Patent Description:
At present, a lot of assays for complex samples of macromolecular usually include two steps. In the first step, molecules that can capture specific target macromolecules are attached to a surface of solid phase. Such attached molecules can capture the target macromolecules from a complex sample in various ways such as hybridization (e.g., in assays based on DNA, RNA) or interactions between antigens and antibodies (in immunoassays). In the second step, molecules required for the detection (hereinafter, "detection molecules") are incubated with and bonded to a compound of the capture molecules and the target, thereby emitting signals such as fluorescent or electromagnetic signals. The target is then quantified based on an intensity of the signals.

Multiplex detection can be carried out by a variety of capture reagents that are specific for different target macromolecules. In a chip-based array multiplex detection, each type of capture reagent is attached to a predetermined position on the chip. Multiple targets in the complex sample are quantified by measuring the signal of the detection molecule at each position of the corresponding type of capture reagent. In a suspension-array-based multiplex detection, particles or microbeads are suspended in a solution required for the detection. Such particles or microbeads include identification elements that can be embedded, printed, or otherwise generated by one or more elements of the particles or microbeads. Each type of capture reagent is fixed to particles having a same identity number, and the signal emitted by the detection molecules on the particles having a specific identity number can reflect the amount of the corresponding target. Through microscope and image recognition algorithms, the particles or microbeads can be identified, so that the signal from the molecules captured by the capture reagent is associated with the identity number to realize multiplex detection.

When particles or microbeads are smaller, a greater number of identity numbers can be detected in a single test, and a larger magnification of the microscope is required for such test. This requires the imaging system to have a large field of view and high-resolution images. Existing optical microscopes are limited by optical design principle, so a spatial bandwidth product are in the scale of millions of pixels, so high resolution and large field of view are very difficult to obtain simultaneously.

Therefore, the existing microscope can obtain clear images of the microbeads/magnetic beads and images of corresponding signals (such as fluorescent signals). However, due to the limitation of the field of view of the microscope, it is extremely difficult to observe all microbeads/magnetic beads in the field of view at one time. To traverse all of the micro-chip microbeads/magnetic beads or to ensure that there are enough microbeads/magnetic beads of a same type, it is needed to repeatedly shift the field of view. The existing optical microscope also needs to perform a lens focusing process, which is not only time-consuming and laborious, but also difficult to find a focus plane. An optical lens system and a slider for moving the field of view result in the detecting system being high cost and not portable. Conventional lens-free microscopic imaging system are known from D1 (<NPL>"), D2 (<NPL>"), D3 (<CIT>), and D4 (<NPL>").

To overcome at least one of the above shortcomings, a lens-free microscopic imaging system and method, and a biochemical substance detection system and method are needed.

In a first aspect, the present disclosure provides a lens-free microscopic imaging system for imaging microbeads with pattern codes. The lens-free microscopic imaging system includes an illumination system and an imaging system. The illumination system includes an illumination light source and an excitation light source. The imaging system includes an image sensor. The illumination light source is configured to emit illumination light to irradiate the microbeads, causing the irradiated microbeads to be imaged on the image sensor. The excitation light source is configured to emit excitation light to excite the microbeads to generate specific signals, the image sensor is configured to collect images of the microbeads and of the specific signals.

Furthermore, the illumination light irradiates the microbeads, causing the images of the irradiated microbeads to be projected on the image sensor.

Furthermore, the illumination light further forms interference fringes on the image sensor, and the interference fringes are collected by the image sensor.

Furthermore, the illumination light source is a monochrome light source.

Furthermore, a pinhole is arranged on an optical path from the illumination light source to the microbeads.

Furthermore, the illumination light source is configured to emit the illumination light from a first orientation to a second orientation to irradiate the microbeads. The excitation light source is configured to emit the excitation light from the first orientation to the second orientation.

Furthermore, the illumination system further includes a total reflection device. The total reflection device includes a total reflection surface, and the total reflection surface is configured to reflect the excitation light emitted by the excitation light source to the microbeads.

Furthermore, the total reflection surface is arranged in the second orientation, and is configured to reflect the excitation light from the second orientation to the first orientation.

Furthermore, the illumination light source is configured to emit the illumination light from a first orientation to a second orientation to irradiate the microbeads, and the excitation light source is configured to emit the excitation light from the second orientation to the first orientation.

Furthermore, a filtering device is further included, which is in front of or on the image sensor.

In a second aspect, the present disclosure provides a lens-free microscopic imaging method for imaging microbeads with pattern codes. The microbeads are configured to capture specific biochemical substances. The lens-free microscopic imaging method including:.

Furthermore, the illumination light irradiates the microbeads, causing the irradiated microbeads to be projected on the image sensor.

In a third aspect, the present disclosure provides a biochemical substance detection system, including:.

Furthermore, the recognition and detection device includes a recognition and detection system. The recognition and detection system includes a microbead recognition module and a biochemical substance detection module. The microbead recognition module is configured to recognize the microbeads in the images, the pattern codes of each of the microbeads, and the identity number of each of the microbeads according to a sequence of the pattern codes of each of the microbeads. The biochemical substance detection module is configured to detect in the images an intensity of one of the specific signals sent by each type of microbead, and detect an amount of one of the specific biochemical substances captured by each type of microbead according to the intensity of the specific signal.

Furthermore, the recognition and detection system also includes an image reconstruction module, the image reconstruction module is configured to reconstruct the images output by the image sensor, and output reconstructed images to the microbead recognition modules.

Furthermore, the image reconstruction module uses a digital holographic reconstruction technology to reconstruct the images.

Furthermore, the recognition and detection system further includes a machine learning module. The machine learning module is configured to automatically learn recognition results from the microbead recognition module and extract characteristic values for subsequent recognitions by the microbead recognition module.

In a fourth aspect, the present disclosure provides a biochemical substance detection method, including:.

Furthermore, in the biochemical substance detection method, recognizing the microbeads in the images, an identity number of each of the microbeads, and an amount of specific biochemical substances captured by each type of microbead further includes recognizing the microbeads in the images, pattern codes of each of the microbeads, and the identity number of each of the microbeads according to a sequence of the pattern codes of each of the microbeads; and detecting in the images an intensity of one of the specific signals sent by each type of microbead, and detecting an amount of one of the specific biochemical substances captured by each type of microbead according to the intensity of the specific signal.

Furthermore, in the biochemical substance detection method, before recognizing the microbeads in the images, an identity number of each of the microbeads, and an amount of specific biochemical substances captured by each type of microbead, the method further includes reconstructing the images output by the image sensor, or reconstructing the images output by the image sensor by a digital holographic reconstruction technology.

Furthermore, the biochemical substance detection method further including automatically learning recognition results and detection results during the recognizing and detecting process and extracting characteristic values.

The lens-free microscopic imaging system and method and the biochemical substance detection system and method according to the embodiment of the present disclosure are used to replace the existing optical microscope to detect the pattern-coded microbeads. The processes of lens focusing and repeatedly shifting the field of view when using the existing optical microscope are avoided. The image processing is only needed to achieve ultra-high resolution, so the imaging speed is faster. Since the optical lens system is omitted, the lens-free microscopic imaging system is more compact and the cost is lower. The field of view is larger. All the pattern-coded microbeads in the reaction vessel can be traversed without the need to rotate the field of view by a slider mechanism.

Implementations of the present technology will now be described, by way of embodiment, with reference to the attached figures. Obviously, the drawings are only some embodiments of the present disclosure. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

Implementations of the disclosure will now be described, by way of embodiments only, with reference to the drawings. The described embodiments are only portions of the embodiments of the present disclosure, rather than all the embodiments. The disclosure is illustrative only, and changes may be made in the detail within the principles of the present disclosure. It will, therefore, be appreciated that the embodiments may be modified within the scope of the claims.

It should be noted that when a component is considered to be "arranged on" another component, the component can be arranged directly on another component, or there may be an intermediate components therebetween. The term "and/or" as used herein includes all or any combination of one or more related listed items.

Some existing microbeads, such as magnetic beads, have opaque substances or fluorescent substances marked therein. Pattern codes with different depths, shapes, and/or intervals are engraved around such microbead. All or a portion of the pattern codes are combined to form an identity number of the microbead. After microscopic imaging, the identity number of the microbead can be obtained by recognizing the pattern codes and their combinations engraved on the microbead. By exciting a fluorescent substance to emit light and detecting the intensity of the fluorescent light, an amount of a specific biochemical substance can be quantified.

Referring to <FIG>, a biochemical substance detection system is provided according to a first embodiment of the present disclosure. The biochemical substance detection system <NUM> is used to detect pattern-coded microbeads <NUM> to obtain an amount of specific biochemical substances captured by the pattern-coded microbeads <NUM>. The biochemical substance detection system <NUM> includes a lens-free microscopic imaging system <NUM> and a recognition and detection device <NUM>. The lens-free microscopic imaging system <NUM>, through microscopic imaging of the microbeads <NUM>, is used to obtain images of the microbeads <NUM> and signals emitted by the microbeads <NUM>. The recognition and detection device <NUM> is used to recognize the microbeads <NUM> and the identity numbers of the microbeads <NUM> from the images, and detect from the images an amount of the specific biochemical substance captured by each type of microbead <NUM>.

The lens-free microscopic imaging system <NUM> includes an illumination system 11a and an imaging system 11b. The illumination system 11a includes an illumination light source <NUM> and an excitation light source <NUM>. The imaging system 11b includes an image sensor <NUM>. The illumination light source <NUM> is used to emit illumination light to irradiate the microbeads <NUM>, so that the irradiated microbeads <NUM> are projected onto the image sensor <NUM> to form the images. The excitation light source <NUM> is used to emit excitation light to irradiate the microbeads <NUM>, and the specific biochemical substances captured by the microbeads <NUM> are excited by the excitation light to respond with signals towards the image sensor <NUM> to form the images. In order to prevent leakage of a portion of the excitation light onto the image sensor <NUM>, in this embodiment, a filtering device <NUM> matching a wavelength of the excitation light is arranged in front of the image sensor <NUM>. The filtering device <NUM> can be a filter device or a filter plate. The imaging system 11b does not require a lens system of lenses. That is, a lens system is not provided between the image sensor <NUM> and the microbeads <NUM>.

In this embodiment, the microbeads <NUM> are pattern-coded magnetic beads of a transparent material. The microbeads <NUM> are placed in a fluorescent reaction container <NUM>. Generally, in order to realize multiplex detection, various types of microbeads <NUM> are placed in the fluorescent reaction container <NUM>, and each type of microbead <NUM> has a same sequence of the pattern codes. Therefore, each type of microbead <NUM> has a same identity number, and different types of microbeads <NUM> have different identity numbers. Each type of microbead <NUM> captures a biochemical substance, and different types of microbeads <NUM> can capture different biochemical substances. When a type of microbead <NUM> captures a specific biochemical substance, the specific biochemical substance captured by the microbeads <NUM> is excited by the excitation light from the excitation light source <NUM> and generates a fluorescent signal. By detecting the intensity of the fluorescent signal from each type of microbead <NUM>, the amount of the specific biochemical substance captured by such type of microbead <NUM> is quantified.

In this embodiment, the microbeads <NUM> are arranged so that a first orientation and a second orientation are defined. The illumination light source <NUM> and the excitation light source <NUM> are both arranged in the first orientation of the microbeads <NUM>. The filtering device <NUM> and the image sensor <NUM> are arranged in the second orientation of the microbeads <NUM>. The illumination light source <NUM> is a point light source or a light source array with limited spatial coherence (i.e., low spatial coherence). An imaging method performed by the lens-free microscopic imaging system <NUM> includes following steps. The illumination light source <NUM> is powered on, which irradiates the microbeads <NUM> from the first orientation to the second orientation, and the irradiated microbeads <NUM> are projected onto the image sensor <NUM>. At the same time, the excitation light source <NUM> is powered on, which emits the excitation light from the first orientation to the second orientation to irradiate the microbeads <NUM>, causing the microbeads <NUM> to emit the fluorescent signals toward the image sensor <NUM>. The image sensor <NUM> collects the projections and the fluorescent signals of the microbeads <NUM>, and generates the images of the microbeads <NUM> and the fluorescent signals. The images are output to the recognition and detection device <NUM>. The recognition and detection device <NUM> recognizes the images to obtain the identity number of each type of microbead <NUM>, and to detect the amount of the specific biochemical substance captured by each type of microbead <NUM>. Specifically, the recognition and detection device <NUM> is a computer device. The recognition and detection device <NUM> includes a processor <NUM>, a memory <NUM>, and a computer program stored in the memory <NUM> and executed by the processor <NUM>, such as a recognition and detection system <NUM>. The recognition and detection system <NUM> is divided into a plurality of functional modules, such as a microbead recognition module <NUM> and a biochemical substance detection module <NUM>, according to its functions. The microbead recognition module <NUM> is used to recognize the microbeads <NUM> in the images and the identity numbers of the microbeads <NUM>. Specifically, the microbead recognition module <NUM> recognizes the microbeads <NUM> according to pre-built models or preset characteristics, recognizes the pattern codes of the microbeads <NUM> according to the pre-built models or preset characteristics, and finally recognizes the identity numbers of the microbeads <NUM> according to the sequences of the pattern codes of the microbeads <NUM>. The biochemical substance detection module <NUM> is used to detect the intensity of the signal from each type of microbead <NUM> in the images, and detect the amount of the specific biochemical substance captured by each type of microbead <NUM> according to the intensity of the signal.

The biochemical substance detection system <NUM> and detection method in this embodiment are applicable to the detection of pattern-coded beads, such pattern-coded beads which have sizes of <NUM> to <NUM>, and a distance between two feature points of the pattern codes is <NUM> or more. Assuming that a distance between the illumination light source <NUM> and the microbeads <NUM> is z1, a distance between the microbeads <NUM> and the image sensor <NUM> is z2. The light carrying the information of the microbeads <NUM>, propagating over the distance z2, is captured by the image sensor <NUM>. In the existing process of semiconductors, a pixel size of the image sensor <NUM> is generally large (a smallest pixel size at present is about <NUM>). Thus, in this embodiment, pixelation exists when the image sensor <NUM> uses projection imaging (the projection imaging forms the images by the projections of the microbeads <NUM>). Furthermore, defocus information of the microbeads <NUM> is directly obtained. Thus, the recognition and detection device <NUM> only needs the projection information of the microbeads <NUM> to recognize the identity numbers of the microbeads <NUM>, without the need for reconstruction of focus information of the microbeads <NUM> from under-sampling images.

Referring to <FIG>, a biochemical substance detection system <NUM> is provided according to a second embodiment of the present disclosure. The biochemical substance detection system <NUM> is used to detect pattern-coded microbeads <NUM> to quantify an amount of specific biochemical substances captured by the pattern-coded microbeads <NUM>. The biochemical substance detection system <NUM> includes a lens-free microscopic imaging system <NUM> and a recognition and detection device <NUM>. The lens-free microscopic imaging system <NUM> is used to obtain images of the microbeads <NUM> and signals emitted by the microbeads <NUM> through microscopic imaging of the microbeads <NUM>. The recognition and detection device <NUM> is used to recognize the microbeads <NUM> in the images and the identity numbers of the microbeads <NUM>, and detect the amount of a specific biochemical substance captured by each type of microbead <NUM>.

The lens-free microscopic imaging system <NUM> includes an illumination system 21a and an imaging system 21b. The illumination system 21a includes an illumination light source <NUM> and an excitation light source <NUM>. The imaging system 21b includes an image sensor <NUM> and a filtering device <NUM> arranged in front of the image sensor <NUM>. The illumination light source <NUM> is a point light source or a light source array with limited spatial coherence. The illumination light source <NUM> is used to emit illumination light to irradiate the microbeads <NUM>, and the irradiated microbeads <NUM> are projected onto the image sensor <NUM> to form the images. In this embodiment, the microbeads <NUM> are made of an opaque material, or a wavelength of light passing through the microbeads <NUM> is not within a detection range of the image sensor <NUM>. The microbeads <NUM> are arranged so that a first orientation and a second orientation are defined. The illumination light source <NUM> is arranged in the first orientation. The excitation light source <NUM>, the image sensor <NUM>, and the filtering device <NUM> are arranged in the second orientation. The illumination light source <NUM> emits the illumination light from the first orientation to the second orientation, which irradiates the microbeads <NUM> so that the irradiated microbeads <NUM> are projected onto the image sensor <NUM>. At the same time, the excitation light source <NUM> irradiates the microbeads <NUM> from the second orientation to the first orientation, so that the excitation microbead <NUM> generates a fluorescent signal that is projected onto the image sensor <NUM>. A lens system is not included in the imaging system 21b. That is, the lens system is not provided between the image sensor <NUM> and the microbeads <NUM>. The image sensor <NUM> directly collects the projections and the fluorescent signals of the microbeads <NUM>, and generates the images of the microbeads <NUM> and the fluorescent signals. The images are output to the recognition and detection device <NUM>. The recognition and detection device <NUM> recognizes the images to obtain the identity number of each type of microbead <NUM>, and to detect the amount of the specific biochemical substance captured by each type of microbead <NUM>. The configuration of the recognition and detection device <NUM> can refer to embodiment <NUM>, which will not be repeated.

The biochemical substance detection system <NUM> and detection method in this embodiment are applicable to the detection of microbeads <NUM>, such microbeads <NUM> which have sizes of <NUM> to <NUM>, and a distance between two feature points of the pattern codes is <NUM> or more.

Referring to <FIG>, a biochemical substance detection system <NUM> is provided according to a third embodiment of the present disclosure. The biochemical substance detection system <NUM> is used to detect pattern-coded microbeads <NUM> to quantify an amount of specific biochemical substances captured by the pattern-coded microbeads <NUM>. The biochemical substance detection system <NUM> includes a lens-free microscopic imaging system <NUM> and a recognition and detection device <NUM>. The lens-free micro imaging system <NUM>, through microscopic imaging of the microbeads <NUM>, is used to obtain images of the identity numbers of the microbeads <NUM> and signal emitted by the microbeads <NUM>. The recognition and detection device <NUM> is used to recognize the microbeads <NUM> in the images and the identity numbers of the microbeads <NUM>, and to detect the amount of the specific biochemical substance captured by each type of microbead <NUM>.

The lens-free microscopic imaging system <NUM> includes an illumination system 31a and an imaging system 31b. The illumination system 31a includes an illumination light source <NUM>, an excitation light source <NUM>, and a total reflection device <NUM>. The imaging system 31b includes an image sensor <NUM> and a filtering device <NUM>. The illumination light source <NUM> is a point light source or a light source array with limited spatial coherence. The microbeads <NUM> are pattern-coded microbeads of an opaque material, or a wavelength of light that the microbeads <NUM> can project is not within a detection range of the image sensor <NUM>. The microbeads <NUM> are arranged so that a first orientation and a second orientation are defined. The illumination light source <NUM> and the excitation light source <NUM> are arranged in the first orientation. The image sensor <NUM>, the filtering device <NUM>, and the total reflection device <NUM> are arranged in the second orientation. The filtering device <NUM> is arranged in front of the image sensor <NUM>. The total reflection device <NUM> is arranged in front of the filtering device <NUM>. The illumination light source <NUM> emits the illumination light from the first orientation to the second orientation to irradiate the microbeads <NUM>, and the irradiated microbeads <NUM> are projected onto the image sensor <NUM>. The excitation light source <NUM> emits the excitation light from the first orientation to the second orientation to irradiate the microbeads <NUM>, causing the microbeads <NUM> to emit the fluorescent signals to the image sensor <NUM>. The total reflection device <NUM> is arranged behind the microbeads <NUM>. The total reflection device <NUM> has a total reflection surface <NUM>. In this embodiment, the total reflection surface <NUM> reflects the excitation light and allows the illumination light to pass through, causing the excitation light to irradiate a side of the microbeads <NUM> facing the image sensor <NUM>. Thus, capture reagents on such side generate fluorescent signals. The fluorescent signals from the microbeads <NUM> are projected to and recorded on the image sensor <NUM>. The image sensor <NUM> generates the images of the microbeads <NUM> and the fluorescent signals. The images are output to the recognition and detection device <NUM>. The recognition and detection device <NUM> recognizes the images to obtain the identity number of each type of microbead <NUM>, and to detect the amount of the specific biochemical substance captured by each type of microbead <NUM>. The setting of the recognition and detection device <NUM> can refer to embodiment <NUM>, which will not be repeated.

It can be understood that in other embodiments, not all parts of the total reflection device <NUM> must be behind the microbeads <NUM>. Only at least a portion of the total reflection surface <NUM> is required to be behind the microbeads <NUM>.

A distance z2 between the microbeads <NUM> and the image sensor <NUM> is a key factor of imaging quality. Compared with the second embodiment, arranging the excitation light source <NUM> in the first orientation can reduce the distance z2 between the microbeads <NUM> and the image sensor <NUM>, so that the images of the projections of the microbeads <NUM> and the fluorescent signals are clearer.

The biochemical substance detection system <NUM> and detection method in this embodiment are applicable to the detection of pattern-coded microbeads <NUM>, such microbeads <NUM> have sizes of <NUM> to <NUM>, and a distance between two feature points of the pattern codes is <NUM> or more.

In this embodiment, a lens-free microscopic imaging system uses an illumination light source with good spatial coherence and time coherence (i.e., high spatial coherence and high time coherence) is used to irradiate microbeads. Interference fringes are formed on an image sensor. The image sensor collects the interference fringes and fluorescent signals excited by an excitation light source, and generates images that are transmitted to a recognition and detection device. Referring to <FIG>, the recognition and detection device <NUM> includes a processor <NUM>, a memory <NUM>, and a computer program stored in the memory <NUM> and executed by the processor <NUM>, such as a recognition and detection system <NUM>. In this embodiment, the recognition and detection system <NUM> includes a microbead recognition module <NUM>, a biochemical substance detection module <NUM>, and an image reconstruction module <NUM>. The image reconstruction module <NUM> is used to reconstruct the images output by the image sensor to obtain reconstructed images with higher resolutions. The microbead recognition module <NUM> is used to recognize the microbeads in the reconstructed images and the identity number of the microbeads. The biochemical substance detection module <NUM> is used to detect the intensity of the signal from each type of microbead in the reconstructed images, and detect the amount of specific biochemical substance captured by each type of microbead according to the intensity of the signal.

Specifically, the image reconstruction module <NUM> applies calculations to the interference fringes to obtain intensity information and phase information of the microbeads at a focus plane, thereby obtaining the identity numbers of the microbeads. The focus plane refers to a plane wherein clear images of the microbeads can be formed. The focus plane is a fictitious plane, not the focusing plane inside a traditional lens.

Images U(x, y) collected by the image sensor are formed by interferences between light U<NUM>(x, y) scattered by the microbeads (object light) and reference light UR(x, y) directly passing through a transparent substrate and the filter without interference: <MAT>.

Wherein, AR and A<NUM>(x, y) are respectively the amplitude information of the reference light and the amplitude information of the object light (from microbeads), and ϕ<NUM>(x, y) is the phase information of the object light (from microbeads). In this embodiment, the image reconstruction module <NUM> uses a digital holographic reconstruction technology to recover the amplitude and phase information of the object light (from microbeads) based on light intensity information I(x, y) directly collected by the image sensor. The transparent substrate refers to a medium between the total reflection device and the filtering device.

The image reconstruction module <NUM> uses digital holographic reconstruction technology to reconstruct the images obtained by the image sensor through algorithms. The reconstruction mainly includes: (<NUM>) phase recovery or conjugate image elimination, which are mainly used to eliminate diffraction effect caused by a spacing between the microbeads and the sensor; (<NUM>) super resolution imaging, which is mainly used to overcome a reduction of resolution caused by a pixel size of the image sensor, thereby achieving sub-pixel resolution without any lens. The two steps can be carried out in sequence, or carried out simultaneously by a phasor propagation method. The phasor propagation method can realize the super-resolution of pixels and phase recovery for the lens-free microscope images, and then realize image reconstruction. For an LED light source with poor spatial coherence, the spatial resolution of the reconstructed images can be further improved by a combination of the phasor propagation method and a point spread function deconvolution method.

In this embodiment, the following technical solutions can be used to obtain an illumination light source with good spatial coherence and temporal coherence.

The above technical solutions can be combined according to needs.

In addition to the use of a different illumination light source, the position of the illumination light source, and the position and function of other components in the lens-free microscopic imaging system in this embodiment can refer to any of the above-mentioned embodiments. For example, for transparent microbeads, the illumination light source, the excitation light source, the image sensor, and filtering device can be set with reference to embodiment <NUM>. For opaque microbeads or microbeads through which a wavelength of light is not within the detection range of the image sensor, the illumination light source, the excitation light source, the image sensor, and the filtering device can be set with reference to embodiment <NUM>, or the illumination light source, the excitation light source, the image sensor, the filtering device, and the total reflection device can be set with reference to embodiment <NUM>.

Compared with the above-mentioned embodiment that use a point light source or a light source array with limited spatial coherence to irradiate the microbeads for projection imaging, in this embodiment, the illumination light source with good spatial and temporal coherence is used to irradiate the microbeads to generate interference fringes. The digital holographic reconstruction image technology is then used to obtain the identity numbers of the microbeads, this has higher resolution and more accurate recognition.

The lens-free microscopic imaging system in this embodiment can refer to embodiments <NUM> to <NUM>. The differences are mainly in respect of the recognition and detection device and the imaging method.

In this embodiment, Fourier laminated imaging is used to replace the above projection imaging to recognize the identity numbers of the microbeads. In this embodiment, the excitation light source is used to excite the microbeads to emit fluorescent signals. The fluorescent signals are collected by the image sensor and finally recognized by a specific computer device to quantify the amount of the captured specific molecules.

In the above-mentioned embodiments <NUM> to <NUM>, accurate modeling for each step is needed in order to recognize the pattern codes of the microbeads and the identity numbers of the microbeads. The above-mentioned "each step" generally includes the placement of optical elements, modeling of the light sources and the illumination process, modeling of the detected objects, or modeling of the imaging process. In this embodiment, in addition to the use of the biochemical substance detection system in any one of embodiments <NUM> to <NUM> to perform the biochemical substance detection, the biochemical substance detection system also includes a machine learning function, which automatically learns from each detection result to extract different characteristic values for the biochemical substance detection system to perform subsequent detections.

Referring to <FIG>, the recognition and detection device <NUM> includes a processor <NUM>, a memory <NUM>, and a computer program stored in the memory <NUM> and executed by the processor <NUM>, such as a recognition and detection system <NUM>. In this embodiment, the recognition and detection system <NUM> includes a microbead recognition module <NUM>, a biochemical substance detection module <NUM>, and a machine learning module <NUM>. The microbead recognition module <NUM> and the biochemical substance detection module <NUM> basically have the same functions as those of the microbead recognition module <NUM> and the biochemical substance detection module <NUM> in embodiment <NUM>, which will not be repeated. The machine learning module <NUM> is used to apply a machine learning method, such as a supervised learning method or an unsupervised learning method, to train through a large number of data sets, thereby realizing the extraction of a large number of characteristic values and the pattern codes of the microbeads. By adding the machine learning module <NUM>, the biochemical material detection system automatically learns from each detection result to obtain the characteristic values. Thus, accurate modeling for each step of the entire imaging process is avoided. The recognition, segmentation, extraction, and counting of the microbeads in a large field of view, as well as the recognition and counting of identity numbers of the microbeads are realized. The supervised learning method substantially includes data preprocessing, neural network construction, network training, and optimal solution preserving.

Referring to <FIG>, a flowchart of a biochemical substance detection method is provided according to a seventh embodiment of the present disclosure. The sequence of some steps in the flowchart may be changed, and some steps may be omitted. For convenience of explanation, only the parts related to the embodiments of the present disclosure are shown.

In this embodiment, the biochemical substance detection method is used to form images of microbeads having pattern codes. The microbeads are used to capture specific biochemical substances. The biochemical substance detection method includes a lens-free microscopic imaging method and a recognition and detection method.

The lens-free microscopic imaging method include following steps.

Step S61, the microbeads are placed between the illumination light source and the image sensor, causing the microbeads to be capable of being illuminated by illumination light from the illumination light source and project their own images on the image sensor.

Step S62, the illumination light source is powered on, which emits the illumination light.

Step S63, the excitation light source is powered on, which emits the excitation light to irradiate the microbeads, so that the specific biochemical substances captured by the microbeads or the biochemical substances generated during the capture process are excited to emit specific signals. The above capture process will generally initialize a series of biochemical reaction processes. The capture process includes but is not limited to a one-step method, sandwich method, or quenching method.

Step S64, the image sensor collects an image of the microbeads and an image of the specific signals. Thus, the images of at least two channels are obtained.

The recognition and detection method include following steps.

Step S65, the images are received. The microbeads in the images and the identity number of each of the microbeads are recognized. The amount of specific biochemical substance captured by each type of microbead is also detected. The microbeads with a same identity number are classified into a single type.

Step S65 may specifically include following steps. The images are received. Then, the microbeads in the images and the identity number of each microbead are recognized in one channel. The position and intensity of the fluorescent signals are recognized in the other channel. The position of the fluorescent signals is registered with the position of the microbeads, or the images of the two channels are registered. The identity number of each microbead, and the presence or absence of the corresponding specific signal and the intensity of such signal are detected. The microbeads with the same identity number are classified into a single type. Through the corresponding signal of multiple microbeads classified in a same type, the intensity and distribution of the signal from this type of microbeads are determined after multiple sampling process.

In another embodiment, the lens-free microscopic imaging method of the biochemical substance detection method also includes configuring the illumination light source as a point light source or a light source array with low spatial coherence.

In another embodiment, the lens-free microscopic imaging method of the biochemical substance detection method also includes irradiating the microbeads by the illumination light emitted by the illumination light source, so that the microbeads are projected on the image sensor.

In another embodiment, the lens-free microscopic imaging method of the biochemical substance detection method also includes configuring the illumination light source to have high temporal coherence and high spatial coherence.

In another embodiment, the lens-free microscopic imaging method of the biochemical substance detection method also includes irradiating the microbeads by the illumination light emitted by the illumination light source, thereby forming interference fringes on the image sensor. The interference fringes are then collected by the image sensor.

In another embodiment, the recognition and detection method of the biochemical substance detection method specifically includes recognizing the microbeads in the images and the pattern codes of each microbead. The identity number of each microbead is recognized according to the sequence of the pattern codes of each microbead. The intensity of the specific signal generated by each type of microbead in the images is detected. The amount of the specific biochemical substance captured by each type of microbead is then detected according to the intensity of the specific signal.

In another embodiment, before "recognizing the microbeads in the images and the identity number of each microbead and detecting the amount of specific biochemical substances captured by each type of microbead", the recognition and detection method of the biochemical substance detection method also includes reconstructing the images output by the image sensor, or reconstructing the images output by the image sensor through a digital holographic reconstruction technology.

In another embodiment, the recognition and detection method of the biochemical substance detection method also includes automatically learning various results of recognition and detection during the recognition and detection processes, and extracting the characteristic values.

In the above-mentioned embodiments <NUM> to <NUM>, in order to further improve the imaging resolution, the following technical solutions or a combination thereof can be used as needed.

Therefore, the lens-free microscopic imaging system and method and the biochemical substance detection system and method according to the embodiment of the present disclosure are used to replace the existing optical microscope to detect the pattern-coded microbeads. The processes of lens focusing and repeatedly shifting the field of view when using the existing optical microscope are avoided. The image processing is only needed to achieve ultra-high resolution, so the imaging speed is faster. Since the optical lens system is omitted, the lens-free microscopic imaging system is more compact and the cost is lower. The field of view is larger. All the pattern-coded microbeads in the reaction vessel can be traversed without the need to rotate the field of view by a slider mechanism.

Claim 1:
A lens-free microscopic imaging system (<NUM>, <NUM>, <NUM>), the lens-free microscopic imaging system (<NUM>, <NUM>, <NUM>) configured for imaging microbeads (<NUM>, <NUM>, <NUM>), each of the microbeads (<NUM>, <NUM>, <NUM>) has opaque substances or fluorescent substances marked therein, the lens-free microscopic imaging system (<NUM>, <NUM>, <NUM>) comprising:
an illumination system (11a, 21a, 31a); and
an imaging system (11b, 21b, 31b);
wherein the illumination system (11a, 21a, 31a) comprises an illumination light source (<NUM>, <NUM>, <NUM>) and an excitation light source (<NUM>, <NUM>, or <NUM>), the imaging system (11b, 21b, 31b) comprises an image sensor (<NUM>, <NUM>, <NUM>), the illumination light source (<NUM>, <NUM>, <NUM>) is configured to emit illumination light to irradiate the microbeads (<NUM>, <NUM>, <NUM>), causing the irradiated microbeads (<NUM>, <NUM>, <NUM>) to be imaged on the image sensor (<NUM>, <NUM>, <NUM>), the excitation light source (<NUM>, <NUM>, or <NUM>) is configured to emit excitation light to excite the microbeads (<NUM>, <NUM>, <NUM>) to generate specific signals, the image sensor (<NUM>, <NUM>, <NUM>) is configured to collect images of the microbeads (<NUM>, <NUM>, <NUM>) and of the specific signals,
characterized in that the system is configured to quantify an amount of specific biochemical substances captured by various types of microbeads (<NUM>, <NUM>, <NUM>), with each of the microbeads (<NUM>, <NUM>, <NUM>) having pattern codes with different depths, shapes, and/or intervals engraved around the microbead (<NUM>, <NUM>, <NUM>), and all or a portion of the pattern codes are combined to form an identity number of the microbead (<NUM>, <NUM>, <NUM>), and each type of microbeads (<NUM>, <NUM>, <NUM>) has a same identity number, each type of microbeads (<NUM>, <NUM>, <NUM>) captures a biochemical substance, different types of microbeads (<NUM>, <NUM>, <NUM>) capture different biochemical substances, the system is further configured to recognize the identity number of the microbead (<NUM>, <NUM>, <NUM>) by the pattern codes in the images, and to quantify an amount of a specific biochemical substance captured by each type of microbeads (<NUM>, <NUM>, <NUM>) by an intensity of the specific signals in the images.