A two-photon microscopy imaging method and apparatus for simultaneous multi-region aberration correction
By splitting the beam and combining it with a multifocal generation and scanning imaging module, aberrations are measured and corrected, solving the problem of slow aberration correction speed in two-photon microscopy under large field of view, and realizing high-resolution imaging of dynamic signals across the entire field of view.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUAZHONG UNIV OF SCI & TECH
- Filing Date
- 2023-09-28
- Publication Date
- 2026-06-30
AI Technical Summary
Traditional two-photon microscopy imaging methods have slow aberration correction speeds in large fields of view, and the dynamic signals of the entire field of view from graded-index lenses cannot be corrected for aberrations, resulting in reduced imaging resolution.
The beam is split by a phase control unit and a phase relay unit, and combined with a multifocal generation module and a scanning imaging module. Aberrations are measured and corrected by modal wavefront detection method, so as to achieve simultaneous aberration correction in multiple regions.
It improves the speed of aberration correction, enables high-resolution imaging of dynamic signals across the entire field of view, and solves the problem of slow aberration correction speed under large field of view.
Smart Images
Figure CN117289444B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microscopic imaging, and more specifically, relates to a two-photon microscopic imaging method and apparatus for simultaneous multi-region aberration correction. Background Technology
[0002] The spatial resolution of two-photon microscopy is limited not only by the bandwidth of the imaging system itself (diffraction-limited resolution) but also by wavefront distortion caused by defects in the optical system and the inhomogeneity of the optical properties of biological samples. To address this issue, aberration correction has been introduced into two-photon microscopy, significantly improving the imaging resolution in living tissues.
[0003] However, traditional aberration correction methods have a small effective field of view (<100 micrometers). Due to the complexity and diversity of biological tissues, aberration characteristics at each location are inconsistent, making aberration correction difficult in large fields of view. Generally, aberration correction needs to be performed in a small field of view, and then stitched together to form a large field of view high-resolution image.
[0004] In neuronal studies of deep brain regions, the self-focusing properties of graded-index lenses allow for imaging of these regions by embedding them. Since two-photon microscopy has a large penetration depth, combining graded-index lenses with two-photon microscopy can acquire three-dimensional dynamic information of deep mouse brain regions. However, due to limitations in manufacturing and design processes, graded-index lenses often exhibit significant aberrations, with inconsistent aberration characteristics at each location. Multiple aberration corrections are required for graded-index lenses, followed by image stitching to correct aberrations across the entire imaging area. This makes it impossible to simultaneously correct aberrations across the entire area of the graded-index lens, thus hindering aberration correction for large field-of-view neuronal dynamic signals. Summary of the Invention
[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a two-photon microscopy imaging method and apparatus for simultaneous multi-region aberration correction, thereby solving the problems of slow aberration correction speed in large-field imaging using traditional two-photon microscopes and the inability to correct aberrations in dynamic signals across the entire field of view during graded refractive index imaging.
[0006] To achieve the above objectives, according to a first aspect of the present invention, a two-photon microscopy imaging device with simultaneous multi-region aberration correction is provided, comprising: an aberration control module, a multifocal generation module, a scanning imaging module, an imaging objective, a fluorescence collection module, and an aberration correction module.
[0007] The aberration control module includes a phase adjustment unit and a phase relay unit;
[0008] After the laser beam undergoes wavefront phase adjustment by the phase modulation unit, it is conjugated to the multifocal generation module by the phase relay unit to form multiple focal points. Then, it is conjugated to the scanning imaging module for scanning and conjugated to the rear pupil of the imaging objective. The imaging objective then focuses the laser beam to form multiple focal points, thereby exciting the object to be imaged to emit fluorescence. The fluorescence collection module collects the fluorescence for imaging.
[0009] The phase control unit is divided into multiple regions, serving as multiple sub-control units. The aberration correction module is used to obtain the optimal amplitude a of sub-phase control unit i in aberration mode j. ij Calculate the corresponding wavefront phase compensation amount. It is then loaded onto the corresponding sub-control unit i to change the wavefront phase control amount of the light beam incident on its surface by the sub-control unit, thereby controlling the aberration of the focal point generated by the beam after focusing, and the imaging is performed again after adjustment to obtain an image after multi-region aberration correction.
[0010] Wherein, the optimal amplitude a ij In aberration mode j, multiple aberration perturbations of different amplitudes are applied to the laser beam through the sub-phase control unit i, and the imaging is performed to obtain the corresponding imaging image. The amplitude corresponding to the best image evaluation index is obtained by fitting all amplitudes and their corresponding imaging image evaluation indexes; i = 1 to n, where n is the number of sub-phase control units.
[0011] According to a second aspect of the present invention, a two-photon microscopy imaging method with simultaneous multi-region aberration correction is provided, applied to the apparatus described in the first aspect, comprising:
[0012] Offline calibration stage:
[0013] Obtain the optimal amplitude a of sub-phase control unit i in aberration mode j. ij Calculate the corresponding wavefront phase compensation amount. And load it onto the corresponding sub-control unit i to change the wavefront phase control amount of the sub-control unit on the beam incident on its surface, thereby controlling the focal point generated by the beam after focusing;
[0014] Wherein, the optimal amplitude a ij In aberration mode j, multiple aberration perturbations of different amplitudes are applied to the laser beam through the sub-phase control unit i and the imaging is performed to obtain the corresponding imaging image. The amplitude corresponding to the best image evaluation index is obtained by fitting all the amplitudes and the evaluation index of the corresponding imaging image.
[0015] Online calibration phase:
[0016] In f iAfter being loaded onto the corresponding sub-control unit i, the imaging is performed again to obtain an image with multi-region aberration correction.
[0017] According to a third aspect of the invention, a computer-readable storage medium is provided, the computer-readable storage medium storing computer instructions for causing a processor to perform the method as described in the second aspect.
[0018] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects:
[0019] The present invention provides a two-photon microscopy imaging method and apparatus for simultaneous multi-region aberration correction. By splitting the beam controlled by the phase modulator into several parts and combining it with a microlens array, a wavefront-phase-controllable multifocal point is generated. After passing through the scanning system, multiple focal points can be generated at the objective lens focal point, providing a basis for simultaneous multi-region aberration correction. On this basis, the existing aberrations are measured and corrected by combining the phase modulator and modal wavefront detection method, thereby realizing aberration correction in multiple imaging regions and obtaining high-resolution images.
[0020] The present invention provides a two-photon microscopy imaging system with simultaneous multi-region aberration correction. Through multi-focal scanning and multi-focal aberration control, the speed of aberration correction is improved. By using the measured wavefront phase, multiple regions are imaged simultaneously, realizing simultaneous aberration correction of multiple imaging regions and achieving aberration correction of dynamic signals across the entire field of view. Attached Figure Description
[0021] Figure 1 A schematic diagram of the structure of a two-photon microscopy imaging device with simultaneous multi-region aberration correction provided in an embodiment of the present invention;
[0022] Figure 2 This is an example diagram of multifocal aberration correction provided in an embodiment of the present invention;
[0023] Figure 3 This is an example image of a 10μm fluorescent sphere imaging with aberration correction using a 3x3 focal array, provided in an embodiment of the present invention.
[0024] Figure 4 A flowchart of a two-photon microscopy method for simultaneous multi-region aberration correction provided in an embodiment of the present invention. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.
[0026] This invention provides a two-photon microscopy imaging device with simultaneous multi-region aberration correction, such as... Figure 1 As shown, it includes: a light source control module, an aberration control module, a multifocal generation module, a scanning imaging module, a scanning relay module, a fluorescence signal collection module, and an aberration correction module.
[0027] The light source control module is mainly used to generate laser light of a certain power, and includes: laser 1, laser power control unit 2, and beam amplification unit 3. The laser power control unit controls the power of the laser source to obtain power suitable for imaging and prevent sample damage. The beam amplification unit enlarges the beam size so that the beam fills the entire chip plane of the phase modulation device.
[0028] Furthermore, the laser power control unit includes a half-wave plate fixed on the rotating device and a polarizing beam splitter. The beam amplification unit includes two achromatic lenses with different focal lengths, requiring the focal length of the first lens to be smaller than that of the second lens; the specific size is determined by the magnification.
[0029] The aberration control module, used to control the wavefront phase of the light beam, includes a phase adjustment unit 5 and a phase relay unit 6. The phase adjustment unit is a liquid crystal spatial light modulator used to modulate the phase of the incident light wave; the phase relay unit is used to conjugate the phase-adjusted light wave to the subsequent multifocal generation module. The phase relay unit consists of two coaxial lenses placed in fixed positions. The distance between the first lens and the phase modulator is equal to the focal length of the first lens, and the distance between the second lens and the first lens is equal to the sum of the focal lengths of the two lenses.
[0030] The multifocal generation module is used to focus a wavefront-phase-adjusted beam to generate multiple aberration-free focal points, including: a small lens group 7 and a relay lens 8. The small lens group is mainly used to focus the phase-adjusted beam to generate multiple focal points, while the relay lens is used to conjugate the phase of the beam controlling the multifocal points to the subsequent scanning relay module.
[0031] The scanning relay module primarily utilizes two scanning galvanometers to perform mechanical scanning in the x and y dimensions. The scanning beam is then transmitted to the imaging objective via the scanning relay unit 10. In other words, the scanning relay module comprises two galvanometer-type galvanometers 9 and the scanning relay unit 10. The two scanning galvanometers are mainly responsible for mechanical scanning in the x and y dimensions. The scanning relay unit conjugates the scanning beam to the rear pupil 11 of the objective, achieving multifocal wavefront phase conjugation. The scanning relay unit includes a scanning lens and a sleeve lens placed in fixed positions. The distance between the scanning lens and the scanning galvanometer is equal to the focal length of that lens, and the distance between the sleeve lens and the scanning lens is equal to the sum of the focal lengths of the two lenses.
[0032] The fluorescence signal collection module is used to collect fluorescence excited by multiple focal points and includes a dichroic mirror 13, a telescope lens 14, and an area array detector 15. The dichroic mirror is used to reflect the fluorescence signal emitted by the sample and collected by the objective lens in a direction perpendicular to the excitation light path; the telescope lens is mainly used to conjugate the fluorescence collected by the objective lens onto the area array detector (e.g., a fluorescence camera); the area array detector is used to collect multiple fluorescence signals excited by multiple focal points and stitch them into an image after scanning.
[0033] In summary, the optical path of the device is as follows: the laser beam emitted by the laser 1 is controlled by the laser power control unit 2, then amplified by the beam expander 3, reflected by the mirror 4 and incident on the phase modulator 4, and then incident on the small lens group 7 through the phase relay unit 6, generating a multifocal point that can be aberration-controlled by the phase modulator. The generated multifocal point is conjugated to the scanning galvanometer group 9 through the relay lens 8, and then conjugated to the rear pupil 11 of the objective lens 12 through the scanning lens 10, focusing to form multiple focal points to excite the object to be imaged to emit fluorescence. The generated fluorescence signal is reflected by the dichroic mirror 13 and conjugated to the area array detector 15 by the barrel lens 14, and collected by the area array detector.
[0034] The aberration control module includes a phase adjustment unit and a phase relay unit. The phase adjustment unit is used to adjust the wavefront phase of the laser beam, and the phase relay unit is used to conjugate the adjusted beam to the multifocal generation module for focusing to generate multiple focal points, and then conjugate the beams from the multiple focal points to the scanning imaging module.
[0035] The pixels on the liquid crystal spatial light modulator are divided into multiple regions, serving as multiple sub-control units. The aberration correction module is used to obtain the optimal amplitude a of sub-phase control unit i in aberration mode j. ij Calculate the corresponding wavefront phase compensation amount. Each f iThe load is applied to the corresponding sub-control unit i to change the wavefront phase control amount of the light beam incident on its surface, thereby controlling the aberration of the focal point generated by the beam after focusing, and the imaging is performed again after adjustment to obtain an aberration-corrected image.
[0036] Wherein, the optimal amplitude a ij In aberration mode j, multiple aberration perturbations of different amplitudes are applied to the laser beam through the sub-phase control unit i, and the imaging is performed to obtain the corresponding imaging image. The amplitude corresponding to the best image evaluation index is obtained by fitting all amplitudes and their corresponding imaging image evaluation indexes; i = 1 to n, where n is the number of sub-phase control units.
[0037] Specifically, the aberration correction module employs a modal wavefront detection method, which is an aberration correction algorithm.
[0038] For the i-th phase adjustment unit, firstly, a series of aberration modes j = 1 to m are selected (for example, if the selected series of aberration modes are: spherical aberration, coma, astigmatism, field curvature, and defocus, then m = 5), where each aberration mode has its own aberration function z, that is, the aberration function of aberration mode j is z. j In the j-th aberration mode, a series of different amplitudes 'a' are artificially applied to the wavefront of the imaging beam through the i-th phase modulation unit. k *z j The aberrations are eliminated and images are formed, resulting in k images. Then, the image evaluation index g corresponding to each image is calculated. k Then, by fitting the function relationship between this index and the aberration mode amplitude, the optimal amplitude of the selected aberration mode j can be obtained. Finally, a corresponding compensation phase is applied to the i-th phase modulation unit of the liquid crystal spatial light modulator. (After compensation, the array detector is used to collect multiple fluorescence signals excited by the multifocal array, and stitches them together into an image after the scan is completed, which is the aberration-corrected image), thus achieving aberration correction.
[0039] For example, for the i-th phase modulation unit, when the aberration mode is spherical aberration mode, three aberration perturbations with different amplitudes a1, a2, and a3 are applied and imaging is performed to obtain three images I. a ,I b Ic, calculate the evaluation metrics A, B, C for each image; fit a, b, c to A, B, C using a Gaussian function to obtain the extreme points (i.e., the optimal evaluation metrics), and obtain the amplitude corresponding to the extreme points as the optimal aberration amplitude (i.e., the target amplitude), which is used as the optimal aberration amplitude a for the spherical aberration mode. i1Similarly, when the aberration mode is coma, the optimal aberration amplitude 'a' for the coma mode is obtained. i2 The optimal amplitude 'a' corresponding to each aberration mode is determined. ij The phase compensation amount is obtained by multiplying it by the phase distribution function of each aberration mode and then superimposing the results. After phase compensation using the i-th phase control unit, imaging is performed again to obtain an aberration-corrected image.
[0040] It is understandable that the series of aberration modes selected by different phase control units can be the same or different, depending on actual needs. For example, the first phase control unit can select four aberration modes: spherical aberration, coma, astigmatism, and field curvature, while the second phase control unit can select five aberration modes: spherical aberration, coma, astigmatism, field curvature, and defocus.
[0041] In summary, the aberration control module tests multiple aberration modes and fits the functional relationship between the image average intensity and the aberration mode amplitude to obtain the amplitude of the selected aberration mode. Finally, a corresponding compensation phase is applied to the liquid crystal spatial light modulator to achieve simultaneous multi-region aberration correction. That is, a series of aberration perturbations of different amplitudes are artificially applied to the wavefront of the imaging beam through the liquid crystal spatial light modulator, and imaging is performed. Then, after calculating the image evaluation index corresponding to each imaging result, the amplitude of the aberration mode to be tested is obtained by fitting the functional relationship between the index and the aberration mode amplitude. Finally, a corresponding compensation phase is applied to the liquid crystal spatial light modulator to achieve simultaneous multi-region aberration correction.
[0042] For implementation schemes of simultaneous multi-region aberration correction, such as Figure 2 As shown, the sub-regions (i.e., sub-control units) e, f, and g on the phase modulator are focused by small lenses to form focal points e', f', and g', respectively. The phase control elements on the three regions of the phase modulator can then control the wavefront phase of these three focal points, thereby achieving simultaneous multi-region aberration correction. Figure 3 As shown, nine focal points were generated in the experiment. By controlling the wavefront phase of each focal point through nine sub-control units, aberration correction in each region can be achieved.
[0043] This invention provides a two-photon microscopy imaging method with simultaneous multi-region aberration correction, applied to the apparatus described in any of the above embodiments, comprising:
[0044] Offline calibration stage:
[0045] Obtain the optimal amplitude a of sub-phase control unit i in aberration mode j. ij Calculate the corresponding wavefront phase compensation amount. Each f iLoaded onto the corresponding sub-control unit i to change the wavefront phase modulation amount of the light beam incident on its surface by the sub-control unit, thereby controlling the focal point generated by the focusing of the light beam;
[0046] Wherein, the optimal amplitude a ij In aberration mode j, multiple aberration perturbations of different amplitudes are applied to the laser beam through the sub-phase control unit i and the imaging is performed to obtain the corresponding imaging image. The amplitude corresponding to the best image evaluation index is obtained by fitting all the amplitudes and the evaluation index of the corresponding imaging image.
[0047] Online calibration phase:
[0048] In each f i After being loaded onto the corresponding sub-control unit i, the imaging is performed again to obtain an image with simultaneous multi-region aberration correction.
[0049] Specifically, such as Figure 4 As shown, in the offline calibration stage, aberration modes are selected, and a phase modulator is used to artificially add biases to the selected aberration modes. The imaging results after each bias addition are recorded, and the average image intensity is calculated as an evaluation function. A Gaussian function is used to fit these biases and the average intensity to obtain the extreme points and the optimal aberration amplitude. Multiple iterations are performed to obtain the final aberration representation. In the online correction stage, a phase modulator is used for aberration modulation to achieve simultaneous correction of aberrations in multiple regions.
[0050] This invention provides a computer-readable storage medium, characterized in that the computer-readable storage medium stores computer instructions, which are used to cause a processor to perform the method described in the above embodiments.
[0051] In summary, the two-photon microscopy imaging method and system for simultaneous multi-region aberration correction provided by this invention achieves simultaneous aberration correction in multiple imaging regions through multifocal scanning and multifocal aberration control, thereby improving the speed of aberration correction and realizing aberration correction for dynamic signals with a large field of view.
[0052] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A two-photon microscopy imaging device with simultaneous multi-region aberration correction, characterized in that, include: The aberration control module, multifocal generation module, scanning imaging module, imaging objective, fluorescence collection module, and aberration correction module are included. The aberration control module includes a phase adjustment unit and a phase relay unit; After the laser beam undergoes wavefront phase adjustment by the phase modulation unit, it is conjugated to the multifocal generation module by the phase relay unit to form multiple focal points. Then, it is conjugated to the scanning imaging module for scanning and conjugated to the rear pupil of the imaging objective. The imaging objective then focuses the laser beam to form multiple focal points, thereby exciting the object to be imaged to emit fluorescence. The fluorescence collection module collects the fluorescence for imaging. The phase control unit is divided into multiple regions as multiple sub-control units, and the aberration correction module is configured to obtain a sub-phase control unit i The optimal amplitude under the aberration mode j a ij , calculate the corresponding wavefront phase compensation amount , and load it to the corresponding sub-control unit i , so as to change the wavefront phase control amount of the sub-control unit to the light beam incident to the surface thereof, thereby controlling the aberration of the focal point generated by focusing the light beam, and performing the imaging again after adjustment to obtain the image after aberration correction of multiple regions; The aberration function is an aberration mode j . Among them, the optimal amplitude a ij For: in aberration mode j Below, through the sub-phase control unit i Multiple aberration perturbations of different amplitudes are applied to the laser beam and the imaging is performed to obtain the corresponding imaging image. The amplitude corresponding to the best image evaluation index is obtained by fitting all the amplitudes and the evaluation index of their corresponding imaging images. i =1~ n , n This represents the number of sub-phase control units.
2. The apparatus as claimed in claim 1, characterized in that, The phase adjustment unit is a liquid crystal spatial light modulator, and the pixels of the liquid crystal spatial light modulator are divided into the plurality of regions; the phase relay unit includes two fixedly placed coaxial lenses.
3. The apparatus as described in claim 1 or 2, characterized in that, The multifocal generation module includes a small lens group and a relay lens; wherein, the small lens group is used for focusing to generate multiple focal points, and the relay lens is used to conjugate the beams from the multiple focal points to the scanning imaging module.
4. The apparatus as described in claim 1 or 2, characterized in that, The scanning imaging module includes a scanning galvanometer group and a scanning relay unit; the scanning galvanometer group is used to scan multiple conjugated focal points, and the scanning relay unit includes a scanning lens and a sleeve lens, used to conjugate the scanning beam to the rear pupil of the imaging objective.
5. The apparatus as described in claim 4, characterized in that, The scanning galvanometer assembly includes two galvanometer-type galvanometers, which are used for mechanical scanning in the x and y dimensions, respectively.
6. The apparatus as claimed in claim 1 or 2, characterized in that, Also includes: A light source control module; the light source control module includes a laser power control unit and a beam amplification unit; the laser power control unit is used to control the power of the laser light source, and the beam amplification unit is used to amplify the size of the light spot.
7. The apparatus as described in claim 1 or 2, characterized in that, The fluorescence collection module includes a dichroic mirror, a telescope lens, and an area array detector. The dichroic mirror is used to reflect the fluorescence, the telescope lens is used to conjugate the fluorescence onto the area array detector, and the area array detector is used to collect the fluorescence and stitch it into an image.
8. A two-photon microscopy imaging method with simultaneous multi-region aberration correction, applied to the apparatus as described in any one of claims 1-7, characterized in that, include: Offline calibration stage: Acquisition of sub-phase control unit i In aberration mode j The optimal amplitude a ij Calculate the corresponding wavefront phase compensation amount. and load it into the corresponding sub-control unit. i The above allows for the modification of the wavefront phase of the beam incident on its surface by the sub-control unit, thereby controlling the focal point generated by the focusing of the beam; Among them, the optimal amplitude a ij For: in aberration mode j Below, through the sub-phase control unit i Multiple aberration perturbations of different amplitudes are applied to the laser beam and the imaging is performed to obtain the corresponding imaging image. The amplitude corresponding to the best image evaluation index is obtained by fitting all the amplitudes and the evaluation index of their corresponding imaging images. Online calibration phase: In Loaded into the corresponding sub-control unit i After that, the imaging is performed again to obtain an image with multi-regional aberration correction.
9. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores computer instructions for causing a processor to perform the method as described in claim 8.