transmitted through the dichroic mirror.

3. A large field-of-view fluorescence microcavity array imaging method for digital PCR, applied to the large field-of-view fluorescence microcavity array imaging system for digital PCR as described in any one of claims 1 to 2, characterized in that: A laser beam is emitted by a laser. The laser beam emitted by the laser is homogenized to obtain a uniform excitation beam. The uniform excitation beam emitted through the microarray lens group is reflected onto the microcavity array of the sample plane, and the fluorescence beam emitted by the microcavity array of the sample plane is transmitted into the subsequent optical path. Imaging of a microcavity array within its field of view.

4. The large field-of-view fluorescence microcavity array imaging method for digital PCR according to claim 3, characterized in that: Homogenizing the laser beam emitted by the laser to obtain a uniform excitation beam includes: The laser beam emitted by the laser is collimated into parallel light; Focusing and diverging the parallel beam; The converged and diverged parallel beams are deflected to obtain a uniform beam.

5. The large field-of-view fluorescence microcavity array imaging method for digital PCR according to claim 4, characterized in that: Also includes: Filter light in the non-excitation band; The interfering light emitted from the fluorescence transmitted through the dichroic mirror onto the sample plane by the microcavity array is filtered out.

6. The large field-of-view fluorescence microcavity array imaging method for digital PCR according to claim 3, characterized in that: It also includes eliminating aberrations generated by large field-of-view imaging through a large field-of-view aberration-correcting lens group.

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