A method for adaptive multi-channel fluorescence detection based on brightfield images

Abstract

This invention provides a method for adaptive multi-channel fluorescence detection based on bright-field images, relating to the fields of optical microscopy and biofluorescence detection. The method includes the following steps: under white Köhler illumination, a camera acquires a bright-field image of the sample and transmits it to a control system; the control software creates a Region of Interest (ROI) on the bright-field image with the center of the field of view as the point; the control system calculates the target variable aperture diameter 2R based on the ROI radius r, and controls the aperture of the motorized variable aperture to open to match 2R; according to a pre-stored zoom lens group position parameter table, the axial position of each lens in the movable lens group is adjusted to smoothly change the total magnification |β| of the zoom lens group, ensuring that the spot diameter on the photocathode of the PMT remains constant while the PMT is in a fixed position. The detection method provided by this invention simultaneously possesses spatial selectivity and multi-channel versatility.

Description

Technical Field

[0001] This invention relates to the fields of optical microscopy imaging and biofluorescence detection technology, and in particular to a method for adaptive multi-channel fluorescence detection based on bright-field images. Background Technology

[0002] In biomedical applications such as high-throughput sorting, cell sorting, and single-cell detection of fluorescently labeled microfluidic droplets, the core requirement is to identify individuals containing target fluorescent labels within a large number of droplets / cells and to accurately locate their positions, thereby triggering the sorting process. Regarding the technical problem of "fluorescence detection of specific droplets or cells on a microfluidic chip," the mainstream technical solutions can currently be divided into the following two main categories: Category 1: Fluorescence imaging detection scheme based on CMOS / CCD cameras. This scheme uses a CMOS or CCD camera to perform fluorescence imaging on droplets / cells on a microfluidic chip, and uses image processing algorithms to identify droplets / cells containing fluorescent labels and extract their position coordinates. The advantages of this scheme are: (1) high spatial resolution, which can simultaneously acquire fluorescence distribution information of all droplets / cells in the field of view; (2) good intuitiveness, what you see is what you get, and researchers can directly observe the fluorescence distribution on the image; (3) fast detection speed, and multiple targets can be analyzed simultaneously in a single frame image. Although this scheme has spatial selectivity and intuitiveness, it is limited by the quantum efficiency of the camera (typically 60-80%), readout noise (typically 1-3 electrons) and dynamic range. It is not sensitive enough to detect weak fluorescence signals (such as low-expression protein labels, single-molecule fluorescence), which may lead to "missed detection" problems.

[0003] The second category: point detector fluorescence detection schemes based on photomultiplier tubes (PMTs). This scheme collects the total fluorescence intensity signal from a specific region of the sample by placing the PMT in the center image plane or exit port of the microscope. Because PMTs have extremely high photoelectric gain (typically 10⁻⁶), this method is highly efficient. 6 -10 7 With extremely low dark noise, its weak light detection sensitivity is 3-5 orders of magnitude higher than that of CMOS / CCD cameras, enabling the detection of weak fluorescence signals at the level of a single fluorescent molecule. The advantages of this scheme are: (1) high sensitivity, capable of detecting weak fluorescence signals that are difficult to detect by CMOS / CCD; (2) fast time response, the time response of PMT is usually on the order of nanoseconds, suitable for high-speed detection; (3) high signal-to-noise ratio, the dark noise of PMT is extremely low. Although this scheme has high sensitivity, PMT is essentially a total light intensity detector without spatial resolution, and can only output a total fluorescence intensity signal. Researchers cannot know which region of the sample the PMT specifically collects the fluorescence signal from, and it lacks spatial information. Therefore, it is difficult to accurately locate and select the region of interest that the user is concerned with.

[0004] In existing technologies, CMOS / CCD imaging and PMT detection schemes are typically designed as two independent detection modes, requiring users to choose between them. This makes it difficult to simultaneously achieve the dual advantages of "spatial selectivity" and "high sensitivity." Furthermore, both CMOS and PMT schemes are usually designed for a single wavelength (single laser or single color filter). Changing the fluorescent label requires readjusting the optical path, lacking the versatility to adaptively switch between multiple channels (DAPI / FITC / TRITC / CY5, etc.), thus limiting their application scope. Additionally, when selecting the detection area, researchers cannot directly see the target location on the bright-field image, making it difficult to determine whether the detection is precisely aligned with the target droplet / cell, resulting in a "blind" operation.

[0005] To address the core shortcomings of PMT (Positive Tube Mechanism) in lacking spatial information and the difficulty in integrating the two approaches, patent CN223742267U offers a solution: its core structure is a "moving laser emitter." A second adjustment structure (a second adjustment slide) moves the laser emitter in the X and Y directions to a position corresponding to the target droplet / cell on the microfluidic chip, enabling the laser to precisely excite the fluorescent marker in the target droplet, thus partially improving the positional alignment accuracy on top of the high sensitivity of PMT. While this solution improves positional alignment accuracy to some extent, it introduces new problems such as device complexity, high cost, cumbersome operation, changes in the excitation optical path, and a lack of multi-channel versatility. Furthermore, it still does not solve the core requirement of "how to allow users to intuitively select the region of interest on a brightfield image and perform multi-channel fluorescence detection on that region with high sensitivity." Therefore, there is an urgent need in the field for an adaptive fluorescence detection device and method that simultaneously possesses spatial selectivity, multi-channel versatility, high automation, high sensitivity, simple device design, and convenient operation. Summary of the Invention

[0006] In view of this, the present invention provides a method for adaptive multi-channel fluorescence detection based on bright-field images. The detection method provided by the present invention can simultaneously possess spatial selectivity and multi-channel versatility, and the device used has a simple structure, is easy to operate, and has high sensitivity.

[0007] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: An adaptive multi-channel fluorescence detection method based on bright-field images includes the following steps: Under white Köhler illumination, a camera is used to capture bright-field images of the sample, which are then transmitted to the control system. Using software, a circular region with radius r is drawn on the bright field image with the center of the field of view as the center point, which is designated as the Region of Interest (ROI). The control system calculates the target variable aperture diameter 2R based on the ROI radius r, and controls the aperture of the motorized variable aperture to open to a size matching 2R; the specific mapping relationship between the motorized variable aperture diameter 2R and the circular region radius r is: r = 2R × 500 / M; where M is the objective lens magnification; Based on the pre-stored zoom lens group position parameter table, adjust the axial position of each lens in the movable lens group to smoothly change the total magnification |β| of the zoom lens group, so that the spot diameter on the PMT photocathode remains constant with the PMT in a fixed position; to ensure that the spot diameter on the PMT photocathode is constant at d_PMT, the total magnification |β| of the zoom system must satisfy: |β| = d_PMT / 2R; The zoom lens group consists of a movable lens group located behind the motorized variable aperture and a fixed cemented doublet lens located behind the movable lens group.

[0008] Preferably, the method for adaptive multi-channel fluorescence detection based on bright-field images uses a detection device comprising: Bright-field imaging unit, The bright-field imaging unit includes a white light source, a white light Köhler illumination assembly, a two-dimensional motorized scanning platform, a microscope objective, an objective focusing assembly, and a camera; Multichannel fluorescence unit, The multi-channel fluorescence unit includes a multi-channel fluorescence filter block group, a reflector, a fluorescence light source, a fluorescence Köhler illumination component, a telescope lens, a beam splitter, a motorized variable aperture, a zoom lens group, and a PMT. The electrically adjustable aperture is located at the center image plane position; The zoom lens group consists of a movable lens group and a cemented doublet lens; the movable lens group is located behind the motorized variable aperture and can move axially; the cemented doublet lens is located behind the movable lens group and its position is fixed. The PMT is located behind the cemented doublet lens and its position is fixed. Control system The control system is connected to the two-dimensional motorized scanning platform, the objective lens focusing assembly, the multi-channel fluorescence filter block group, the camera, the motorized variable aperture, the movable lens group, and the PMT.

[0009] Preferably, the two-dimensional electric scanning platform includes an X-axis electric slide and a Y-axis electric slide, used to adjust the position of the microfluidic chip by displacement in the X and Y directions, so that the area to be measured enters the center of the field of view.

[0010] Preferably, the objective lens focusing assembly includes a Z-axis motorized slide and a position feedback element.

[0011] Preferably, the multi-channel fluorescent filter block group includes at least two sets of filter blocks and a Y-axis motorized slide.

[0012] Preferably, the multi-channel fluorescence filter block group includes multiple filter block groups with different wavelengths, and each filter block group contains an excitation filter, a dichroic mirror and an emission filter.

[0013] Preferably, the electrically adjustable aperture is driven by an iris aperture in conjunction with a stepper motor / servo motor, or by a liquid crystal spatial light modulator to achieve continuous adjustment.

[0014] Preferably, the effective diameter of the photocathode of the PMT is 8-25 mm.

[0015] Preferably, the camera is a CCD or CMOS camera.

[0016] Preferably, the white light source is an LED white light source or a halogen lamp.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The adaptive multi-channel fluorescence detection method based on bright-field images provided by this invention marks the ROI on the bright-field image and performs spatially selective PMT detection through an electrically adjustable aperture + zoom lens group. It simultaneously achieves the "spatial selectivity" of CMOS fluorescence imaging and the "high sensitivity" of PMT detection, overcoming the dilemma of insufficient sensitivity of existing CMOS imaging schemes and lack of spatial information in PMT detection schemes. It combines spatial selectivity and high sensitivity.

[0018] The method provided by this invention allows users to simply draw a circle on a brightfield image, and the control system automatically controls the coordinated action of the motorized variable aperture and zoom lens group, achieving a fully automated workflow of "detection upon drawing a circle." By using the position parameter table of the zoom lens group, the size of the light spot on the PMT photocathode can be precisely ensured to remain constant, avoiding detection errors caused by light spot variations and resulting in more accurate detection. Furthermore, since the sensitivity of PMT is 3-5 orders of magnitude higher than that of CMOS / CCD cameras, this invention can detect weak fluorescence signals (such as low-expression protein labels and single-molecule fluorescence) that are difficult to detect with CMOS / CCD, improving detection accuracy and reducing "missed detection" problems. High detection sensitivity is achieved.

[0019] Furthermore, the adaptive multi-channel fluorescence detection device based on bright-field images provided by this invention achieves adaptive switching of multi-channel fluorescence detection on a single hardware platform without the need to replace hardware or readjust the optical path, making it highly versatile and widely applicable. The PMT is the most precise component in this device; fixing its position avoids mechanical vibration, signal drift, and registration errors caused by PMT displacement, significantly improving the long-term stability of the system and reducing dependence on the vibration isolation system. With the laser position fixed, the geometric relationship of the excitation optical path remains constant, fundamentally solving the problem of under-excitation or over-excitation caused by "moving the laser to change the optical path collimation," significantly improving fluorescence excitation efficiency and uniformity.

[0020] This invention adopts a "PMT fixed + lens moving" design, which eliminates the need for complex mechanical structures such as moving lasers and laser precision guide rails, greatly simplifying the device structure and significantly reducing costs; the fixed position of the laser also avoids the problem of excitation optical path collimation caused by laser movement.

[0021] The device provided by this invention is easy to operate and highly efficient: users do not need to understand the complex relationships between optical parameters such as aperture, lens, and PMT, nor do they need to perform complex optical path alignment operations. They only need to draw a circle on the bright field image to complete high-sensitivity multi-channel fluorescence detection of any position and any size area, which greatly improves the ease of operation and is particularly suitable for high-throughput microfluidic microdroplet sorting and other scenarios.

[0022] The device provided by this invention supports both manual and automatic modes: it supports both manual ROI labeling by users and automatic ROI labeling after target identification based on image processing algorithms. This preserves the autonomy of researchers while supporting high-throughput automatic screening, resulting in high application flexibility. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the overall structure of the device provided by the present invention; Figure 2 A flowchart illustrating the detection method provided by this invention; Figure 3 This is a schematic diagram of the position parameters of the zoom lens group provided by the present invention; Among them: 1-White light source, 2-White light Köhler illumination assembly, 3-Microfluidic chip, 4-Two-dimensional motorized scanning platform, 5-Microscope objective, 6-Objective focusing assembly, 7-Multi-channel fluorescence filter block group, 8-Reflector, 9-Fluorescence light source, 10-Eye tube lens, 11-Camera, 12-Beam splitter, 13-Motorized variable aperture, 14-Movable lens group, 15-Cemented doublet lens, 16-PMT, 17-Control system, 18-Fluorescence Köhler illumination assembly. Detailed Implementation

[0024] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0025] Obviously, the accompanying drawings described below are merely some examples or embodiments of the present invention. Those skilled in the art can apply the present invention to other similar scenarios based on these drawings without any inventive effort. Furthermore, it is understood that although the efforts made in this development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this invention, modifications to design, manufacturing, or production based on the technical content disclosed in this invention are merely conventional technical means and should not be construed as insufficient disclosure of the present invention.

[0026] In this invention, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described in this invention may be combined with other embodiments without conflict.

[0027] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. The terms "a," "an," "an," "the," and similar words used in this invention do not indicate quantity limitation and may indicate singular or plural. The terms "comprising," "including," "having," and any variations thereof used in this invention are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or device that includes a series of steps or modules (units) is not limited to the listed steps or units, but may also include steps or units not listed, or may include other steps or units inherent to these processes, methods, products, or devices. The terms "connected," "linked," "coupled," and similar words used in this invention are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "A plurality" in this invention refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships may exist; for example, "A and / or B" can represent: A alone, A and B simultaneously, and B alone. The character " / " generally indicates that the preceding and following objects have an "or" relationship. The terms "first," "second," and "third" used in this invention are merely to distinguish similar objects and do not represent a specific ordering of the objects.

[0028] This invention provides an adaptive multi-channel fluorescence detection method based on bright-field images, comprising the following steps: Under white Köhler illumination, a camera is used to capture bright-field images of the sample, which are then transmitted to the control system. Using software, a circular region with radius r is drawn on the bright field image with the center of the field of view as the center point, which is designated as the Region of Interest (ROI). The control system calculates the target variable aperture diameter 2R based on the ROI radius r, and controls the aperture of the motorized variable aperture to open to a size matching 2R; the specific mapping relationship between the motorized variable aperture diameter 2R and the circular region radius r is: r = 2R × 500 / M; where M is the objective lens magnification; Based on the pre-stored zoom lens group position parameter table, adjust the axial position of each lens in the movable lens group to smoothly change the total magnification |β| of the zoom lens group, so that the spot diameter on the PMT photocathode remains constant with the PMT in a fixed position; to ensure that the spot diameter on the PMT photocathode is constant at d_PMT, the total magnification |β| of the zoom system must satisfy: |β| = d_PMT / 2R; The zoom lens group consists of a movable lens group located behind the motorized variable aperture and a fixed cemented doublet lens located behind the movable lens group.

[0029] The core idea of ​​the adaptive multi-channel fluorescence detection method based on bright-field images provided by this invention is to integrate the bright-field imaging optical path, the multi-channel fluorescence excitation optical path, and the PMT fluorescence collection optical path into the same inverted microscope optical path structure. The control system automatically controls the aperture size of the motorized variable aperture and the position of the movable lens in the zoom lens group based on the position and size of the region of interest (a circle with radius r) marked by the user on the bright-field image. This allows the PMT to efficiently collect fluorescence signals from the region of interest while ensuring a constant spot size on the PMT photocathode (matching the effective photocathode size), thus achieving "what you see is what you get" adaptive high-sensitivity multi-channel fluorescence detection. The flowchart of the adaptive multi-channel fluorescence detection method based on bright-field images provided by this invention is shown below. Figure 2 As shown.

[0030] The adaptive multi-channel fluorescence detection method based on bright-field images described in the above-described scheme of the present invention uses a detection device including a bright-field imaging unit, a multi-channel fluorescence unit, and a control system.

[0031] like Figure 1 As shown, the above bright-field imaging unit includes a white light source 1, a white light Köhler illumination assembly 2, a two-dimensional motorized scanning platform 4, a microscope objective 5, an objective focusing assembly 6, and a camera 11.

[0032] In this invention, the white light source 1 is used to generate white light for bright field illumination. In some embodiments, the white light source 1 is further an LED white light source or a halogen lamp.

[0033] In this invention, the white light Köhler illumination component 2 is used to shape the light from the white light source into a uniform Köhler illumination path, illuminating the sample to form bright-field illumination. This invention does not specifically limit the structure of the white light Köhler illumination component 2; conventional products in the art can be used.

[0034] In this invention, the two-dimensional motorized scanning platform 4 is used to move and adjust the position of the microfluidic chip 3 in the X and Y directions, so that the test area in the sample on the microfluidic chip 3 enters the center of the field of view. In some embodiments, the two-dimensional motorized scanning platform further includes an X-axis motorized slide and a Y-axis motorized slide to facilitate the movement of the microfluidic chip 3.

[0035] In this invention, the microscope objective 5 (the objective of an inverted microscope) is used to collect imaging light signals from the sample. The light signals are bright-field transmitted light or fluorescence signals.

[0036] In this invention, the objective lens focusing assembly 6 is used to precisely adjust the axial position of the objective lens in the Z direction to achieve sample focusing. In some embodiments, the objective lens focusing assembly 6 further includes a Z-axis motorized slide and a position feedback element. This invention does not impose any particular limitation on the specific structure of the motorized slide and the position feedback element; conventional commercially available products in the art can be used.

[0037] In this invention, the camera 11 is used to acquire bright-field images of the sample under bright-field illumination and transmit them to the control system. In some embodiments, the camera 11 is further a CCD or CMOS camera.

[0038] like Figure 1 The aforementioned multi-channel fluorescence unit includes a multi-channel fluorescence filter block group 7, a reflector 8, a fluorescence light source 9, a fluorescence Köhler illumination component 18, a lens barrel 10, a beam splitter 12, an electrically adjustable aperture 13, a zoom lens group, and a PMT 16.

[0039] It should be noted that: Figure 1 The red double-headed arrows indicate that the component can be precisely moved by a motor or electric slide, including a two-dimensional electric scanning platform (X, Y directions), an objective lens focusing assembly (Z direction), a multi-channel fluorescence filter block group (Y direction switching), an electric aperture (aperture), and a movable lens group (X direction movement).

[0040] In this invention, the multi-channel fluorescence filter block group 7 of the multi-channel fluorescence unit is used to switch between the bright-field observation channel and fluorescence excitation / emission channels of different wavelengths. It achieves motorized switching of multiple fluorescence channels without readjusting the optical path, and is the core execution unit for realizing "multi-channel adaptive switching" in this invention. In some embodiments, the multi-channel fluorescence filter block group 7 further includes at least two sets of filter blocks and a Y-axis motorized slide. In some embodiments, the multi-channel fluorescence filter block group 7 further includes multiple filter block groups of different wavelengths (such as DAPI, FITC, TRITC, and CY5 filter block groups), each containing an excitation filter, a dichroic mirror, and an emission filter.

[0041] In this invention, the reflector 8 is used to reflect the bright-field transmitted light path 90° to the lens barrel.

[0042] In this invention, the fluorescent light source 9 and the fluorescent Köhler illumination assembly 18 are used to generate excitation light of a specific wavelength (such as ultraviolet, blue, green, etc.), which, after being shaped by Köhler illumination, enters the sample from the objective lens end to excite the fluorescent markers in the sample. This invention does not impose any particular limitation on the fluorescent light source; conventional products in the art can be used.

[0043] In this invention, the barrel lens 10 is used to image the light signal collected by the objective lens onto the intermediate image plane, and further transmit the light signal to the camera 11 and the PMT 16 channel. In this embodiment, it is represented by a single schematic lens.

[0044] In this invention, the beam splitter 12 is used to split the light from the lens barrel 10 according to wavelength or polarization state, reflect bright field visible light to the camera 11, and transmit fluorescence signals to the PMT16 channel; or vice versa.

[0045] In this invention, the electrically adjustable aperture 13 is located at the intermediate image plane in the PMT fluorescence collection optical path. Its aperture size 2R can be adjusted in real time by the control system, physically defining the source area of ​​the fluorescence signal collected by the PMT 16, so that it corresponds one-to-one with the ROI marked by the user on the bright field image. By adjusting the aperture, the PMT can collect all fluorescence signals within the field of view, achieving spatial selectivity.

[0046] The motorized variable aperture 13 is one of the core execution units of this invention. The aperture adjustment range of the motorized variable aperture 13 is: 2R_min = 2mm, 2R_max = 6.9mm, corresponding to the range of the user's circle radius r (related to the objective lens magnification M): when the objective lens magnification M = 5×: the range of r is 500-3450μm (i.e., 0.5-3.45mm); when the objective lens magnification M = 10×: the range of r is 250-1725μm; when the objective lens magnification M = 20×: the range of r is 125-863μm. The specific mapping relationship is: r(μm) = 2R×500 / M In some embodiments, the electrically adjustable aperture 13 is further driven by an iris aperture in conjunction with a stepper motor / servo motor, or by a liquid crystal spatial light modulator to achieve continuous adjustment. Furthermore, using an iris aperture offers advantages such as good mechanical stability, low cost, and fast response speed.

[0047] In this invention, the zoom lens group consists of a movable lens group 14 and a cemented doublet lens 15; the movable lens group 14, as the first part of the zoom lens group, is located behind the electrically adjustable aperture 13 and consists of one or more axially movable lenses; the control system adjusts the axial position z1, z2, … of each lens through an electric slide, thereby changing the total magnification of the zoom lens group so that the spot size on the PMT photocathode matches the effective photocathode of the PMT.

[0048] The cemented doublet lens 15, as the second part of the zoom lens group, is fixed in position and works with the movable lens group 14 to form a zoom system, providing the main imaging function; at the same time, it serves as a "PMT interface" to stably image the relay image of the motorized variable aperture 13 onto the PMT photocathode.

[0049] In this invention, the movable lens group 14 and the fixed cemented doublet lens 15 together constitute the zoom system of this invention. Its core design is "under the premise that the PMT6 is fixed in a fixed position, by moving the lens in the movable lens group 14 axially, the total magnification |β| of the zoom system is smoothly changed, thereby ensuring that the spot size on the PMT photocathode is constant".

[0050] In this invention, the zoom system design parameters are as follows: L1: Achromatic doublet lens, focal length f1=80mm L2: Achromatic doublet lens, focal length f2=60mm The PMT is fixed 220mm behind the stage rail. Covering the ROI range of 2R = 2mm - 6.9mm Total magnification |β| range: 1.014-3.5 The zoom system position parameters are shown in Table 1: Table 1

[0051] In this invention, the key design principle of the zoom system is: to ensure that the spot diameter on the PMT photocathode remains constant at d_PMT (matching the effective photocathode diameter of the PMT, for example, d_PMT = 7mm), the total magnification |β| of the zoom system must satisfy: |β| = d_PMT / 2R Where 2R is the aperture diameter of the motorized variable aperture (i.e., the diameter of the ROI corresponding to the intermediate image plane).

[0052] For a dual-lens zoom system consisting of L1 (focal length f1 = 80 mm) and L2 (focal length f2 = 60 mm), based on the thin lens imaging equation and the combined magnification equation, the axial positions (z1, z2) of L1 and L2 and their corresponding values ​​in 2R can be calculated under the premise of a fixed PMT position (220 mm from the motorized variable aperture), as shown in Table 1. In this invention, a schematic diagram of the zoom lens group position parameters is shown below. Figure 3 As shown, this illustrates the principle that the axial positions of L1 and L2 and the PMT spot size remain constant under different 2R conditions.

[0053] In this invention, the electric slide table is selected as follows: L1 travel requirement: 17mm (range 31-48mm), accuracy 0.1mm, electric slide driven by stepper motor.

[0054] L2 travel requirement: 82mm (range 55-136mm), accuracy 0.1mm, electric slide driven by stepper motor.

[0055] In this invention, the PMT (photomultiplier tube) 16 is located behind the cemented doublet lens 15 and its position is fixed. The photomultiplier tube is used to collect fluorescence signals from the ROI with high sensitivity and convert them into electrical signals. In some embodiments, the effective diameter of the photocathode of the PMT 16 is further 8-25 mm. The PMT is a highly sensitive device, and mechanical vibration will significantly increase its dark noise and signal fluctuations. In this invention, fixing the PMT can avoid mechanical vibration and signal drift caused by PMT displacement; fixing the PMT is the key to ensuring detection accuracy.

[0056] In this invention, the control system 17 is communicatively connected to the two-dimensional motorized scanning platform 4, the objective lens focusing assembly 6, the multi-channel fluorescence filter block group 7, the camera 11, the motorized variable aperture 13, the movable lens group 14, and the PMT 16. It coordinates and controls the operation of each component. The control system 17 is the "brain" of this invention, communicating with each motorized component to achieve coordinated control of the entire device. It converts the user's ROI markings on the brightfield image into collaborative control commands for the aperture, lens group, and filter block group, realizing a fully automated workflow of "drawing a circle equals multi-channel detection." In this invention, the connection method can be wired or wireless.

[0057] In this invention, the hardware components of the control system 17 include: a main control computer (including CPU, memory, and display); an embedded motion controller (for controlling a multi-axis electric slide); a data acquisition card (for acquiring PMT signals); and a communication interface card (for communicating with the camera and various electric components). It may also include a programmable logic controller (PLC) as a slave device. In this invention, the control system 17 has the following functions: real-time display of the bright-field image acquired by the camera 11; receiving the ROI (circle with radius r) marked by the user on the image; calculating the target variable aperture diameter 2R = 2rM / 500 based on r and the objective lens magnification M; searching for the corresponding (z1, z2) according to the pre-stored zoom system position parameter table; simultaneously driving: the motorized variable aperture 13 to 2R, the motorized slide L1 to z1, and the motorized slide L2 to z2; switching the multi-channel fluorescence filter block group 7 to the corresponding fluorescence channel (DAPI / FITC / TRITC / CY5, etc.); controlling the fluorescence light source switch; acquiring, displaying, saving, and analyzing PMT signals; supporting multiple working modes such as single measurement, continuous measurement, and scanning measurement; and supporting manual / automatic marking of ROIs by the user. This invention does not impose any special limitations on the specific program of the control system; conventional programs in the field can be used.

[0058] Communication protocols: Motorized slide: USB, RS-232, RS-485, EtherCAT, etc.; Motorized variable aperture: USB, RS-232, analog signal; Camera: USB3.0, GigE, Camera Link, etc.; PMT: Analog signal input to data acquisition card; Multi-channel fluorescent color filter block group: USB, RS-232.

[0059] In this invention, a bright-field imaging optical path is formed by: white light source 1 → white light Köhler illumination assembly 2 → microfluidic chip 3 (sample) → microscope objective 5 → multi-channel fluorescence filter block group 7 (switched to bright-field position) → reflector 8 → lens 10 → beam splitter 12 (reflecting bright-field light to camera) → camera 11 → control system 17. This path is used to acquire bright-field images of the sample under white light Köhler illumination, serving as the "what you see is what you get" basis for user-marked ROIs. In this optical path, the bright-field image acquired by the camera is displayed in real time on the control software interface; the user draws a circular area with radius r on the image with the center of the field of view as the ROI using a mouse, touchscreen, or external input device.

[0060] In this invention, a multi-channel fluorescence excitation optical path is formed by a fluorescent light source 9 → a fluorescent Köhler illumination component 18 → a multi-channel fluorescence filter block group 7 (switched to the corresponding fluorescence channel position, including an excitation filter and a dichroic mirror) → a microscope objective 5 → a microfluidic chip 3 (sample). This path is used to excite the sample's ROI region with corresponding wavelengths after the user completes ROI labeling and selects a fluorescence channel. The excitation light is focused by the objective lens and irradiates the sample, exciting the fluorescent markers in the ROI region of the sample to generate fluorescence signals. In this invention, "multi-channel" refers to a multi-channel fluorescence filter block group that includes at least a combination of different excitation / emission wavelengths such as DAPI, FITC, TRITC, and CY5. The control system electrically switches to the corresponding filter block group position according to the fluorescence channel selected by the user in the control software, enabling sequential detection of different fluorescent markers without readjusting the optical path.

[0061] In this invention, the microfluidic chip 3 (sample) → microscope objective 5 → multi-channel fluorescence filter block group 7 (including emission filter to filter out excitation light) → beam splitter 12 (transmitting fluorescence to the PMT channel) → motorized variable aperture 13 → movable lens group 14 (zoom) → cemented doublet lens 15 (fixed) → PMT 16 (fixed) form a PMT fluorescence collection optical path for collecting fluorescence signals from the ROI region and efficiently coupling them to the PMT photocathode for high-sensitivity detection. In this optical path, the motorized variable aperture is located at the middle image plane, and its aperture size 2R determines the size of the sample ROI region corresponding to the fluorescence signal passing through the aperture. The movable lens group 14 and the fixed cemented doublet lens 15 form a zoom system, which adaptively adjusts the total magnification |β| according to the ROI size to ensure that the spot diameter on the PMT photocathode is constant (e.g., 7 mm) and matches the effective photocathode diameter of the PMT.

[0062] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1 Multichannel adaptive fluorescence detection in microfluidic microdroplet sorting Application scenarios This embodiment will be applied to a high-throughput sorting system for microfluidic microdroplets based on fluorescent labeling. Under multi-channel fluorescent labeling (DAPI / FITC / TRITC / CY5) conditions, the fluorescence signal intensity within a specific droplet on the microfluidic chip will be detected, and the sorting electrode will be triggered based on the fluorescence intensity to sort the droplets.

[0064] System Configuration Microscope structure: Inverted fluorescence microscope as the basic platform Objective lens: 10× objective lens (NA=0.3), M=10 Camera: High-sensitivity CMOS camera, 2048×2048 pixels, pixel size 6.5μm Fluorescence channels: DAPI / FITC / TRITC / CY5 four channels (excitation / emission wavelengths are 405 / 460, 488 / 525, 561 / 595, 640 / 670nm respectively) Multi-channel fluorescence filter block set: includes four filter blocks: DAPI, FITC, TRITC, and CY5, with motorized switching. PMT: High-sensitivity photomultiplier tube, with an effective photocathode diameter of 8mm. Motorized variable aperture: Motorized iris aperture + stepper motor driven, aperture range 0.5-12mm Movable lens group L1: Ø25.4mm achromatic doublet lens, f1=80mm, mounted on a stepper motor-driven electric slide. Movable lens group L2: Ø25.4mm achromatic doublet lens, f2=60mm, mounted on a stepper motor-driven electric slide. Fixed cemented bilayer lens: Ø25.4mm achromatic bilayer lens, f=60mm, fixed installation. PMT Position: Fixed 220mm behind the motorized variable aperture. Work steps Step S1: Bright-field image acquisition The white LED light source is illuminated, and the multi-channel fluorescence filter group switches to the bright field position (without filters). The white light Köhler illumination component shapes the white light into uniform illumination light, which is then projected onto the sample on the microfluidic chip. The microscope objective collects the bright field transmitted light from the sample, which is then imaged onto a CMOS camera after passing through the multi-channel fluorescence filter group, a mirror, a lens in the microscope tube, and a beam splitter. The CMOS camera acquires bright field images in real time at a frame rate of 10fps and transmits them to the PC of the control system via a USB 3.0 interface. The control software on the PC displays the bright field images in real time.

[0065] Step S2: ROI Marking and Multi-Channel Selection Researchers used the mouse to drag a circular region with a radius of r = 500 μm (corresponding to 50 pixels) on the bright-field image of the control software interface, using the center of the field of view as the ROI. They then selected the target fluorescence channel as FITC from the software's drop-down menu. This ROI corresponds to a droplet region with a diameter of approximately 50 μm on the microfluidic chip. The control software used (r = 500 μm, M = 10, channel = FITC) as input parameters.

[0066] Step S3: Parameter Calculation (Simultaneous Solution) The control software calculates the target variable aperture diameter based on r and M: 2R = 2r × M / 500 = 2 × 500 × 10 / 500 = 20mm However, since the effective diameter of the PMT photocathode is 8mm and the spot diameter d_PMT=7mm (smaller than the photocathode diameter, leaving a margin), the maximum supported 2R_max=7mm / 1.014=6.9mm. When 2R=20mm exceeds the maximum value supported by the aperture stop, the control software automatically prompts the user to reduce the ROI range or switch to a higher magnification objective lens.

[0067] Step S4: Coordinated control of electric components (synchronous drive) The control software queries the pre-stored zoom lens group position parameter table and obtains the following: when 2R=2mm, β=3.5, L1 position z1=39.8mm, L2 position z2=54.6mm (using the minimum 2R=2mm value supported by this embodiment). The control software simultaneously drives the following via motion control software: the stepper motor of the electric variable aperture to adjust the aperture diameter to 2R=2mm; the electric slide (driving L1) to move L1 to z1=39.8mm; and the electric slide (driving L2) to move L2 to z2=54.6mm. After the drive is completed, the control software confirms that each component has reached the target position through position feedback signals.

[0068] Step S5: Multi-channel switching The control software drives the multi-channel fluorescent filter block group to switch to the FITC channel position (including a 488nm excitation filter, a 505nm dichroic mirror, and a 525nm emission filter), and illuminates the 488nm fluorescent light source.

[0069] Step S6: PMT fluorescence detection The 488nm excitation light, after passing through a fluorescent Köhler illumination assembly, a multi-channel fluorescent filter group, and an objective lens, illuminates the sample ROI region on the microfluidic chip. The fluorescent markers within the ROI region are excited to generate a 525nm fluorescence signal. This fluorescence signal is then imaged onto the PMT photocathode after passing through the objective lens, the multi-channel fluorescent filter group, a beam splitter, a motorized variable aperture (limited to an aperture of 2R=2mm), a movable lens group (L1+L2 zooming to β=3.5), and a fixed cemented doublet lens. Due to the zoom system, the spot diameter on the PMT photocathode is 2R×β=2×3.5=7mm, matching the effective photocathode of the PMT.

[0070] The PMT converts the fluorescence signal into an electrical signal, which is then acquired by a data acquisition card (sampling rate 1kHz) and transmitted to the control software. The control software displays the PMT signal intensity curve over time in real time and calculates statistical parameters such as average fluorescence intensity, peak-to-peak value, and standard deviation.

[0071] Step S7: Multichannel sequence detection (if required) If the user needs to detect the same ROI in multiple channels sequentially (e.g., FITC→TRITC→CY5), the control software repeats steps S5 and S6, sequentially switching the multi-channel fluorescence filter block group to the corresponding channel and acquiring the PMT signal. In this embodiment, after the FITC channel detection is completed, the control software automatically switches to the TRITC channel and repeats S5-S6, finally obtaining the fluorescence intensity of the ROI in both the FITC and TRITC channels.

[0072] Example Effects This embodiment achieves adaptive high-sensitivity fluorescence detection by "drawing a circle for multi-channel detection": researchers draw a circle with r=25μm on a bright-field image, select the FITC channel, and the PMT can perform high-sensitivity fluorescence detection on that area under the FITC channel; if it is necessary to switch channels, there is no need to readjust the optical path, just select the new channel in the software; the light spot on the PMT photocathode is stable at 7mm, which matches the effective photocathode of the PMT, resulting in high detection sensitivity and good stability.

[0073] Example 2 ROI scanning in single-cell multichannel fluorescence quantitative analysis Application scenarios This embodiment applies the present invention to single-cell multichannel fluorescence quantitative analysis, measuring the fluorescence intensity of different subcellular regions (nucleus, cytoplasm, cell membrane) under multiple fluorescence channels (DAPI / FITC / TRITC) separately.

[0074] Workflow Steps S1-S4: Same as in Example 1, the user sequentially marks three ROIs on the bright field image: cell nucleus (r=10μm), cytoplasm (r=30μm), and cell membrane (r=50μm), and selects the target channel DAPI.

[0075] Step S5: The control software calculates 2R for each ROI, looks up the position parameter table, and drives the motorized variable aperture and movable lens group.

[0076] Step S6: Perform PMT fluorescence detection on each ROI sequentially and record the fluorescence intensity of the DAPI channel.

[0077] Step S7: The control software switches the target channel to FITC and TRITC, and repeats steps S5-S6 to obtain the fluorescence intensity matrix of the three ROIs in the three channels of DAPI / FITC / TRITC.

[0078] Data Analysis: The control software outputs single-cell multi-channel fluorescence quantitative analysis results such as "nucleo-cytoplasmic ratio" based on the fluorescence intensity ratio of the cell nucleus, cytoplasm, and cell membrane in the three channels.

[0079] Example Effects This embodiment demonstrates the application potential of the present invention in single-cell multi-channel fluorescence quantitative analysis: by drawing circles on different subcellular regions on a brightfield image and switching the multi-channel fluorescence filter blocks in sequence, the fluorescence intensity matrix of each region in each channel can be obtained quickly without complicated optical path switching or realignment.

[0080] Example 3 Automated ROI labeling and multichannel detection in high-throughput microdroplet screening Application scenarios This embodiment applies the present invention to high-throughput microdroplet screening. The target droplets on the microfluidic chip are automatically identified by a target detection algorithm based on deep learning, and the ROI is automatically marked. The control software sequentially switches the multi-channel fluorescent filter block group (DAPI / FITC / TRITC / CY5) to perform high-sensitivity detection on each droplet in multiple channels.

[0081] Workflow Step S1: The camera acquires a bright-field image.

[0082] Step S2 (Automatic Mode): The control software runs the pre-trained YOLOv8 target detection model, automatically identifies target droplets in the bright field image, and automatically marks the ROI at each target droplet (radius r is automatically calculated from the droplet size); at the same time, according to the preset multi-channel detection sequence (DAPI→FITC→TRITC→CY5), S3-S6 are executed sequentially.

[0083] Steps S3-S6: Perform parameter calculation, motor component control, multi-channel switching, and PMT fluorescence detection sequentially for each automatically marked ROI.

[0084] Example Effects This embodiment demonstrates the application capability of the present invention in high-throughput multi-channel automatic screening: the combination of deep learning-based automatic ROI marking, electric component collaborative control, multi-channel adaptive switching, and PMT high-sensitivity detection can achieve a throughput of tens to hundreds of droplets per second, and the fluorescence information of each droplet under multi-channel conditions can be obtained simultaneously, far exceeding the existing manual alignment scheme.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for adaptive multi-channel fluorescence detection based on brightfield images, characterized in that, Includes the following steps: Under white Köhler illumination, a camera is used to capture bright-field images of the sample, which are then transmitted to the control system. Using software, a circular region with radius r is drawn on the bright-field image with the center of the field of view as the radius, which is designated as the Region of Interest (ROI). The control system calculates the target variable aperture diameter 2R based on the ROI radius r, and controls the aperture of the motorized variable aperture to open to a size matching 2R; the specific mapping relationship between the motorized variable aperture diameter 2R and the circular region radius r is: r = 2R×500 / M; where M is the objective lens magnification; Based on the pre-stored zoom lens group position parameter table, adjust the axial position of each lens in the movable lens group to smoothly change the total magnification |β| of the zoom lens group, so that the spot diameter on the PMT photocathode remains constant with the PMT in a fixed position; to ensure that the spot diameter on the PMT photocathode is constant at d_PMT, the total magnification |β| of the zoom system must satisfy: |β| = d_PMT / 2R; The zoom lens group consists of a movable lens group located behind the motorized variable aperture and a fixed cemented doublet lens located behind the movable lens group.

2. The method for adaptive multi-channel fluorescence detection based on bright-field images according to claim 1, characterized in that, The testing equipment used during the testing includes: Bright-field imaging unit, The bright-field imaging unit includes a white light source, a white light Köhler illumination assembly, a two-dimensional motorized scanning platform, a microscope objective, an objective focusing assembly, and a camera; Multichannel fluorescence unit, The multi-channel fluorescence unit includes a multi-channel fluorescence filter block group, a reflector, a fluorescence light source, a fluorescence Köhler illumination component, a telescope lens, a beam splitter, a motorized variable aperture, a zoom lens group, and a PMT. The electrically adjustable aperture is located at the center image plane position; The zoom lens group consists of a movable lens group and a cemented doublet lens; the movable lens group is located behind the motorized variable aperture and can move axially; the cemented doublet lens is located behind the movable lens group and its position is fixed. The PMT is located behind the cemented doublet lens and its position is fixed. Control system The control system is connected to the two-dimensional motorized scanning platform, the objective lens focusing assembly, the multi-channel fluorescence filter block group, the camera, the motorized variable aperture, the movable lens group, and the PMT.

3. The method according to claim 2, characterized in that, The two-dimensional electric scanning platform includes an X-axis electric slide and a Y-axis electric slide, which are used to adjust the position of the microfluidic chip by displacement in the X and Y directions, so that the area to be measured enters the center of the field of view.

4. The method according to claim 2, characterized in that, The objective lens focusing assembly includes a Z-axis motorized slide and a position feedback element.

5. The method according to claim 2, characterized in that, The multi-channel fluorescent filter block group includes at least two sets of filter blocks and a Y-axis motorized slide.

6. The method according to claim 5, characterized in that, The multi-channel fluorescence filter block group includes multiple filter block groups with different wavelengths. Each filter block group contains an excitation filter, a dichroic mirror, and an emission filter.

7. The method according to claim 2, characterized in that, The electrically adjustable aperture is driven by an iris aperture in conjunction with a stepper motor / servo motor, or by a liquid crystal spatial light modulator to achieve continuous adjustment.

8. The method according to claim 2, characterized in that, The effective diameter of the photocathode of the PMT is 8-25 mm.

9. The method according to claim 2, characterized in that, The camera is a CCD or CMOS camera.

10. The method according to claim 2, characterized in that, The white light source is an LED white light source or a halogen lamp.

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