CN115077872A - Resolution detection system and method suitable for annular scanning two-photon imaging - Google Patents
Resolution detection system and method suitable for annular scanning two-photon imaging Download PDFInfo
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- CN115077872A CN115077872A CN202210741960.8A CN202210741960A CN115077872A CN 115077872 A CN115077872 A CN 115077872A CN 202210741960 A CN202210741960 A CN 202210741960A CN 115077872 A CN115077872 A CN 115077872A
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- G01M—TESTING STATIC OR DYNAMIC BALANCE OF MACHINES OR STRUCTURES; TESTING OF STRUCTURES OR APPARATUS, NOT OTHERWISE PROVIDED FOR
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- G01M11/0242—Testing optical properties by measuring geometrical properties or aberrations
- G01M11/0257—Testing optical properties by measuring geometrical properties or aberrations by analyzing the image formed by the object to be tested
Abstract
The invention discloses a resolution detection system and a method suitable for annular scanning two-photon imaging, wherein the system comprises: the device comprises a light source module, a dichroic mirror, an annular glass container, a reflecting element with a paraboloid, a rotary driving mechanism for driving the reflecting element to rotate around a Z axis, a linear driving mechanism for driving the annular glass container to perform linear motion along the Z direction, a photomultiplier, an upper computer and a software module. The invention drives the reflection element to rotate around the Z axis through the rotary driving mechanism, synchronously carries out annular scanning on the annular glass container filled with the fluorescent beads, drives the reflection element to move along the Z axis through the linear driving mechanism to realize the axial scanning of the annular glass container, thereby realizing the omnibearing scanning of the annular glass container, obtaining the imaging result of the fluorescent beads through image reconstruction, and finally, calculating the resolution ratio of the annular scanning two-photon imaging system through analyzing the number of pixels occupied by the fluorescent beads and the real size of the fluorescent beads.
Description
Technical Field
The invention relates to the field of optical imaging resolution detection, in particular to a resolution detection system and method suitable for annular scanning two-photon imaging.
Background
Among many high-resolution optical microscopic imaging technologies using fluorescent markers, the two-photon imaging technology has natural tomography capability, submicron-level spatial resolution and millisecond-level real-time performance, and the penetration depth can reach nearly 1mm, which is one of the best methods for the study of the in vivo nerve activity signals of animals at present. Two-photon imaging technology has been widely used in biomedical research in recent decades, especially in neuroscience, for in vivo nerve function research. Two-photon imaging has the unique advantage over other fluorescence imaging techniques in studying the complete live brain neural activity of accuracy in highly scattering and densely fluorescently labeled brain tissue, acquiring images pixel-by-pixel with minimal background signal interference, and thus directly achieving optical resolution near the diffraction limit without the need for deconvolution or other complex mathematical reconstructions.
Almost all two-photon microscopic imaging systems, regardless of their morphology and imaging principles, are limited by the most basic design paradigm, with the field of view limited to a limited range of viewing angles directly in front of the optical objective. The annular scanning two-photon microscope makes original technical innovation on the basic design mode of micro-optics, can realize 360-degree continuous rotation lateral cylindrical surface scanning, and can realize surrounding panoramic mesoscopic imaging in the depth range of several centimeters or even tens of centimeters continuously covered in the direction of an endoscopic pipeline with submicron precision.
In the conventional two-photon microscope imaging system research, a USAF1951 resolution plate is usually used as a standard template to detect the transverse resolution of the system, and the test method is simple and easy to operate. However, this method is not applicable to the ring scanning two-photon microscope imaging system, and therefore, it is necessary to design a new lateral resolution detection method suitable for the ring scanning two-photon imaging system.
Disclosure of Invention
The present invention provides a resolution detection system and method suitable for ring scanning two-photon imaging, aiming at the above deficiencies in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: a resolution detection system suitable for ring scanning two-photon imaging, the system comprising: the device comprises a light source module, a dichroic mirror, an annular glass container, a reflecting element with a paraboloid, a rotary driving mechanism for driving the reflecting element to rotate around a Z axis, a linear driving mechanism for driving the annular glass container to perform linear motion along the Z direction, a photomultiplier, an upper computer and a software module embedded in the upper computer;
the inside of the annular glass container is filled with a carrier, and fluorescent small balls are uniformly distributed in the carrier; the cavity in the middle of the annular glass container forms a scanning cavity, and the reflecting element is arranged in the scanning cavity and can rotate around a Z axis so as to perform annular scanning on the fluorescent small balls in the annular glass container;
the laser emitted by the light source module penetrates through the dichroic mirror and then irradiates onto the paraboloid of the reflecting element, the laser is reflected by the paraboloid and then irradiates onto the small fluorescent balls in the annular glass container, the fluorescent light emitted by the small fluorescent balls is reflected by the paraboloid and the dichroic mirror in sequence and then is subjected to annular scanning of the reflecting element on the annular glass container by the photomultiplier tube in cooperation with rotation of the reflecting element around the Z axis and linear motion of the annular glass container along the Z axis, then an imaging diagram of all the small fluorescent balls in the annular glass container is obtained through reconstruction of the upper computer connected with the photomultiplier tube, and the resolution of the annular two-photon imaging system is finally obtained through the imaging diagram.
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 having a spot size capable of completely covering the paraboloid.
Preferably, the reflective element is an off-axis parabolic mirror.
Preferably, the carrier is agar.
Preferably, a filter is further disposed between the photomultiplier and the dichroic mirror.
Preferably, the system further comprises a code wheel provided on the motor for locating a home position of rotation of the reflective element and counting a number of revolutions of the reflective element.
Preferably, the ring-shaped glass container is arranged on an object stage, the linear driving mechanism comprises a linear guide rail, a slide block arranged on the linear guide rail and a linear motor for driving the slide block to move on the linear guide rail, and the object stage is arranged on the slide block.
Preferably, the software module comprises an imaging module, a code disc feedback module, a control module and a data processing module, and the upper computer is connected with the light source module, the rotary driving mechanism, the linear driving mechanism, the photomultiplier and the code disc.
Preferably, the system is used for ring scanning two-photon imaging resolution detection, and the steps comprise:
1) the laser emitted by the light source module is transmitted by the dichroic mirror and then irradiates the paraboloid of the reflecting element;
2) the rotary driving mechanism drives the reflecting element to rotate, the coded disc counts the number of rotating circles of the reflecting element, and the focus position of the paraboloid of the reflecting element performs annular scanning on the annular glass container filled with the fluorescent small balls;
3) the linear driving mechanism drives the objective table to perform linear movement, so that the annular glass container performs Z-direction movement relative to the reflecting element, and the paraboloid focal position of the reflecting element performs axial scanning on the annular glass container;
4) the rotary driving mechanism and the linear driving mechanism work simultaneously, so that the reflecting element performs rotary motion around the Z axis and linear motion along the Z axis simultaneously, the small fluorescent balls are excited at the focus of the paraboloid to emit fluorescent light, and the fluorescent light is reflected to the dichroic mirror;
5) the fluorescence is continuously reflected by the dichroic mirror, is collected by the photomultiplier after passing through the optical filter, converts optical signals into electric signals and transmits the electric signals to the upper computer, and an imaging module in the software module is used for carrying out image reconstruction to obtain imaging images of all the small fluorescent balls in the annular glass container;
6) the data processing module analyzes the number of pixels occupied by the diameter of a single fluorescent small ball in an imaging picture of the fluorescent small ball, calculates the size of the single fluorescent small ball, compares the size with the real size of the fluorescent small ball, and analyzes the size to obtain the minimum resolution which can be distinguished by the annular two-photon imaging system.
Preferably, the method for reconstructing an image in step 5) includes: and taking each acquired turn of data as a line of the image, sequentially splicing the acquired data to form a plurality of lines, arranging and reconstructing the image, and then filtering and smoothing the image to obtain an imaging image of all the fluorescent spheres.
The beneficial effects of the invention are:
the resolution detection system suitable for annular scanning two-photon imaging provided by the invention has the advantages that a rotary driving mechanism drives a reflecting element to rotate around a Z axis to carry out annular scanning on an annular glass container filled with fluorescent beads, a linear driving mechanism drives the reflecting element to move along the Z axis to realize axial scanning of the annular glass container, the rotary driving mechanism and the linear driving mechanism are matched to realize all-dimensional scanning of the annular glass container, an imaging result of the fluorescent beads is obtained through image reconstruction, and finally, the measurement value of the size of the fluorescent beads and the real size of the fluorescent beads are calculated through the number of pixels occupied by the fluorescent beads to carry out contrastive analysis, so that the imaging resolution of an annular scanning two-photon system can be calculated;
the invention can use the fluorescent beads with different diameters for testing, and can use the system for testing the resolution ratio only by making samples of fluorescent bead solutions with different specifications and packaging the samples in the annular container, thereby having simple and convenient operation.
Drawings
FIG. 1 is a schematic diagram of a resolution detection system suitable for ring scanning two-photon imaging according to the present invention;
FIG. 2 is an image of a 50 μm fluorescent bead tested in an embodiment of the invention.
Description of reference numerals:
1-a light source module; 2-photomultiplier tube; 3-a linear drive mechanism; 4-a rotary drive mechanism; 5-code disc; 6-a reflective element; 60-paraboloid; 7-ring-shaped glass container; 8, an objective table; 9-dichroic mirror; 10-an optical filter; 30-linear guide rail; 31-a slide block; 32-linear motor.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
As shown in fig. 1, a resolution detection system suitable for ring scanning two-photon imaging of the present embodiment includes: the device comprises a light source module 1, a dichroic mirror 9, an annular glass container 7, a reflecting element 6 with a paraboloid 60, a rotary driving mechanism 4 for driving the reflecting element 6 to rotate around a Z axis, a linear driving mechanism 3 for driving the annular glass container 7 to perform linear motion along the Z direction, a photomultiplier 2, an upper computer and a software module embedded in the upper computer;
the inside of the annular glass container 7 is filled with a carrier, and fluorescent small balls are uniformly distributed in the carrier; a scanning cavity is formed in a cavity in the middle of the annular glass container 7, and the reflecting element 6 is arranged in the scanning cavity and can rotate around the Z axis so as to perform annular scanning on the fluorescent small balls in the annular glass container 7;
laser emitted by the light source module 1 penetrates through the dichroic mirror 9 and then irradiates onto a paraboloid 60 of the reflecting element 6, the laser is reflected by the paraboloid 60 and then irradiates onto small fluorescent balls in the annular glass container 7, fluorescent light emitted by the small fluorescent balls is reflected by the paraboloid 60 and the dichroic mirror 9 in sequence and then is subjected to annular scanning of the reflecting element 6 on the annular glass container 7 by the photomultiplier tube 2 in cooperation with rotation of the reflecting element 6 around the Z axis and linear movement of the annular glass container 7 along the Z axis, then imaging images of all the small fluorescent balls in the annular glass container 7 are obtained through reconstruction of an upper computer connected with the photomultiplier tube 2, and the resolution of the annular two-photon imaging system is finally obtained through the imaging images.
In a preferred embodiment, the light source module 1 includes a femtosecond laser and a beam expanding element, and the femtosecond laser emitted from the femtosecond laser is expanded by the beam expanding element into a beam with a spot size capable of completely covering the paraboloid 60. In a further preferred embodiment, the light source is a femtosecond laser with a wavelength of 920nm, the light beam is expanded through a light path, the spot size of the light beam is 6.35mm, the energy distribution of the spot is uniform, and the light beam can completely cover the paraboloid 60 and is used for exciting fluorescence.
In a preferred embodiment, the carrier is agar, the glass annular container 7 has an inner diameter of 10.60mm, an outer diameter of 17.00mm, a length of 40.00mm and a wall thickness of 1mm, and is closed at one end and open at the other end for storing a mixture of fluorescent beads and agar.
In a further preferred embodiment, the used fluorescent beads have four specifications, the diameters of the fluorescent beads are 1 μm, 2 μm, 10 μm and 50 μm respectively, the color of the fluorescent beads is highlight green, the excitation wavelength is 488nm, the emission wavelength is 525nm, the agar is purified agar powder, agar solution is prepared by pouring 33g of agar powder into 1000ml of water, the agar is completely melted by heating and is transparent colloidal solution, 5ml of agar solution is added into 500ml of fluorescent bead solution for fully mixing, mixed solution of the fluorescent beads and the agar is prepared, and the solidified agar after cooling fixes the fluorescent beads, so that the fluorescent beads are uniformly distributed and are convenient for imaging observation.
In a preferred embodiment, the reflective element 6 is an off-axis parabolic 60 mirror. In a further preferred embodiment, off-axis paraboloid 60 reflects a model Edmund Optics 37-282 with an effective focal length of 6.35mm for reflecting and focusing the expanded laser light.
In a preferred embodiment, a filter 10 is further disposed between the photomultiplier tube 2 and the dichroic mirror 9, and the filter 10 is used for filtering out the excitation light and transmitting the fluorescence.
In a preferred embodiment, the rotary drive mechanism 4 is a brushless motor, and in a further preferred embodiment, the brushless motor is a fly BLDC4260, and the rotation speed is set to 7200 rpm for rotating the paraboloid 60 to realize circular scanning of the paraboloid 60 in the circular glass container 7.
In a preferred embodiment, the ring-shaped glass container 7 is disposed on the stage 8, the linear driving mechanism 3 includes a linear guide 30, a slider 31 disposed on the linear guide 30, and a linear motor 32 for driving the slider 31 to move on the linear guide 30, and the stage 8 is disposed on the slider 31. In a further preferred embodiment, the linear driving mechanism 3 is a Z-axis linear ball guide, which is of a type of KZL06075, and is used for driving the object stage 8 to move so as to realize Z-axis scanning of the annular glass container 7. The stage 8 is a custom-made Z-stage 8 for placing the sample of the glass annular container 7, the Z-stage being capable of balancing the height of the sample placement so that the sample, the light source and the paraboloid 60 are in a horizontal plane.
In a preferred embodiment, the system further comprises a code wheel 5 arranged on the motor, the code wheel 5 being used to locate the starting position of rotation of the reflective element 6 and to count the number of revolutions of the reflective element 6. In a further preferred embodiment, the code wheel 5 has 1000 circles of grids, and ABZ three-phase pulses can be output and used for aligning the acquisition starting positions. When the brushless motor rotates for one circle, the coded disc 5 outputs a high-level pulse signal once at a fixed position, thereby realizing the counting function.
In a preferred embodiment, the software module comprises an imaging module, a code wheel 5 feedback module, a control module and a data processing module, the upper computer is connected with the light source module 1, the rotary driving mechanism 4, the linear driving mechanism 3, the photomultiplier tube 2 and the code wheel 5, and the control module controls the mechanisms. The information fed back by the code wheel 5 is transmitted into the control module.
The upper computer is a DELL graphical workstation, has better processing capacity, is provided with a plurality of PCIe card slots, and can be inserted with a collection card, a GPU, a signal generator card and the like. The system is used for realizing image acquisition and display, and realizing imaging and data processing by matching with an internally embedded software module so as to finally realize the detection of the annular scanning two-photon imaging resolution.
In a preferred embodiment, the step of using the system for ring scanning two-photon imaging resolution detection comprises:
1) laser (wavelength 920nm, light beam spot size 6.35mm) emitted by the light source module 1 is transmitted through the dichroic mirror 9 and then irradiated onto the paraboloid 60 of the reflecting element 6;
2) the rotary driving mechanism 4 drives the reflecting element 6 to rotate, the coded disc 5 counts the number of rotating circles of the reflecting element 6, and the focus position of the paraboloid 60 of the reflecting element 6 carries out annular scanning on the annular glass container 7 filled with the fluorescent small balls;
3) the linear driving mechanism 3 drives the object stage 8 to perform linear movement, so that the annular glass container 7 performs Z-direction movement relative to the reflecting element 6, and the focal position of the paraboloid 60 of the reflecting element 6 performs axial scanning on the annular glass container 7;
4) the rotary drive mechanism 4 and the linear drive mechanism 3 operate simultaneously, so that the reflecting element 6 performs rotary motion around the Z axis and linear motion along the Z axis simultaneously, the small fluorescent balls are excited at the focal point of the paraboloid 60 to emit fluorescent light, and the fluorescent light is reflected to the dichroic mirror 9;
5) the fluorescence is continuously reflected by the dichroic mirror 9, is collected by the photomultiplier 2 after passing through the optical filter 10 (filtering out excitation light with the wavelength of 920nm and emitting fluorescence with the wavelength of 525 nm), converts an optical signal into an electric signal and transmits the electric signal to an upper computer, and image reconstruction is carried out through an imaging module in the software module to obtain an imaging picture of all the small fluorescent balls in the annular glass container 7;
the image reconstruction method comprises the following steps: and sequentially splicing each acquired circle of data as a first line of the image and a second circle of data as a second line to form a plurality of lines to obtain a final two-dimensional image, and then filtering and smoothing the image to obtain an imaging picture of all the fluorescent beads with high signal-to-noise ratio.
6) The data processing module analyzes the number of pixels occupied by the diameter of a single fluorescent small ball in an imaging graph of the fluorescent small ball, calculates the size corresponding to each pixel by using the scanning length of each circle and the sampling number of data of each circle, calculates the size of the single fluorescent small ball by using the value, compares the size with the real size of the fluorescent small ball, and analyzes to obtain the minimum resolution which can be distinguished by the annular two-photon imaging system.
Referring to fig. 2, an image of a 50 μm fluorescent bead tested by the system of the present invention is shown, wherein the bright spots in the image are fluorescent beads with a diameter of 50 μm, the number of corresponding pixels of a plurality of fluorescent beads is measured by system software, and the minimum resolution of the lateral resolution of the ring scanning two-photon imaging system can be obtained by comparing the actual size.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. A resolution detection system suitable for ring scanning two-photon imaging, the system comprising: the device comprises a light source module, a dichroic mirror, an annular glass container, a reflecting element with a paraboloid, a rotary driving mechanism for driving the reflecting element to rotate around a Z axis, a linear driving mechanism for driving the annular glass container to perform linear motion along the Z direction, a photomultiplier, an upper computer and a software module embedded in the upper computer;
the inside of the annular glass container is filled with a carrier, and fluorescent small balls are uniformly distributed in the carrier; the cavity in the middle of the annular glass container forms a scanning cavity, the reflecting element is arranged in the scanning cavity and can rotate around a Z axis, and the reflecting element is used for circularly scanning the fluorescent small balls in the annular glass container;
the laser emitted by the light source module penetrates through the dichroic mirror and then irradiates onto the paraboloid of the reflecting element, the laser is reflected by the paraboloid and then irradiates onto the small fluorescent balls in the annular glass container, the fluorescent light emitted by the small fluorescent balls is sequentially reflected by the paraboloid and the dichroic mirror and then collected by the photomultiplier tube, the annular scanning of the reflecting element on the annular glass container is realized by matching with the rotation of the reflecting element around the Z axis and the linear motion of the annular glass container along the Z axis, then, the imaging images of all the small fluorescent balls in the annular glass container are obtained through the reconstruction of the upper computer connected with the photomultiplier tube, and the resolution of the annular two-photon imaging system is finally obtained through the imaging images.
2. The resolution detection system suitable for annular scanning two-photon imaging according to claim 1, wherein the light source module comprises a femtosecond laser and a beam expanding element, and the femtosecond laser emitted by the femtosecond laser is expanded into a light beam with a spot size capable of completely covering the paraboloid by the beam expanding element.
3. The resolution detection system suitable for annular scanning two-photon imaging according to claim 1, wherein the reflective element is an off-axis parabolic mirror.
4. A resolution detection system suitable for use in ring scanning two-photon imaging according to claim 1, wherein said carrier is agar.
5. The resolution detection system suitable for annular scanning two-photon imaging according to claim 1, wherein a light filter is further arranged between the photomultiplier and the dichroic mirror.
6. A resolution detection system suitable for ring scanning two-photon imaging according to claim 5, further comprising a code wheel disposed on said motor, said code wheel being configured to position a home position of rotation of said reflective element and count a number of rotations of said reflective element.
7. A resolution detection system suitable for ring scanning two-photon imaging according to claim 6, wherein said ring-shaped glass container is disposed on a stage, said linear driving mechanism comprises a linear guide, a slide block disposed on said linear guide, and a linear motor for driving said slide block to move on said linear guide, and said stage is disposed on said slide block.
8. The resolution detection system suitable for annular scanning two-photon imaging according to claim 7, wherein the software module comprises an imaging module, a code disc feedback module, a control module and a data processing module, and the upper computer is connected with the light source module, the rotary driving mechanism, the linear driving mechanism, the photomultiplier and the code disc.
9. The resolution detection system suitable for ring scanning two-photon imaging according to claim 8, wherein the resolution detection of ring scanning two-photon imaging is performed by the system, comprising the steps of:
1) the laser emitted by the light source module is transmitted by the dichroic mirror and then irradiates the paraboloid of the reflecting element;
2) the rotary driving mechanism drives the reflecting element to rotate, the coded disc counts the number of rotating circles of the reflecting element, and the focus position of the paraboloid of the reflecting element performs annular scanning on the annular glass container filled with the fluorescent small balls;
3) the linear driving mechanism drives the objective table to perform linear movement, so that the annular glass container performs Z-direction movement relative to the reflecting element, and the paraboloid focal position of the reflecting element performs axial scanning on the annular glass container;
4) the rotary driving mechanism and the linear driving mechanism work simultaneously, so that the reflecting element performs rotary motion around the Z axis and linear motion along the Z axis simultaneously, the small fluorescent balls are excited at the focus of the paraboloid to emit fluorescent light, and the fluorescent light is reflected to the dichroic mirror;
5) the fluorescence is continuously reflected by the dichroic mirror, is collected by the photomultiplier after passing through the optical filter, converts optical signals into electric signals and transmits the electric signals to the upper computer, and an imaging module in the software module is used for carrying out image reconstruction to obtain imaging images of all the small fluorescent balls in the annular glass container;
6) the data processing module analyzes the number of pixels occupied by the diameter of a single fluorescent small ball in an imaging picture of the fluorescent small ball, calculates the size of the single fluorescent small ball, compares the size with the real size of the fluorescent small ball, and analyzes the size to obtain the minimum resolution which can be distinguished by the annular two-photon imaging system.
10. The resolution detection system suitable for annular scanning two-photon imaging according to claim 9, wherein the method for image reconstruction in the step 5) is as follows: and taking each acquired turn of data as a line of the image, sequentially splicing the acquired data to form a plurality of lines, arranging and reconstructing the image, and then filtering and smoothing the image to obtain an imaging picture of all the fluorescent beads.
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