Resolution detection system and method suitable for annular scanning two-photon imaging

Abstract

The present invention discloses a resolution detection system and method suitable for annular scanning two-photon imaging. The system comprises: a light source module, a dichroic mirror, an annular glass container, a reflective element having a parabola, a rotary drive mechanism for driving the reflective element to rotate about the Z axis, a linear drive mechanism for driving the annular glass container to perform linear motion in the Z direction, a photomultiplier tube, a host computer, and a software module. The present invention uses the rotary drive mechanism to drive the reflective element to rotate about the Z axis, while simultaneously performing an annular scanning of the annular glass container containing fluorescent spheres. The linear drive mechanism drives the reflective element to move along the Z axis to achieve axial scanning of the annular glass container, thereby achieving omnidirectional scanning of the annular glass container. Imaging results of the fluorescent spheres are obtained through image reconstruction. Finally, the resolution of the annular scanning two-photon imaging system is calculated by analyzing the number of pixels occupied by the fluorescent spheres and the actual size of the fluorescent spheres.

Description

Technical Field

[0001] The present invention relates to the field of optical imaging resolution detection, and in particular to a resolution detection system and method suitable for annular scanning two-photon imaging. Background Art

[0002] Among the many high-resolution optical microscopy techniques that use fluorescent markers, two-photon imaging offers inherent tomographic capabilities, submicron spatial resolution, and millisecond real-time performance. Furthermore, its penetration depth can reach nearly 1 mm, making it one of the best methods for studying neural activity signals in animals in vivo. In recent decades, two-photon imaging has been widely used in biomedical research, particularly among neuroscientists for in vivo studies of neural function. Compared to other fluorescence imaging techniques, two-photon imaging's unique advantage in studying neural activity in intact, living brains lies in its accuracy in highly scattering and densely fluorescently labeled brain tissue. It acquires images pixel by pixel with minimal background signal interference, thus directly achieving optical resolution close to the diffraction limit without the need for deconvolution or other complex mathematical reconstruction techniques.

[0003] Currently, nearly all two-photon microscopy systems, regardless of their form and imaging principle, are constrained by a fundamental design paradigm, limiting their field of view to a limited viewing angle directly in front of the optical objective lens. The annular scanning two-photon microscope represents an original technological innovation within this fundamental design paradigm for microscopic optics. It enables 360-degree continuous lateral cylindrical scanning, achieving panoramic mesoscopic imaging with submicron precision across depths of up to several centimeters or even tens of centimeters in the direction of the endoscopic canal. This two-photon imaging device is particularly suitable for in vivo imaging of neural morphology and functional activity with single-cell and subcellular precision in large animals, including non-human primates.

[0004] In conventional two-photon microscopy imaging system research, the USAF1951 resolution plate is often used as a standard sample to test the system's lateral resolution. This simple and easy-to-use test method is not suitable for annular scanning two-photon microscopy imaging systems. Therefore, a new lateral resolution test method suitable for annular scanning two-photon imaging systems is needed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a resolution detection system and method suitable for annular scanning two-photon imaging in response to the above-mentioned deficiencies in the prior art.

[0006] To solve the above technical problems, the present invention adopts a technical solution: a resolution detection system suitable for annular scanning two-photon imaging, comprising: a light source module, a dichroic mirror, an annular glass container, a reflective element having a parabola, a rotational drive mechanism for driving the reflective element to rotate about the Z axis, a linear drive mechanism for driving the annular glass container to perform linear motion along the Z direction, a photomultiplier tube, a host computer, and a software module embedded in the host computer;

[0007] The interior of the annular glass container is filled with a carrier, and fluorescent beads are evenly distributed in the carrier; the cavity in the middle of the annular glass container forms a scanning cavity, and the reflective element is arranged in the scanning cavity and can rotate about the Z axis to perform an annular scanning of the fluorescent beads inside the annular glass container;

[0008] The laser light emitted by the light source module transmits through the dichroic mirror and illuminates the parabola of the reflective element. After being reflected by the parabola, it illuminates the fluorescent spheres in the annular glass container. The fluorescence emitted by the fluorescent spheres is reflected in sequence by the parabola and the dichroic mirror. The fluorescence is then reflected by the photomultiplier tube. In conjunction with the rotation of the reflective element around the Z axis and the linear motion of the annular glass container along the Z axis, the reflective element performs an annular scanning of the annular glass container. Then, an image of all the fluorescent spheres in the annular glass container is reconstructed by the host computer connected to the photomultiplier tube, and the resolution of the annular two-photon imaging system is ultimately obtained through this image.

[0009] Preferably, the light source module includes a femtosecond laser and a beam expanding element, and the femtosecond laser emitted by the femtosecond laser is expanded by the beam expanding element into a beam with a spot size capable of completely covering the parabola.

[0010] Preferably, the reflective element is an off-axis parabolic reflector.

[0011] Preferably, the carrier is agar.

[0012] Preferably, a filter is further provided between the photomultiplier tube and the dichroic mirror.

[0013] Preferably, the system further comprises a code disc arranged on the motor, wherein the code disc is used to locate the starting position of the rotation of the reflective element and to count the number of rotations of the reflective element.

[0014] Preferably, the annular glass container is arranged on a stage, the linear drive mechanism includes a linear guide rail, a slider arranged on the linear guide rail, and a linear motor for driving the slider to move on the linear guide rail, and the stage is arranged on the slider.

[0015] Preferably, the software module includes an imaging module, a code disk feedback module, a control module and a data processing module, and the host computer is connected to the light source module, the rotary drive mechanism, the linear drive mechanism, the photomultiplier tube and the code disk.

[0016] Preferably, the system is used to perform annular scanning two-photon imaging resolution detection, the steps of which include:

[0017] 1) The laser light emitted by the light source module passes through the dichroic mirror and then irradiates the parabola of the reflective element;

[0018] 2) The rotary drive mechanism drives the reflective element to rotate, the code disk counts the number of revolutions of the reflective element, and the focal position of the parabola of the reflective element performs a circular scan on the annular glass container containing the fluorescent balls;

[0019] 3) The linear drive mechanism drives the stage to move linearly, causing the annular glass container to move in the Z direction relative to the reflective element, so that the focal position of the parabola of the reflective element performs axial scanning on the annular glass container;

[0020] 4) The rotational drive mechanism and the linear drive mechanism operate simultaneously, causing the reflective element to simultaneously perform rotational motion around the Z axis and linear motion along the Z axis, and the focus of the parabola excites the fluorescent sphere to emit fluorescence, which is then reflected to the dichroic mirror;

[0021] 5) The fluorescence is further reflected by the dichroic mirror, passes through the filter, and is collected by the photomultiplier tube. The optical signal is converted into an electrical signal and transmitted to the host computer. The imaging module in the software module performs image reconstruction to obtain an image of all fluorescent beads in the annular glass container.

[0022] 6) The data processing module analyzes the number of pixels occupied by the diameter of a single fluorescent ball in the imaging image of the fluorescent ball, calculates the size of the single fluorescent ball, and compares it with the actual size of the fluorescent ball to analyze and obtain the minimum resolution that the annular two-photon imaging system can distinguish.

[0023] Preferably, the method for image reconstruction in step 5) is: taking each circle of collected data as a row of the image, sequentially splicing to form several rows, arranging and reconstructing the image, and then filtering and smoothing the image to obtain an imaging image of all fluorescent spheres.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a resolution detection system suitable for annular scanning two-photon imaging. A rotary drive mechanism drives a reflective element to rotate about the Z axis to perform annular scanning of an annular glass container containing fluorescent beads. A linear drive mechanism drives the reflective element to move along the Z axis to achieve axial scanning of the annular glass container. The rotary drive mechanism and the linear drive mechanism cooperate to achieve omnidirectional scanning of the annular glass container, and an imaging result of the fluorescent beads is obtained through image reconstruction. Finally, the imaging resolution of the annular scanning two-photon system can be calculated by comparing and analyzing the measured value of the fluorescent bead size calculated based on the number of pixels occupied by the fluorescent bead and the actual size of the fluorescent bead.

[0026] The present invention can use fluorescent beads of different diameters for testing. It only needs to prepare samples of fluorescent bead solutions of different specifications and encapsulate them in an annular container to perform resolution testing using the system. The operation is simple and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the structure of the resolution detection system suitable for annular scanning two-photon imaging of the present invention;

[0028] Figure 2 This is an imaging diagram of a 50 μm fluorescent bead tested in an embodiment of the present invention.

[0029] Description of reference numerals:

[0030] 1—light source module; 2—photomultiplier tube; 3—linear drive mechanism; 4—rotary drive mechanism; 5—code disk; 6—reflective element; 60—parabola; 7—annular glass container; 8—stage; 9—dichroic mirror; 10—filter; 30—linear guide rail; 31—slider; 32—linear motor. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0032] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0033] like Figure 1 As shown, a resolution detection system suitable for annular scanning two-photon imaging according to this embodiment includes: a light source module 1, a dichroic mirror 9, an annular glass container 7, a reflective element 6 having a parabola 60, a rotational drive mechanism 4 for driving the reflective element 6 to rotate about the Z axis, a linear drive mechanism 3 for driving the annular glass container 7 to perform linear motion along the Z direction, a photomultiplier tube 2, a host computer, and a software module embedded in the host computer;

[0034] The interior of the annular glass container 7 is filled with a carrier, in which fluorescent beads are evenly distributed. The cavity in the middle of the annular glass container 7 forms a scanning cavity. The reflective element 6 is disposed in the scanning cavity and can rotate about the Z axis to perform an annular scanning of the fluorescent beads inside the annular glass container 7.

[0035] The laser light emitted by the light source module 1 passes through the dichroic mirror 9 and irradiates the parabola 60 of the reflective element 6. After being reflected by the parabola 60, it irradiates the fluorescent balls in the annular glass container 7. The fluorescence emitted by the fluorescent balls is reflected by the parabola 60 and the dichroic mirror 9 in sequence, and then is reflected by the photomultiplier tube 2. In conjunction with the rotation of the reflective element 6 around the Z axis and the linear motion of the annular glass container 7 along the Z axis, the reflective element 6 performs an annular scanning of the annular glass container 7. Then, the upper computer connected to the photomultiplier tube 2 reconstructs the image of all the fluorescent balls in the annular glass container 7, and finally obtains the resolution of the annular two-photon imaging system through this image.

[0036] In a preferred embodiment, the light source module 1 includes a femtosecond laser and a beam expander. The femtosecond laser light emitted by the femtosecond laser is expanded by the beam expander to a beam with a spot size that can completely cover the parabola 60. In a further preferred embodiment, the light source is a femtosecond laser with a wavelength of 920 nm. After optical path expansion, the spot size of the beam is 6.35 mm, the energy distribution of the spot is uniform, and the beam can completely cover the parabola 60 for exciting fluorescence.

[0037] In a preferred embodiment, the carrier is agar, the annular glass container 7 has an inner diameter of 10.60 mm, an outer diameter of 17.00 mm, a length of 40.00 mm, and a wall thickness of 1 mm. One end is sealed and the other end is open, and is used to store a mixture of fluorescent beads and agar.

[0038] In a further preferred embodiment, the fluorescent beads used are of four specifications, with diameters of 1 μm, 2 μm, 10 μm and 50 μm, respectively, and are bright green in color. The excitation wavelength is 488 nm, and the emission wavelength is 525 nm. Purified agar powder is used as agar, and an agar solution is prepared by pouring 33 g of agar powder into 1000 ml of water. After heating and dissolving, the agar is completely melted to form a transparent colloidal solution. 5 ml of the agar solution is added to 500 ml of the fluorescent bead solution and mixed thoroughly to prepare a mixed solution of fluorescent beads and agar. After the agar cools down and solidifies, the fluorescent beads will be fixed, so that the fluorescent beads are evenly distributed, which is convenient for imaging observation.

[0039] In a preferred embodiment, the reflective element 6 is an off-axis parabola 60. In a further preferred embodiment, the off-axis parabola 60 is an Edmund Optics 37-282 reflector with an effective focal length of 6.35 mm, and is used to reflect and converge the expanded laser beam.

[0040] In a preferred embodiment, a filter 10 is further provided between the photomultiplier tube 2 and the dichroic mirror 9 . The filter 10 is used to filter out the excitation light and transmit the fluorescence.

[0041] In a preferred embodiment, the rotary drive mechanism 4 is a brushless motor. In a further preferred embodiment, the model of the brushless motor is Chihai BLDC4260, and the speed is set to 7200 rpm, which is used to drive the parabola 60 to rotate and realize the annular scanning of the parabola 60 in the annular glass container 7.

[0042] In a preferred embodiment, the annular glass container 7 is placed on a stage 8. The linear drive mechanism 3 includes a linear guide 30, a slider 31 mounted on the linear guide 30, and a linear motor 32 for driving the slider 31 on the linear guide 30. The stage 8 is mounted on the slider 31. In a further preferred embodiment, the linear drive mechanism 3 is a Z-axis linear ball guide, model KZL06075, which is used to drive the stage 8 to achieve Z-axis scanning of the annular glass container 7. The stage 8 is a custom-made Z-shaped stage 8 for placing the sample in the annular glass container 7. The Z-shape can balance the height of the sample placement, ensuring that the sample, light source, and parabola 60 are on the same horizontal plane.

[0043] In a preferred embodiment, the system also includes a code disk 5 mounted on the motor. This code disk 5 is used to locate the starting position of the reflective element 6 and count the number of revolutions of the reflective element 6. In a further preferred embodiment, the code disk 5 has 1000 grids per revolution and can output ABZ three-phase pulses for aligning the acquisition starting position. With each revolution of the brushless motor, the code disk 5 outputs a high-level pulse signal at a fixed position, thereby implementing the counting function.

[0044] In a preferred embodiment, the software module includes an imaging module, a code disk 5 feedback module, a control module, and a data processing module. The host computer is connected to the light source module 1, the rotary drive mechanism 4, the linear drive mechanism 3, the photomultiplier tube 2, and the code disk 5, and each mechanism is controlled by the control module. Information fed back by the code disk 5 is transmitted to the control module.

[0045] The host computer is a Dell graphical workstation with excellent processing capabilities and multiple PCIe slots for acquisition cards, GPUs, signal generator cards, etc. It is used for image acquisition and display, and works with internally embedded software modules to perform imaging and data processing, ultimately achieving detection with circular scanning two-photon imaging resolution.

[0046] In a preferred embodiment, the steps of performing annular scanning two-photon imaging resolution detection using the system include:

[0047] 1) The laser light (wavelength 920nm, beam spot size 6.35mm) emitted by the light source module 1 passes through the dichroic mirror 9 and irradiates the parabola 60 of the reflective element 6;

[0048] 2) The rotary drive mechanism 4 drives the reflective element 6 to rotate, and the code disk 5 counts the number of revolutions of the reflective element 6. The focal position of the parabola 60 of the reflective element 6 performs a circular scan on the annular glass container 7 containing the fluorescent beads;

[0049] 3) The linear drive mechanism 3 drives the stage 8 to move linearly, causing the annular glass container 7 to move in the Z direction relative to the reflective element 6, so that the focal position of the parabola 60 of the reflective element 6 performs an axial scan on the annular glass container 7;

[0050] 4) The rotary drive mechanism 4 and the linear drive mechanism 3 operate simultaneously, causing the reflective element 6 to simultaneously rotate about the Z axis and linearly move along the Z axis. The focus of the parabola 60 excites the fluorescent beads to emit fluorescence, which is then reflected to the dichroic mirror 9.

[0051] 5) The fluorescence is further reflected by the dichroic mirror 9, passes through the filter 10 (which filters out the 920nm excitation light and emits the 525nm fluorescence), and is then collected by the photomultiplier tube 2. The optical signal is converted into an electrical signal and transmitted to the host computer. The imaging module in the software module reconstructs the image to obtain an image of all the fluorescent beads in the annular glass container 7;

[0052] Among them, the method for image reconstruction is: each circle of collected data is used as the first row of the image, the second circle as the second row, and they are sequentially spliced ​​to form several rows to obtain the final two-dimensional image, and then the image is filtered and smoothed to obtain an imaging map of all fluorescent spheres with a high signal-to-noise ratio.

[0053] 6) The data processing module analyzes the number of pixels occupied by the diameter of a single fluorescent bead in the imaging image of the fluorescent bead, calculates the size corresponding to each pixel using the scanning length of each circle and the number of samples of each circle of data, uses this value to calculate the size of a single fluorescent bead, and compares it with the actual size of the fluorescent bead to analyze and obtain the minimum resolution that can be distinguished by the annular two-photon imaging system.

[0054] Reference Figure 2 , which is an image of a 50μm fluorescent sphere tested using the system of the present invention. The bright spots in the image are fluorescent spheres with a diameter of 50μm. By measuring the corresponding pixel numbers of multiple fluorescent spheres with the system software and comparing them with the actual sizes, the minimum lateral resolution size of the annular scanning two-photon imaging system can be obtained.

[0055] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A resolution detection system suitable for annular scanning two-photon imaging, characterized in that: The system includes: a light source module, a dichroic mirror, an annular glass container, a reflective element having a parabola, a rotation drive mechanism for driving the reflective element to rotate about a Z axis, a linear drive mechanism for driving the annular glass container to perform linear motion along the Z direction, a photomultiplier tube, a host computer, and a software module embedded in the host computer; The interior of the annular glass container is filled with a carrier, and fluorescent beads are evenly distributed in the carrier; the cavity in the middle of the annular glass container forms a scanning cavity, and the reflective element is arranged in the scanning cavity and can rotate about the Z axis to perform an annular scanning on the fluorescent beads inside the annular glass container; The laser light emitted by the light source module transmits through the dichroic mirror and illuminates the parabola of the reflective element. After being reflected by the parabola, it illuminates the fluorescent spheres in the annular glass container. The fluorescence emitted by the fluorescent spheres is reflected in sequence by the parabola and the dichroic mirror, and then collected by the photomultiplier tube. In conjunction with the rotation of the reflective element around the Z axis and the linear motion of the annular glass container along the Z axis, the reflective element performs an annular scanning of the annular glass container. Then, an image of all the fluorescent spheres in the annular glass container is reconstructed by the host computer connected to the photomultiplier tube, and the resolution of the annular two-photon imaging system is ultimately obtained through this image.

2. The resolution detection system for annular scanning two-photon imaging according to claim 1, characterized in that: The light source module includes a femtosecond laser and a beam expansion element. The femtosecond laser emitted by the femtosecond laser is expanded by the beam expansion element into a beam with a spot size capable of completely covering the parabola.

3. The resolution detection system for annular scanning two-photon imaging according to claim 1, characterized in that: The reflective element is an off-axis parabolic reflector.

4. The resolution detection system for annular scanning two-photon imaging according to claim 1, characterized in that: The carrier is agar.

5. The resolution detection system for annular scanning two-photon imaging according to claim 1, characterized in that: A filter is also provided between the photomultiplier tube and the dichroic mirror.

6. The resolution detection system for annular scanning two-photon imaging according to claim 5, characterized in that: The system further comprises a code disk arranged on a motor used as a rotation driving mechanism, wherein the code disk is used to locate a starting position of rotation of the reflective element and to count the number of revolutions of the reflective element.

7. The resolution detection system for annular scanning two-photon imaging according to claim 6, characterized in that: The annular glass container is arranged on a stage, the linear drive mechanism includes a linear guide rail, a slider arranged on the linear guide rail, and a linear motor for driving the slider to move on the linear guide rail, and the stage is arranged on the slider.

8. The resolution detection system for annular scanning two-photon imaging according to claim 7, characterized in that: The software module includes an imaging module, a code disk feedback module, a control module and a data processing module. The host computer is connected to the light source module, the rotary drive mechanism, the linear drive mechanism, the photomultiplier tube and the code disk.

9. The resolution detection system for annular scanning two-photon imaging according to claim 8, characterized in that: The system is used to perform resolution detection of annular scanning two-photon imaging, and the steps include: 1) The laser light emitted by the light source module passes through the dichroic mirror and then irradiates the parabola of the reflective element; 2) The rotary drive mechanism drives the reflective element to rotate, the code disk counts the number of revolutions of the reflective element, and the focal position of the parabola of the reflective element performs a circular scan on the annular glass container containing the fluorescent balls; 3) The linear drive mechanism drives the stage to move linearly, causing the annular glass container to move in the Z direction relative to the reflective element, so that the focal position of the parabola of the reflective element performs axial scanning on the annular glass container; 4) The rotary drive mechanism and the linear drive mechanism operate simultaneously, causing the reflective element to simultaneously perform rotational motion around the Z axis and linear motion along the Z axis, and the focus of the parabola excites the fluorescent sphere to emit fluorescence, which is then reflected to the dichroic mirror; 5) The fluorescence is further reflected by the dichroic mirror, passes through the filter, and is collected by the photomultiplier tube. The optical signal is converted into an electrical signal and transmitted to the host computer. The imaging module in the software module performs image reconstruction to obtain an image of all fluorescent beads in the annular glass container. 6) The data processing module analyzes and obtains the number of pixels occupied by the diameter of a single fluorescent bead in the imaging image of the fluorescent bead, calculates the size of the single fluorescent bead, and compares it with the actual size of the fluorescent bead to analyze and obtain the minimum resolution that can be distinguished by the annular two-photon imaging system.

10. The resolution detection system for annular scanning two-photon imaging according to claim 9, characterized in that: The method for image reconstruction in step 5) is as follows: each circle of collected data is taken as a row of the image, and several rows are sequentially spliced ​​together to reconstruct the image; then, the image is filtered and smoothed to obtain an image of all fluorescent spheres.

Images