CN117405635A - dPCR film reading system - Google Patents

Abstract

The application provides a piece system is read to dPCR, the piece system is read to dPCR includes image acquisition subsystem (1) and light source subsystem (2), wherein, image acquisition subsystem (1) includes microscope and liquid lens, the visual field of microscope is less than the area of waiting to detect micro-droplet area in the biochip, light source subsystem (2) set up as the bypass of image acquisition subsystem (1), be convenient for realize the switching of multi-wavelength light source, and, make the radiation scope of light source subsystem (2) with the visual field of microscope is equivalent or slightly less than the visual field of microscope, liquid lens can realize automatic focusing to improve and solve the problem that microscope focuses inaccurately in the continuous removal in-process.

Description

dPCR film reading system

Technical Field

The application belongs to the field of biological detection equipment, and particularly relates to a dPCR film reading system.

Background

Digital PCR (dPCR) is an absolute quantitative technique of nucleic acid molecules, which can directly measure the number of DNA molecules based on microdroplets, and is an absolute quantification of a starting sample, compared with real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR). The basic principle of digital PCR is that a sample is divided into tens to tens of thousands of parts, and then the parts are distributed into different reaction units, so that each micro-droplet contains one or more copies of nucleic acid molecules (namely DNA templates), each micro-droplet can amplify target molecules, and then the statistics and calculation of fluorescent signals are carried out on each micro-droplet.

In the prior art, in the process of quantitatively analyzing a micro-droplet area, an image of the micro-droplet area is acquired, and then the ratio of fluorescent micro-droplets in the total amount of the micro-droplets is acquired through image processing, so that quantitative analysis is performed on target DNA.

Generally, the field of view of the image acquisition subsystem (1) for acquiring the image of the micro-droplet area covers the whole area of the micro-droplet area, and under the condition that the sizes of pictures are the same, the diameter of each micro-droplet unit occupies too few pixels in an imaging target surface, so that the number of micro-droplets is difficult to accurately identify, and the accuracy of quantitative analysis is low.

Disclosure of Invention

In order to solve the problems existing in the prior art, the application provides a dPCR film reading system, the film reading system comprises an image acquisition subsystem 1 and a light source subsystem 2, wherein the image acquisition subsystem 1 comprises a micro lens and a liquid lens, the field of view of the micro lens is smaller than the area of a micro-droplet area in a biochip to be detected, thereby improving the resolution of the acquired image and further improving the accuracy of quantitative analysis, however, if the area to be detected is not acquired in time after being excited by fluorescence, the area to be detected is subjected to fluorescence bleaching, the fluorescence bleaching is a phenomenon that a fluorescent group loses fluorescence due to damage caused by light, once fluorescence bleaching occurs, the fluorescent signal loss of a sample is caused, further the detection accuracy is reduced, in order to solve the derivative problem, the application sets the light source subsystem 2 as a bypass of the image acquisition subsystem 1, and enables the radiation range of the light source subsystem 2 to be equal to or slightly smaller than the field of view of the micro lens, thereby enabling the micro-droplet area to be detected to be only excited by the micro lens, and the fluorescence area to be excited by the micro lens to be greatly matched with the excitation area to be detected; further, in the continuous moving process of the microscope lens, the object distance may exceed the depth of field to cause focusing inaccuracy, and in order to solve the derivative problem, the front end of the microscope lens is provided with the liquid lens, so that automatic focusing is realized, and the problem that the microscope lens is not focused in the continuous moving process is solved.

An object of the present application is to provide a dPCR film reading system, the dPCR film reading system includes an image acquisition subsystem 1 and a light source subsystem 2, wherein, the image acquisition subsystem 1 includes a camera 11, a liquid lens 12, a micro lens 13 and a lens switchable light filter module 14 that coaxially set up in proper order, the light source subsystem 2 includes a light source 21, a beam shaping module 22 and a light source switchable light filter module 23 that coaxially set up in proper order, the optical axis of the light source subsystem 2 with the optical axis of the image acquisition subsystem 1 does not coincide, and, intersect in the image acquisition area of waiting to detect biochip, the field of view of the micro lens 13 is less than the area of the micro droplet area in waiting to detect biochip.

In one possible way, the field of view of the micro-lens 13 is small, typically only one tenth, or even one hundredth, of the area of the micro-droplet area in the biochip to be tested.

In one implementation, the lens switchable filter module 14 matches the filter wavelengths of the light source switchable filter module 23.

In one possible way, the field of view of the camera 11 is equal to or slightly larger than the radiation range of the light source 21.

In one implementation, the dPCR film reading system further includes a biochip moving platform 3 for carrying a biochip, where a moving plane of the biochip moving platform 3 is perpendicular to an optical axis of the camera 11, and can move in a plane where the biochip is located and can reciprocate in a direction perpendicular to the biochip plane.

The applicant finds that the biochip has a simple structure and a small volume, and the volume of a platform used for carrying the biochip is correspondingly small, so that moving the biochip to be detected can reduce the whole volume of the dPCR film reading system and is convenient to operate.

In one implementation, the lens switchable filter module 14 includes at least two wavelength filters, the light source switchable filter module 23 includes at least two wavelength filters, and the filter wavelength of the filter in the light source switchable filter module 23 corresponds to the filter wavelength of the filter in the lens switchable filter module 14, so that the lens switchable filter module 14 and the light source switchable filter module 23 can synchronously obtain monochromatic light with corresponding wavelengths.

In one implementation, the lens switchable filter module comprises a rotatable filter stage rotatable about its own central axis, optionally provided with a plurality of adjustment gears, each corresponding to one filter.

Further, the structure of the light source switchable filter module is similar to or the same as that of the lens switchable filter module.

In one realisable way, a beam shaping module 22 for beam collimation is also provided between the light source 21 and the light source switchable filter module 23.

Compared with the prior art, the dPCR film reading system provided by the application adopts the microscope lens to collect the image of the to-be-detected area of the to-be-detected biochip, the view field range of the microscope lens is smaller than the area of the to-be-detected biochip, the object space resolution of the system is improved, the detection precision is improved, the by-pass light source is used for irradiating the to-be-detected area, the irradiation range of the light source is equal to or smaller than the view field range of the microscope lens, and the front end of the microscope lens is provided with the liquid lens, so that the problem of inaccurate focusing caused by exceeding of the depth of field of the microscope lens by the unavoidable ground object distance of the biochip in the continuous moving process is solved, and further, the application is provided with the lens switchable light filtering module and the light source switchable light filtering module respectively at the front end of the liquid lens, so that the switching of the multi-wavelength excitation light source is realized, and the simultaneous detection of different marks in the same biochip is realized.

Drawings

FIG. 1 shows a schematic diagram of a preferred dPCR read system of the present application.

Description of the reference numerals

The device comprises a 1-image acquisition subsystem, a 11-camera, a 12-liquid lens, a 13-microscope lens, a 14-lens switchable filter module, a 141-rotatable filter carrier, a 2-light source subsystem, a 21-light source, a 22-light beam shaping module, a 23-light source switchable filter module and a 3-biochip moving platform.

Detailed Description

Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples do not represent all implementations consistent with the invention. Rather, they are merely examples of methods consistent with aspects of the invention as detailed in the accompanying claims.

The following describes in detail the structure of the dPCR slide reading system provided herein and its method of use by way of specific examples.

First, a brief description will be given of a usage scenario of the present solution.

The dPCR slide system provided herein is used to count partition units in a biochip. In particular, the counting object comprises a partition unit displaying fluorescence and a partition unit not displaying fluorescence. The amount of target DNA in the original sample is quantified by calculating and displaying information such as the number proportion of fluorescent partition units.

A biochip for implementing dPCR is generally provided with a microdroplet region in which a large number of microdroplets with almost uniform volumes are distributed, each microdroplet being a partition unit, which can be generated on line by a mobile phase and a dispersed phase, for example, can be a water-in-oil microdroplet, the volume of each microdroplet can be 1nL, and each partition unit may contain a target DNA fragment, i.e., a reaction substrate of digital PCR reacts independently in each partition unit during PCR reaction, without interfering with each other, and if the partition unit contains a target DNA fragment, the partition unit emits fluorescence when irradiated by an excitation beam after PCR reaction is completed, and is denoted as a positive droplet; and the partition units which do not contain target DNA fragments do not emit fluorescence after the PCR reaction is completed and are irradiated by the excitation light beam, the partition units are marked as negative liquid drops, and the sum of the number of positive liquid drops and the number of negative liquid drops is the total number of micro liquid drops in the biochip, namely the total number of partition units in the biochip.

Fig. 1 shows a schematic structural diagram of a dPCR film reading system according to the present application, as shown in fig. 1, where the dPCR film reading system includes an image acquisition subsystem 1 and a light source subsystem 2, and the light source subsystem 2 is disposed at a bypass of the image acquisition subsystem 1, that is, an optical axis of the light source subsystem 2 is neither parallel nor coincident with an optical axis of the image acquisition subsystem 1.

In this example, a preferable scheme is that the optical axis of the light source subsystem 2 and the optical axis of the image acquisition subsystem 1 intersect in an image acquisition area of the biochip to be detected.

It will be appreciated that in a typical dPCR assay, the image acquisition area of the biochip to be tested is located in the microdroplet area of the biochip, but in individual cases the image acquisition area of the biochip to be tested may also be located beyond the microdroplet area of the biochip and in other areas of the biochip to be tested.

As further shown in fig. 1, the image acquisition subsystem 1 includes a camera 11, a liquid lens 12, a micro lens 13, and a lens switchable filter module 14, which are coaxially arranged in order.

The camera 11 is not particularly limited in this application, and any one of the cameras available for image acquisition in the related art may be used.

In this example, the micro lens 13 makes each partition unit diameter in the camera field of view occupy about 20 pixels in the imaging target surface, so as to realize high-definition shooting of the micro droplet area in the biochip to be detected.

It will be appreciated that the field of view of the micro-lens 13 is small compared to a conventional imaging lens, typically only one tenth, or even one hundredth, of the area of the micro-droplet area in the biochip to be tested, and therefore, the use of the micro-lens as an imaging lens requires that the micro-lens be continuously moved in the micro-droplet area in the biochip to be tested to obtain a finished image of the micro-droplet area.

Further, compared with the biochip, the precision of the optical device is high, and frequent movement of the optical device easily causes the consequences of reduced precision of the optical device, and the like, so that the biochip to be detected is selectively moved, and the loss of the optical device is reduced.

Furthermore, the biochip has simple structure and small volume, and the volume of the platform used for carrying the biochip is correspondingly smaller, so that the biochip to be detected is moved instead of the optical device, the whole volume of the dPCR film reading system can be minimized, and the operation is convenient.

As mentioned before, the dPCR film reading system provided by the present application further includes a biochip moving platform 3 for carrying a biochip, where a moving plane of the biochip moving platform 3 is perpendicular to an optical axis of the camera 11, and can move in a plane where the biochip is located, so that the biochip can move under the driving of the biochip moving platform 3, and further, the biochip moving platform 3 can also reciprocate along a direction perpendicular to the biochip plane, so that the biochip can also be vertically close to or far away from the image acquisition subsystem 1 under the driving of the biochip moving platform 3.

It will be appreciated that the biochip to be tested may itself have quality defects, e.g. the surface of its microdroplet region is not flat; or the surface of the micro-droplet area is not parallel to the movable plane of the movable platform, so that the distance between the surface of the micro-droplet area and the micro-lens of the biochip to be detected can be changed in the continuous moving process of the movable platform; still alternatively, the movable plane of the movable platform is not perpendicular to the optical axis of the lens, so that it is difficult to ensure the parallelism between the biochip to be detected and the microscope lens 13 during the movement of the biochip to be detected, especially for the case that the micro-droplet area is far greater than the field of view of the microscope lens, the parallelism between the two is more difficult to ensure.

Further, the depth of field of the micro-lens 13 is generally smaller, and there is a stricter limit on the imaging distance, so that in the case that the parallelism between the biochip to be detected and the micro-lens 13 is difficult to ensure, the distance between the biochip to be detected and the micro-lens 13 is easy to exceed the depth of field, thereby causing inaccurate focusing, in the case, the acquired image is blurred, each partition unit cannot be identified, and finally quantitative analysis cannot be performed.

Based on this, the liquid lens 12 is disposed at the front end of the micro lens 13, and the liquid lens 12 is not particularly limited in this application, and any liquid lens available in the prior art may be used.

In the present application, the liquid lens can adjust the focal plane of the micro lens 13 by adjusting the curvature of the lens, so as to realize an auto-focusing function.

In this application, the front end of the liquid lens is further provided with a lens switchable filter module 14, the lens switchable filter module 14 includes a rotatable filter stage 141, the rotatable filter stage 141 may rotate around its own central axis, the rotatable filter stage 141 is provided with a plurality of adjustment gears, and each adjustment gear corresponds to one filter, so that the lens switchable filter module 14 may select a filter with a proper filter wavelength according to needs.

In the present application, the field of view of the camera 11 is equal to or slightly larger than the radiation range of the light source 21, so as to avoid fluorescence bleaching caused by the advanced excitation of the partition units, thereby improving the accuracy of quantification.

As further shown in fig. 1, in the present application, the light source subsystem 2 includes a light source 21, a beam shaping module 22, and a light source switchable filter module 23 coaxially arranged in this order.

In this application, the light emitted by the light source 21 is preferably white light, so that the filter in the light source switchable filter module 23 can be used to screen out monochromatic light with a target wavelength.

In this application, the light source switchable filter module 23 includes filters with at least two wavelengths, it is understood that the number of filters in the light source switchable filter module 23 is equal to the number of filters in the lens switchable filter module 14, and the filter wavelengths of the filters in the light source switchable filter module 23 are in one-to-one correspondence with the filter wavelengths of the filters in the lens switchable filter module 14, for example, the excitation wavelength of the maximum excitation efficiency of FAM is 493nm, the wavelength of the maximum light intensity detected is 519nm, the light source switchable filter module includes a filter with a filter wavelength of 470±25nm, and the lens switchable filter module includes a filter with a filter wavelength of 520±20 nm.

Further, the light source switchable filter module 23 is the same as or similar to the structure of the lens switchable filter module 14.

In the present application, a beam shaping module 22 is arranged between the light source 21 and the light source switchable filter module 23, the beam shaping module 22 being configured to achieve beam collimation.

The structure of the beam shaping module 22 is not particularly limited in this application, and any beam shaping module applicable to the scene described in this application in any of the prior art may be used.

The following describes the use of the dPCR reader system provided herein, taking the dPCR reader system illustrated in fig. 1 as an example.

Adding a sample into a biochip to be detected, placing the biochip after the sample addition on a biochip moving platform of the biochip to be detected, moving the biochip moving platform to drive the biochip to be detected to move to a preset position, and processing according to a preset program, so that a large number of micro droplets are distributed in a micro droplet area in the biochip to be detected, and each micro droplet relatively and independently completes a PCR reaction, and starting the dPCR film reading system provided by the application after the PCR reaction is completed, specifically:

and moving the micro-droplet area of the biochip to be detected to the front end of the image acquisition subsystem 1, and adjusting the rotatable optical filtering device of the lens and the rotatable optical filtering device of the light source to enable the optical wavelengths filtered by the two optical filtering devices to be matched, so that the micro-droplet area can be clearly imaged in the image acquisition subsystem 1. And continuously moving the biochip to be detected according to a preset time interval and a preset distance interval, so that the image acquisition subsystem 1 traverses the whole range of the micro-droplet area, and therefore the images acquired by the image acquisition subsystem 1 are spliced, and the finished image of the micro-droplet area can be obtained.

It can be appreciated that in moving the biochip to be tested, the micro-droplet area is not coincident with the focal plane of the microscope lens, however, the liquid lens can adjust the imaging position of the micro-droplet area to be located on the imaging plane of the camera, thereby realizing automatic focusing of the micro-droplet area.

Further, the light spot range irradiated by the light source subsystem 2 is just equal to or slightly smaller than the field of view range of the image acquisition subsystem 1, so that the micro-droplet area cannot be excited to fluoresce in a large area in the image acquisition process, and inaccurate quantification caused by the bleaching phenomenon generated by over excitation of the micro-droplet is avoided.

The foregoing detailed description has been provided for the purposes of illustration in connection with specific embodiments and exemplary examples, but such description is not to be construed as limiting the application. Those skilled in the art will appreciate that various equivalent substitutions, modifications and improvements may be made to the technical solution of the present application and its embodiments without departing from the spirit and scope of the present application, and these all fall within the scope of the present application. The scope of the application is defined by the appended claims.

Claims (10)

1. The dPCR film reading system is used for collecting images of micro-droplet areas in a biochip, and comprises an image collecting subsystem (1) and a light source subsystem (2), wherein the image collecting subsystem (1) comprises a camera (11), a liquid lens (12), a micro-lens (13) and a lens switchable light filtering module (14) which are coaxially arranged in sequence, the light source subsystem (2) comprises a light source (21), a beam shaping module (22) and a light source switchable light filtering module (23) which are coaxially arranged in sequence, the optical axis of the light source subsystem (2) is not coincident with the optical axis of the image collecting subsystem (1), and the light source subsystem is intersected with the image collecting area of the biochip to be detected, and the field of view of the micro-lens (13) is smaller than the area of the micro-droplet areas in the biochip to be detected.

2. The dPCR slide reading system according to claim 1, characterized in that the field of view of the microscope (13) is less than one tenth of the area of the micro-droplet area in the biochip to be detected.

3. The dPCR film reading system according to claim 1 or 2 wherein the lens switchable filter module (14) matches the filter wavelength of the light source switchable filter module (23).

4. A dPCR slide reading system according to any one of claims 1 to 3, characterized in that the field of view of the camera (11) is equal to or slightly larger than the radiation range of the light source (21).

5. The dPCR slide reading system according to any one of claims 1 to 4, further comprising a biochip moving stage (3) for carrying a biochip, the moving plane of the biochip moving stage (3) being perpendicular to the optical axis of the camera (11), being movable in the plane of the biochip and being reciprocally movable in a direction perpendicular to the biochip plane.

6. The dPCR slide viewing system according to any one of claims 1 to 5, wherein the lens-switchable filter module (14) comprises filters of at least two wavelengths, the light source-switchable filter module (23) comprises filters of at least two wavelengths, and the filter wavelengths of the filters in the light source-switchable filter module (23) correspond to the filter wavelengths of the filters in the lens-switchable filter module (14).

7. The dPCR slide reading system according to any one of claims 1 to 6 wherein the lens switchable filter module (14) includes a rotatable filter stage (141) rotatable about its own central axis.

8. The dPCR slide reading system of claim 7, wherein the rotatable filter stage (141) is provided with a plurality of adjustment gears, each adjustment gear corresponding to a piece of filter.

9. The dPCR slide reading system according to any one of claims 1 to 8 wherein the light source switchable filter module (23) is the same as or similar in structure to the lens switchable filter module (14).

10. The dPCR slide reading system according to any one of claims 1 to 9, characterized in that a beam shaping module (22) for beam collimation is further provided between the light source (21) and the light source switchable filter module (23).