Real-time imaging system integrating bright field and fluorescence functions

Abstract

The invention discloses a real-time imaging system integrating bright field and fluorescence functions. The real-time imaging system comprises a digital micro-fluidic chip base, a light-shielding heat preservation module, an excitation module, a bright field module, a receiving and imaging module, a modulation control module and a data acquisition and processing module, by optimizing light source control and light path design, the influence of external interference on the imaging effect is reduced, meanwhile, the response speed and the detection precision of the system are improved, the system has high integration level and good portability and is suitable for rapid and efficient biological sample analysis and detection, the system provides an overall scheme from a hardware light path to a software algorithm, and the system is suitable for large-scale popularization and application. All the modules are integrated, and data exchange and processing are carried out in a full-automatic mode.

Description

Technical Field

[0001] The present invention belongs to the technical field of digital microfluidics, and in particular relates to a real-time imaging system integrating bright field and fluorescence functions. Background Art

[0002] Digital microfluidics (DMF) is a microfluidic technology that precisely manipulates tiny droplets through the electrowetting effect. Unlike traditional continuous flow microfluidics, digital microfluidics can control the generation, splitting, merging, and transmission of droplets on an open two-dimensional chip surface by applying an electric field. Due to its high flexibility and precise droplet control capabilities, digital microfluidics is widely used in biomedical analysis, drug screening, genetic testing, and other fields.

[0003] Digital microfluidics technology based on the principle of electrowetting has become a research hotspot in the field of on-chip laboratories due to its advantages such as portability, low cost and high efficiency. In order to achieve real-time monitoring of samples in microfluidic chips, an imaging system integrating bright field and fluorescence functions has emerged, aiming to provide fast and efficient sample detection and analysis capabilities. This imaging system combines the advantages of bright field imaging for structural display and fluorescence imaging for molecular specific detection, allowing samples in tiny droplets or tiny channels in microfluidic chips to be observed and recorded in real time and accurately.

[0004] Digital microfluidics can manipulate trace amounts of biological samples and combine bright field imaging to display the overall structure with high-contrast marker detection using fluorescence imaging to identify specific biological molecules or reaction processes (such as PCR reactions). To achieve the high efficiency of this system, light source control and optical path design are key technical challenges. It is necessary to ensure that it has high sensitivity and rapid response instant detection capabilities on the microfluidics platform, thereby providing comprehensive support for the precise detection of biological samples.

[0005] Existing digital microfluidics technologies usually rely on the human eye, conventional cameras or microscopes for real-time imaging detection. First, these systems have some limitations in imaging field of view, resolution and detection sensitivity. It is difficult to meet the needs of accurately observing changes in tiny droplets while taking into account high-throughput biological reaction processes. Secondly, the existing systems are relatively complex in light source control and optical path design, and are easily affected by external interference, which in turn affects the accuracy and response speed of imaging. Furthermore, the integration of multifunctional imaging systems is low, and separate equipment is usually required for bright field and fluorescence imaging. This results in a bulky system, lack of portability, inflexible use, low software integration, and multi-software collaboration that needs to be completed manually. Summary of the invention

[0006] In view of the above defects, the present invention provides a real-time imaging system integrating bright field and fluorescence functions, including a digital microfluidic chip base for carrying and fixing a digital microfluidic chip, a light-proof and heat-insulating module for independent reaction without external interference, an excitation module for excitation and irradiation of the whole chip in fluorescence mode and detection of fluorescent labeled liquid on the chip, a bright field module for real-time observation in non-fluorescence mode, a receiving and imaging module for receiving real-time imaging signals of the bright field module and fluorescence signals detected by the excitation module, a modulation control module for exchanging data with a host computer and responsible for modulation and control of a system light source motor, and a data acquisition and processing module for driving an imaging camera, analyzing the acquired data and executing instructions issued;

[0007] The data acquisition and processing module includes a control port unit, a camera driving unit and a data processing unit. The control port unit is used to communicate with the modulation control module and provide an operation interface. The camera driving unit is used to activate, test and adjust the imaging device and store image data. The data processing unit is used to browse, analyze and manipulate the stored data.

[0008] Furthermore, the digital microfluidic chip base includes a driving circuit connection unit and a magnetically controlled temperature control unit, and the magnetically controlled temperature control unit includes two customizable temperature areas.

[0009] Furthermore, the excitation module includes an LED surface light source, a collimating lens and a filter lens group.

[0010] Furthermore, the receiving and imaging module includes a photosensitive chip, a filter lens group and a lens which are combined for imaging, and is provided with a plurality of switchable filter holes.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. Integrating bright field and fluorescence imaging into the same system reduces the size of the device, simplifies the operation, facilitates multimodal detection, and is suitable for portable applications.

[0013] 2. The integrated light source design of collimation, uniform light and filtering, and the symmetrical oblique incident light path reduce the interference of stray light. Combined with the high-sensitivity imaging sensor, it can achieve high-resolution real-time imaging to meet the needs of precise observation of tiny droplets or biological reaction processes.

[0014] 3. Use the detector's extreme wavelength region for bright field illumination observation in fluorescence experiments to avoid interference from bright field excitation reagents. This can meet users' real-time observation needs for samples when using fluorescent markers, and can effectively locate samples without affecting fluorescence detection.

[0015] 4. Data acquisition and processing can receive and analyze image data from imaging sensors in real time, and quickly output information such as droplet movement and fluorescence intensity changes, making sample analysis more efficient and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the system composition of the present invention.

[0017] Figure 2 It is a schematic diagram of the excitation module in the present invention.

[0018] Figure 3 It is a schematic diagram of a conventional bright field in the bright field module of the present invention.

[0019] Figure 4 It is a schematic diagram of a special low-energy bright field in the bright field module of the present invention.

[0020] Figure 5 It is a schematic diagram of the receiving and imaging module in the present invention.

[0021] Figure 6 Schematic diagram of the control timing of two-step PCR performed by the system of the present invention.

[0022] Figure 7 It is a schematic diagram of bright field switching between high and low temperature zones of two samples in two-step PCR performed by the system of the present invention.

[0023] Figure 8 The schematic diagram of the fluorescence collection of the low temperature region excitation state switching in the two-step PCR performed by the system of the present invention is shown.

[0024] Figure 9 This is a schematic diagram of the change in fluorescence intensity during the process of gradually decreasing the high temperature to the low temperature of the final test solution of the two-step PCR system of the present invention.

[0025] Figure 10 Schematic diagram of importing collected fluorescence data into the digital microfluidic fluorescence annotation detection system.

[0026] Figure 11 PCR fluorescence intensity curve after the collected fluorescence data is imported into the digital microfluidic fluorescence labeling detection system.

[0027] In the figure: 1. Digital microfluidic chip base; 2. Light-proof and heat-insulating module; 3. Excitation module; 31. LED surface light source; 32. Collimating lens; 33. Filter lens group; 4. Bright field module; 5. Receiving and imaging module; 51. Photosensitive chip; 52. Filter lens group; 53. Lens; 6. Modulation control module; 7. Data acquisition and processing module. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present invention, the device of the present invention will be described more fully below with reference to the relevant drawings. Embodiments of the device are given in the drawings. However, the device can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0029] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "set" should be understood in a broad sense, for example, they can be fixedly connected or set, or detachably connected or set, or integrally connected or set. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0030] Example

[0031] like Figure 1 As shown, this embodiment provides a real-time imaging system integrating bright field and fluorescence functions, including a host computer, and also includes a digital microfluidic chip base 1 for carrying and fixing a digital microfluidic chip, the digital microfluidic chip base 1 includes a driving circuit connection unit and a magnetically controlled temperature control unit, and the magnetically controlled temperature control unit includes two customizable temperature areas;

[0032] Light-shielding and heat-insulating module 2 is used for light-shielding and active heat-insulating functions of the open platform to ensure that the overall reaction is independent and not interfered by the outside world;

[0033] The excitation module 3 is used for excitation and illumination of the whole chip in the fluorescence mode to detect the liquid with fluorescent markers on the chip. The excitation module 3 includes an LED surface light source 31, a collimating lens 32 and a filter lens group 33, that is, Figure 2 As shown, the LED surface light source 31 is modulated by the collimating lens 32, and then emitted by the filter lens group 32 for excitation. By configuring multiple filter holes, multi-channel switching is achieved by relying on a motor, and symmetrical incidence is used to ensure the irradiation range and uniformity;

[0034] Bright field module 4 is used to illuminate the entire chip in non-fluorescence mode, and real-time observation avoids result deviation caused by improper sample handling, providing a reliable basis for subsequent experiments. Bright field module 4 is equipped with conventional bright field and special low-energy bright field for real-time observation in different application scenarios, such as Figure 3 As shown, conventional bright field imaging uses a white light source, which can effectively observe and locate samples without using fluorescent labels. Special low-energy bright field is used in fluorescence experiments to avoid interference from bright field excitation reagents, meet users' real-time observation needs for samples when using fluorescent labels, and can effectively locate samples without affecting fluorescence detection.

[0035] like Figures 4 - 5As shown, the receiving and imaging module 5 is used to receive the real-time imaging signal of the bright field module 4 and the fluorescence signal detected by the excitation module 3 (i.e., for real-time imaging in the bright field mode and fluorescence detection in the fluorescence mode, and for data acquisition and storage), and is used to exchange data with the host computer communication. The receiving and imaging module 5 is used for imaging, and includes a photosensitive chip 51, a filter lens group 52 and a lens 53 arranged in sequence from top to bottom, and is provided with a plurality of filter holes that can be switched by motor drive, and real-time data collection can be realized through the data acquisition and processing module 7;

[0036] The modulation control module 6, which is responsible for the modulation and control of the system light source motor, communicates with the host computer through the integrated wireless transmission module for data transmission, and cooperates with the digital microfluidic software to realize bright field observation, fluorescence switching, brightness adjustment and other functions. In order to realize a variety of automatic and manual control methods, a control interface can be installed on the host computer for selection and use;

[0037] The data acquisition and processing module 7 is used to drive the imaging camera, analyze the collected data and execute the instructions issued. The data acquisition and processing module 7 includes a control port unit, a camera driving unit and a data processing unit, wherein the control port unit, the camera driving unit and the data processing unit are all embodied in the operating interface of the host computer in the form of software, and the control port unit, the camera driving unit and the data processing unit can also be directly replaced by the control port software, the camera driving software and the data processing software directly integrated into the host computer. The control port unit is used to communicate with the modulation control module 6 and provide an operating interface. The camera driving unit is a separate call to the camera specific function test unit for loading and adjusting the stored data. The data processing unit is used for system storage data browsing and data manipulation. There are automatic and manual functions for fluorescence detection data, and support customized pixel-level fluorescence acquisition.

[0038] The real-time imaging system integrating bright field and fluorescence functions described in this embodiment is used to perform two-step PCR operation, two-step PCR on a digital microfluidic chip, to achieve real-time bright field monitoring of two droplets, and automatic fluorescence data collection, and finally use data processing software to generate a data set to draw a PCR curve. Figure 6 As shown, the specific steps are:

[0039] The two-step method in PCR combines the annealing and extension steps into one step to simplify the reaction process. Usually, the first step is high-temperature denaturation to separate the DNA double strands; the second step is to complete primer annealing and DNA extension at a moderate temperature. This method helps to improve the specificity and efficiency of PCR.

[0040] The two-step method implemented in the real-time imaging system of this embodiment is that the temperature zone is changed by the movement of the droplet, and the modulation control module controls the bright field and excitation light sources to light up alternately. When detecting fluorescence, the excitation light source is turned on briefly and multiple times, and the bright field light source is always on at other times to monitor the entire experimental process. In particular, the high and low temperature zones are switched to bright field observation. Figure 7 As shown, the fluorescence acquisition of the low temperature region excitation state switching is as follows Figure 8 As shown in the figure, the bright field and fluorescence data are collected in this way for multiple times, and the fluorescence data is transmitted to the host computer through wireless transmission or wiring harness for subsequent processing. After being imported into the digital microfluidic fluorescence labeling detection system, Figure 10 As shown, the labeling is performed manually or automatically, and finally a PCR curve is drawn (such as Figure 11 shown).

[0041] It can be concluded that this real-time imaging system not only has the ability of real-time observation in bright field, but also has the ability of real-time observation in fluorescence mode. The melting curve is an important tool for evaluating the specificity of amplification products in real-time fluorescence quantitative PCR (qPCR). Its principle is based on the denaturation characteristics of double-stranded DNA. By gradually increasing the temperature, double-stranded DNA will be melted into single strands, causing the fluorescent dye (such as SYBR Green I) to be released from the DNA, and the fluorescence signal will decrease. The melting curve reflects the relationship between temperature and fluorescence intensity. The basic principle is that the fluorescence intensity is inversely proportional to the temperature. Therefore, in the process of the PCR final test solution gradually decreasing from high temperature to low temperature, the fluorescence intensity gradually increases. Figure 9 shown.

[0042] It should be noted that the structure described in the present invention can be implemented in a variety of different forms and is not limited to the described embodiments. Any equivalent transformations made by ordinary technicians in this field using the contents of the present invention description and drawings, or directly or indirectly applied to other related technical fields, such as the loading and unloading of other items, are included in the protection scope of the present invention.

Claims

1. A real-time imaging system integrating bright field and fluorescence functions, characterized in that: It includes a digital microfluidic chip base for carrying and fixing the digital microfluidic chip, a light-proof and heat-insulating module for independent reaction without external interference, an excitation module for full-chip excitation and irradiation in fluorescence mode and detection of fluorescent-labeled liquid on the chip, a bright field module for real-time observation in non-fluorescence mode, a receiving and imaging module for receiving real-time imaging signals from the bright field module and fluorescent signals detected by the excitation module, a modulation control module for data exchange with the host computer communication and responsible for the modulation and control of the system light source motor, and a data acquisition and processing module for driving the imaging camera, analyzing the collected data and executing the instructions issued; The data acquisition and processing module includes a control port unit, a camera driving unit and a data processing unit. The control port unit is used to communicate with the modulation control module and provide an operation interface. The camera driving unit is used to activate, test and adjust the imaging device and store image data. The data processing unit is used to browse, analyze and manipulate the stored data.

2. A real-time imaging system integrating bright field and fluorescence functions as claimed in claim 1, characterized in that: The digital microfluidic chip base includes a driving circuit connection unit and a magnetically controlled temperature control unit, and the magnetically controlled temperature control unit includes two customizable temperature areas.

3. A real-time imaging system integrating bright field and fluorescence functions as claimed in claim 1, characterized in that: The excitation module comprises an LED surface light source, a collimating lens and a filter lens group.

4. The real-time imaging system integrating bright field and fluorescence functions as claimed in claim 1, characterized in that: The receiving and imaging module comprises a photosensitive chip, a filter lens group and a lens which are combined for imaging, and is provided with a plurality of switchable filter apertures.

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