Quantitative phase microscopy apparatus and imaging method based on non-rotationally symmetric light source illumination

Abstract

The application discloses a quantitative phase microscope device based on non-rotationally symmetric light source illumination and an imaging method, and belongs to the technical field of optical imaging. The device comprises a cylindrical lens optical system, an electric rotation control unit, an image acquisition unit and a computer processing unit. The cylindrical lens optical system is arranged in a device shell and comprises an optical lens group for converting a microscope light source into an asymmetric illumination light beam and a lens barrel for fixing the optical lens group. The electric rotation control unit drives the lens barrel to rotate around an optical axis to realize multi-directional asymmetric illumination. The image acquisition unit is used for acquiring a light intensity image modulated by a sample. The computer processing unit is used for controlling rotation and acquisition, and performing phase inversion and three-dimensional topography reconstruction on the image. The device has simple structure, high illumination efficiency and strong compatibility, and is suitable for high-precision quantitative phase imaging of transparent and semi-transparent samples.

Description

Technical Field

[0001] This invention relates to the field of optical imaging technology, and in particular to a quantitative phase microscopy device and imaging method based on non-rotationally symmetric light source illumination. Background Technology

[0002] In the field of optical microscopy, transparent and semi-transparent samples (such as biological cells, microlens arrays, and transparent devices) often exhibit extremely weak light absorption, making it difficult to achieve sufficient structural contrast under traditional bright-field microscopy, thus hindering the clear presentation of their morphology and phase information. To address this issue, phase microscopy was proposed and developed. Its core principle is to convert invisible phase differences into detectable changes in light intensity through optical means, thereby enabling the visualization of the structure of transparent samples.

[0003] Traditional phase-contrast microscopy was one of the earliest and most widely used phase imaging techniques. It enhances contrast by introducing an annular aperture and a matching phase ring at the rear focal plane of the objective lens, converting the phase delay caused by the sample into intensity differences. However, this technique has several limitations: First, the system structure is complex and has low adaptability, requiring dedicated phase-contrast objectives and strictly matched phase ring assemblies. Installation and debugging are cumbersome, and specific objectives must be replaced at different magnifications, resulting in poor versatility. Second, illumination efficiency is low; the annular aperture blocks most of the incident light, significantly reducing light throughput and placing higher demands on the image sensor's performance. Third, the high overall system cost, constrained by the precision-machined phase ring and dedicated objectives, makes it difficult to widely apply on ordinary bright-field microscope platforms. Finally, the fixed imaging mode prevents flexible adjustment of illumination direction or phase modulation strategies, limiting its adaptability in measuring the characteristics of different types of transparent samples.

[0004] In recent years, some research has focused on employing digital phase imaging methods, such as digital holography and digital phase contrast techniques, to reduce the complexity of optical systems. However, these methods typically require complex light source modulation, rely on extensive algorithmic processing, or suffer from low system integration, and have yet to achieve low-cost, fast, and universal quantitative phase imaging.

[0005] To address the shortcomings of the existing technologies, it is necessary to propose a quantitative phase microscopy device and imaging method based on non-rotationally symmetric light source illumination. Summary of the Invention

[0006] The purpose of this invention is to provide a quantitative phase microscopy device and imaging method based on non-rotationally symmetric light source illumination, which has the advantages of simple structure, high illumination efficiency, and flexible adjustment of illumination direction, in order to solve the problems of existing phase contrast microscopy imaging systems such as complex structure, low illumination efficiency, high cost, poor flexibility, and insufficient compatibility with general microscopes.

[0007] To achieve the above objectives, in one aspect, the present invention provides a quantitative phase microscopy device based on non-rotationally symmetric light source illumination, comprising: a cylindrical lens optical system disposed within a device housing, including an optical lens group that converts the microscope light source into an asymmetric illumination beam and a microscope tube for fixing the optical lens group; an electric rotation control unit, drivenly connected to the microscope tube, driving the microscope tube to rotate around the optical axis to achieve multi-directional asymmetric illumination; an image acquisition unit for acquiring light intensity images modulated by the sample; and a computer processing unit for controlling the electric rotation control unit and the image acquisition unit, and performing phase inversion and three-dimensional topography reconstruction on the acquired images.

[0008] Furthermore, the optical lens assembly includes a condenser lens assembly and at least one replaceable cylindrical lens, the condenser lens assembly and the microscope light source together forming a Köhler illumination structure.

[0009] Furthermore, the lens barrel is fixed inside the device housing by a lower lens barrel fixing bearing and an upper lens barrel fixing bearing.

[0010] Furthermore, the electric rotation control unit includes a worm gear, a worm, and a stepper motor. The worm gear is fixed to the lens barrel, and the worm meshes with the worm gear and is connected to the output shaft of the stepper motor.

[0011] Furthermore, the electric rotation control unit also includes a microcontroller control unit for controlling the rotation angle and speed of the stepper motor.

[0012] Furthermore, the computer processing unit communicates with the microcontroller control unit via a USB interface to realize the setting of the lighting angle and the synchronous triggering of image acquisition.

[0013] Furthermore, the image acquisition unit employs a CCD or CMOS camera, and its mounting position is aligned with the optical path of the microscope eyepiece.

[0014] Furthermore, the quantitative phase microscopy device based on non-rotationally symmetric light source illumination also includes a microscope condenser lens retaining ring for mounting the entire device at the condenser lens position of the microscope.

[0015] In another aspect, the present invention also provides a quantitative phase microscopy imaging method based on non-rotationally symmetric light source illumination, using the quantitative phase microscopy apparatus based on non-rotationally symmetric light source illumination as described in any of the preceding claims, the imaging method comprising the following steps:

[0016] The quantitative phase microscopy device is installed at the condenser lens position of the microscope.

[0017] The computer processing unit sets the illumination azimuth sequence and controls the electric rotation control unit to drive the cylindrical lens optical system to rotate to the target angle.

[0018] At each illumination azimuth angle, the image acquisition unit is triggered to acquire the light intensity image of the sample, forming a multi-angle light intensity image sequence;

[0019] The light intensity image sequence is preprocessed, and the phase transfer function sequence is calculated based on the phase-intensity modulation model;

[0020] A phase inversion algorithm is used to perform phase inversion and obtain the quantitative phase distribution of the sample;

[0021] The phase distribution is converted into a three-dimensional topography distribution.

[0022] In some imaging methods of the present invention, the illumination azimuth angle includes 0°, 45°, 90°, and 135°.

[0023] Compared with the prior art, the present invention discloses at least the following beneficial effects:

[0024] This invention enables multi-directional asymmetric illumination through electric rotation control, modulates and encodes the phase information of transparent and semi-transparent samples, and combines computer inversion algorithms to achieve rapid and quantitative phase reconstruction, thereby obtaining a high-precision three-dimensional morphology distribution. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the quantitative phase microscopy device based on non-rotationally symmetric light source illumination according to the present invention.

[0027] Figure 2 This is an exploded view of the quantitative phase microscopy device based on non-rotationally symmetric light source illumination according to the present invention;

[0028] Figure 3 This is a schematic diagram of the installation of the quantitative phase microscopy device based on non-rotationally symmetric light source illumination according to the present invention.

[0029] Figure 4 This is a system structure diagram of the quantitative phase microscopy device based on non-rotationally symmetric light source illumination according to the present invention;

[0030] Figure 5 This is a sequence of light intensity images collected at different angles in an embodiment of the present invention;

[0031] Figure 6 This is a quantitative phase distribution map of a sample reconstructed according to an embodiment of the present invention;

[0032] Figure 7 This is a three-dimensional morphological distribution diagram of the sample reconstructed according to an embodiment of the present invention.

[0033] In the diagram: 1. Upper housing of the device; 2. Lower housing of the device; 3. Microscope tube; 4. USB interface; 5. Lower bearing for fixing the microscope tube; 6. Bearing for fixing the stepper motor; 7. Shaft for fixing the stepper motor; 8. Stepper motor; 9. Motor control circuit unit; 10. Worm gear; 11. Microcontroller control unit; 12. Worm wheel; 13. Upper bearing for fixing the microscope tube; 100. Microscope light source; 200. Quantitative phase microscopy device; 300. Microscope condenser lens fixing ring. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] Reference Figures 1 to 4 As shown, this embodiment of the invention provides a quantitative phase microscopy device based on non-rotationally symmetric light source illumination, including a cylindrical lens optical system for generating asymmetric illumination, an electrically driven rotation control unit, an image acquisition unit, and a computer processing unit. Specifically, as... Figure 1 and Figure 2 As shown, the device includes an upper housing 1, a lower housing 2, a lens barrel 3, a USB interface 4, a lower lens barrel fixing bearing 5, a stepper motor fixing bearing 6, a stepper motor fixing shaft 7, a stepper motor 8, a motor control circuit unit 9, a worm gear 10, a microcontroller control unit 11, a worm wheel 12, and an upper lens barrel fixing bearing 13. The lens barrel 3 and the optical lens assembly installed within it constitute a cylindrical lens optical system for generating asymmetric illumination. The lower lens barrel fixing bearing 5, the stepper motor fixing bearing 6, the stepper motor fixing shaft 7, the stepper motor 8, the motor control circuit unit 9, the worm gear 10, the microcontroller control unit 11, the worm wheel 12, and the upper lens barrel fixing bearing 13 together constitute an electric rotation control unit.

[0037] like Figure 3As shown, in use, the quantitative phase microscopy device 200 is installed at the microscope condenser and fixed by the microscope condenser retaining ring 300. The lower housing 2 of the device is directly connected to the microscope condenser interface, and the upper housing 1 is connected to the lower housing by a snap-fit ​​structure for easy disassembly and maintenance. The microscope tube 3 is supported in the housing by the upper bearing 13 and the lower bearing 5, ensuring the accuracy and stability of its rotation around the optical axis. An optical lens group is installed inside the microscope tube 3. This optical lens group consists of cylindrical lenses and a condenser lens group, which is used to uniaxially focus the light emitted by the microscope light source 100 to form an asymmetric illumination spot.

[0038] In this embodiment, the optical lens assembly is installed at the center of the optical axis of the microscope tube 3 to focus the parallel light generated by the microscope light source 100 along a single axis, forming a one-dimensional asymmetric illumination distribution with a specific axis. This optical lens assembly consists of cylindrical lenses and a condenser lens assembly, where the condenser lens assembly and the microscope light source 100 together form a Köhler illumination structure, making the incident light approximately parallel, thereby ensuring a controllable and uniform illumination distribution. It should be understood that in practical applications, the optical lens assembly can be modified by replacing the cylindrical lenses with different focal lengths, materials, or sizes according to experimental requirements to adjust the optical parameters of the asymmetric illumination. For example, a 25mm focal length BK7 cylindrical lens can be used to obtain a typical phase modulation effect. The illumination light passes through the optical lens assembly to form an elliptical spot, i.e., asymmetric illumination light. The angle between the major axis of the elliptical spot and the parallel direction of the short side (or long side) of the target surface of the industrial camera in the image acquisition unit is defined as the illumination azimuth angle. By rotating the lens barrel 3, the illumination direction can be switched at different azimuth angles (such as 0°, 45°, 90°, and 135°), achieving multi-directional asymmetric illumination. The asymmetric illumination characteristic originates from the rotatable optical axis formed by the optical system, and the illumination direction can be changed by rotating the lens barrel 3.

[0039] like Figure 2 As shown, in one specific embodiment, the outer wall of the lens barrel 3 is connected to the worm gear 12 by fastening screws. The worm gear 12 meshes with the worm 10, forming a worm gear 12-worm 10 transmission mechanism to achieve precise angle rotation control of the lens barrel 3. In one specific embodiment, the transmission ratio of the worm gear 12 to the worm 10 is 2:1. The stepper motor 8 is connected to the worm 10 through the stepper motor fixed shaft 7, driving the worm 10 to rotate, thereby driving the lens barrel 3 and the optical lens assembly to rotate around the optical axis. The stepper motor 8 is provided with microstepping pulse drive and regulated power by the motor control circuit unit 9. The motor control circuit unit 9 includes a motor drive module, a power management module, and a communication interface module, used to provide microstepping pulse drive, regulated power supply, and communication with the microcontroller control unit 11 for the stepper motor 8. The motor control circuit unit 9 is connected to the microcontroller control unit 11 to realize closed-loop control of the motor angle. The microcontroller control unit 11 communicates with the computer processing unit through the USB interface 4, receives illumination angle commands, and provides feedback on the rotation status.

[0040] The external power supply and data communication of the entire device can be completed through the USB interface 4. The USB interface 4 can provide power input and also be used for bidirectional data transmission between the host computer and the microcontroller, realizing synchronous triggering of lighting angle setting, rotation status reading and imaging acquisition.

[0041] The electric rotation control unit in this embodiment consists of a stepper motor 8, a worm gear 12-worm 10 transmission mechanism, a bearing support assembly, a motor control circuit unit 9, and a microcontroller control unit 11. The system control flow is as follows: the computer processing unit sends the target rotation angle and illumination sequence commands to the microcontroller control unit 11 via the USB interface 4. After parsing the commands, the microcontroller sends a stepping signal to the motor control circuit unit 9. The motor control circuit unit 9 drives the stepper motor 8 to generate microstepping pulses, achieving high-precision angle rotation. The worm 10 drives the worm gear 12 to rotate, thereby achieving rotation of the lens barrel 3 around the optical axis. The microcontroller control unit 11 monitors the angle feedback signal in real time to ensure closed-loop accuracy. When the cylindrical lens rotates to the preset azimuth angle, the image acquisition unit is automatically triggered to perform exposure and shooting. Through this control flow, automatic switching of the illumination angle and synchronous acquisition by the camera can be achieved, ensuring the consistency and time synchronization of imaging data from various angles.

[0042] The image acquisition unit consists of an industrial camera and its accompanying software. The industrial camera is mounted on the imaging end of the microscope objective and is directly connected to the microscope body (for acquiring image data of the sample after asymmetric illumination, but not directly connected to this device). It connects to the computer processing unit via a USB or Gigabit Ethernet interface to acquire light intensity images modulated by the sample. The computer processing unit runs control and data processing programs to perform image acquisition, storage, preprocessing, phase inversion, and 3D topography reconstruction.

[0043] Therefore, the quantitative phase microscopy device based on non-rotationally symmetric light source illumination proposed in this invention has a simple structure, high illumination efficiency, flexible illumination direction adjustment, and seamless compatibility with conventional microscope systems. Combined with the proposed imaging method, it generates asymmetric illumination through optical lens groups and utilizes an electric rotation drive system to achieve multi-angle switching of the illumination direction, thereby realizing rapid quantitative phase imaging of transparent samples. It has significant application potential in multiple fields such as industrial testing, biomedicine, and materials science.

[0044] This invention also provides a quantitative phase microscopy imaging method based on non-rotationally symmetric light source illumination. Based on the quantitative phase microscopy device based on non-rotationally symmetric light source illumination described in the above embodiments, the method includes the following steps:

[0045] First, the quantitative phase microscopy device 200 is installed at the microscope condenser position, ensuring that the optical assembly is in the illumination path. The illumination azimuth sequence (e.g., 0°, 45°, 90°, 135°) is set via the computer processing unit, where the 0° azimuth is defined as the direction parallel to the short (or long) side of the industrial camera target surface. The command is transmitted via USB interface 4 to the microcontroller control unit 11, which controls the stepper motor 8 to drive the microscope barrel 3 to rotate to the target angle.

[0046] At each illumination azimuth angle, an industrial camera is automatically triggered to acquire light intensity images of the sample, forming a multi-angle light intensity image sequence, specifically as follows: Figure 5 As shown, image data is transmitted to a computer processing unit in real time for preprocessing such as intensity normalization and noise reduction.

[0047] Next, based on the phase-intensity modulation model of asymmetric illumination, the corresponding phase transfer function is calculated according to the system parameters and the current illumination direction, obtaining a phase transfer function sequence corresponding to the light intensity image sequence. Combining the light intensity image sequence, the Tikhonov regularization algorithm is used for phase inversion, suppressing noise and improving the stability and accuracy of phase reconstruction, thus obtaining the quantitative phase distribution of the sample.

[0048] Given the refractive index of the sample material, the reconstructed phase distribution is converted into a high-precision three-dimensional morphology distribution, enabling morphology measurement of transparent or semi-transparent samples.

[0049] In one specific application of this embodiment, the device is mounted on a conventional bright-field microscope, using a white LED light source, a cylindrical lens focal length of 25mm, an industrial camera exposure time of 100ms, and an objective lens magnification of 10×. Light intensity images are acquired at four azimuth angles: 0°, 45°, 90°, and 135°, with a total acquisition time of less than 1 second. After computer inversion processing, a quantitative phase map and three-dimensional morphology map of the microlens array are obtained, as shown below. Figure 6 and Figure 7 As shown, compared with the theoretical design value, the error is within ±3%, which verifies the high precision and feasibility of the method of the present invention.

[0050] It should be understood that this imaging method is applicable to the three-dimensional morphology measurement of transparent and translucent samples, including but not limited to microlens arrays, cells and biological tissues, thin film materials and transparent devices.

[0051] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A quantitative phase microscopy apparatus based on non-rotationally symmetric light source illumination, characterized in that, The quantitative phase microscopy device (200) comprises a column lens optical system arranged in a device housing, a motorized rotation control unit, and an image acquisition unit. The column lens optical system comprises a mirror tube (3) for fixing an optical lens group for converting a microscope light source (100) into an asymmetric illumination light beam, and the optical lens group comprising a condenser lens group and at least one replaceable column lens, wherein the condenser lens group and the microscope light source (100) jointly form a Kohler illumination structure. The motorized rotation control unit is in driving connection with the mirror tube (3) and drives the mirror tube (3) to rotate around an optical axis to realize multi-directional asymmetric illumination. The motorized rotation control unit comprises a worm wheel (12), a worm (10), a stepping motor (8), and a single-chip microcomputer control unit (11). The worm wheel (12) is fixed to the mirror tube (3), the worm (10) is in engagement with the worm wheel (12) and connected with an output shaft of the stepping motor (8), and the single-chip microcomputer control unit (11) is used for controlling the rotation angle and speed of the stepping motor (8).

2. The quantitative phase microscopy apparatus based on non-rotationally symmetric light source illumination of claim 1, wherein, The image acquisition unit is used for acquiring a light intensity image modulated by a sample.

3. The quantitative phase microscopy apparatus based on non-rotationally symmetric light source illumination of claim 1, wherein, The computer processing unit is used for controlling the motorized rotation control unit and the image acquisition unit, and performing phase inversion and three-dimensional topography reconstruction on the acquired image.

4. The quantitative phase microscopy apparatus based on non-rotationally symmetric light source illumination of claim 1, wherein, The mirror tube (3) is fixed in the device housing through a mirror tube fixed lower bearing (5) and a mirror tube fixed upper bearing (13).

5. The quantitative phase microscopy apparatus based on non-rotationally symmetric light source illumination of claim 1, wherein, The computer processing unit communicates with the single-chip microcomputer control unit (11) through a USB interface (4) to realize setting of an illumination angle and synchronous triggering of image acquisition.

6. A quantitative phase microscopy imaging method based on non-rotationally symmetric illumination, based on the quantitative phase microscopy apparatus based on non-rotationally symmetric illumination according to any one of claims 1 to 5, characterized in that, The image acquisition unit adopts a CCD or CMOS camera, and the installation position of the camera is aligned with a microscope eyepiece light path. The microscope condenser lens fixing ring (300) is used for mounting the entire device at a condenser lens position of a microscope. The quantitative phase microscopy device (200) is mounted at a condenser lens position of a microscope. An illumination azimuth angle sequence is set by the computer processing unit, and the motorized rotation control unit is controlled to drive the column lens optical system to rotate to a target angle. At each illumination azimuth angle, the image acquisition unit is triggered to acquire a light intensity image of a sample to form a multi-angle light intensity image sequence. The light intensity image sequence is preprocessed, and a phase transfer function sequence is calculated based on a phase-light intensity modulation model. A phase inversion algorithm is used for phase inversion to obtain a quantitative phase distribution of the sample.

7. The quantitative phase microscopy imaging method based on non-rotationally symmetric light source illumination of claim 6, wherein, The phase distribution is converted into a three-dimensional topography distribution. The illumination azimuth angle comprises 0°, 45°, 90°, and 135°.

Images