CN109188667B - Multi-beam array multi-photon heavy scanning microscopic imaging device - Google Patents
Multi-beam array multi-photon heavy scanning microscopic imaging device Download PDFInfo
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Abstract
The invention provides a multi-beam array multi-photon rescanning microscopic imaging device, which comprises: a light source module for generating laser light usable for multi-photon excitation; the beam splitter is used for converting the laser into a plurality of two-dimensional laser arrays with equal angular intervals; the two-dimensional scanning head is used for scanning the two-dimensional laser array; the focusing module is used for converting the two-dimensional laser array into a plurality of two-dimensional focusing light spot arrays with equal intervals and focusing the two-dimensional focusing light spot arrays to a sample so as to excite fluorescence or generate a multi-photon high-order harmonic signal; the rescanning module is used for enabling the fluorescence or multi-photon high-order harmonic signal to be incident to the two-dimensional scanning head so as to perform rescanning; and the imaging module is used for collecting the rescanned fluorescence or multi-photon high-order harmonic signals and imaging. The device provided by the invention can realize rescanning and improve the spatial resolution by emitting fluorescent or multi-photon high-order harmonic signals to the same two-dimensional scanning head.
Description
Technical Field
The invention relates to the technical field of optics, in particular to a multi-beam array multi-photon rescan microscopic imaging device.
Background
With the development of scientific technology, optical microscopy imaging technology has become an important technology and measurement tool in biology, medicine and related interdisciplinary research. The optical microscopic imaging technology has the characteristics of no damage, non-invasiveness, quick acquisition and the like, is very suitable for imaging of living cells, biological tissues and non-biological systems, and has the advantage that other technologies cannot be replaced. Therefore, in recent years, optical microscopy imaging technology has been the leading edge and hot spot of technology development, and is continuously making new development and breakthrough, and the development trend thereof is higher spatial resolution, faster speed, dynamic biological information acquisition and the like.
Therefore, how to significantly improve the time and spatial resolution of microscopic imaging is a problem to be solved.
Disclosure of Invention
To overcome the above-described deficiencies of the prior art, the present invention provides a multibeam array multiphoton rescan microscopy imaging apparatus.
The invention provides a multi-beam array multi-photon rescanning microscopic imaging device, which comprises:
a light source module for generating laser light usable for multi-photon excitation;
the beam splitter is used for converting the laser into a plurality of two-dimensional laser arrays with equal angular intervals;
the two-dimensional scanning head is used for receiving the two-dimensional laser arrays with the equiangular intervals and scanning the two-dimensional laser arrays with the equiangular intervals;
the focusing module is used for converting the scanned two-dimensional laser array with the equiangular intervals into a two-dimensional focusing light spot array with the equiangular intervals and focusing the two-dimensional focusing light spot array to a sample so as to excite fluorescence or generate a multi-photon high-order harmonic signal;
the rescanning module is used for collecting the fluorescence or multiphoton high-order harmonic signals and enabling the fluorescence or multiphoton high-order harmonic signals to be incident to the two-dimensional scanning head so as to perform rescanning;
the imaging module is used for collecting and imaging the rescanned fluorescence or multi-photon high-order harmonic signal;
wherein a rescanning angle of the fluorescent or multiphoton higher-order harmonic signal is proportional to a scanning angle of the plurality of equiangularly spaced two-dimensional laser arrays.
Preferably, the scanning angle of the fluorescence or multiphoton higher-order harmonic signal is proportional to the scanning angle of the multi-beam equiangular-spaced two-dimensional laser array, specifically:
the scanning angle of the fluorescence or multiphoton high-order harmonic signal is 2 times of that of the two-dimensional laser array with the equiangular spacing.
Preferably, the beam splitter comprises a beam expander, a two-dimensional light shaping element and a scanning lens.
Preferably, the two-dimensional light shaping element comprises: a microlens array, a spatial light modulator, a digital microlens, or a diffractive optical element.
Preferably, the focusing module comprises, in order along the laser light path direction: the device comprises a first scanning lens, a first imaging lens, a first dichroic mirror and an objective lens.
Preferably, the rescan module includes, sequentially arranged along the detection light path direction: the device comprises an objective lens, a reflecting unit, a second imaging lens, a second scanning lens, a second dichroic mirror and a two-dimensional scanning head.
Preferably, the imaging module comprises, arranged in sequence along the detection light path direction: the two-dimensional scanning head, the third dichroic mirror, the third imaging lens and the camera.
Preferably, the physical size of a single pixel of the camera satisfies the shannon sampling theorem, the physical size of the single pixel being no greater than 1/2 for spatial resolution.
Preferably, the two-dimensional scanning head comprises a resonant galvanometer scanning head, a galvanometer scanning head, or a piezo-piezo scanning head.
Preferably, at least one relay module is further included in the optical path, and the relay module is used for adjusting the beam diameter of the two-dimensional laser array with the plurality of beams in equal angular spacing.
The invention provides a multi-beam array multi-photon rescanning microscopic imaging device, which realizes multi-beam optical scanning by adopting a two-dimensional scanning head and enables fluorescence or multi-photon high-order harmonic signals excited by a sample to be incident to the same two-dimensional scanning head, thereby realizing rescanning and improving spatial resolution.
Drawings
FIG. 1 is a schematic structural diagram of a multi-beam array multi-photon rescan microscopic imaging apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a beam splitter according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a two-dimensional multi-beam laser array scan according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a two-dimensional multi-beam laser array scan according to another embodiment of the present invention;
FIG. 5 is a schematic diagram of a multi-beam array multi-photon rescan microscopic imaging apparatus according to another embodiment of the present invention;
FIG. 6 is a schematic view of a scanning dynamic optical path according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
It should be clear that the laser, probe and collimation optical paths mentioned herein are all conventional meanings as understood by those skilled in the art.
The invention provides a multi-beam array multi-photon rescanning microscopic imaging device based on the development trend that the current optical microscopic imaging technology tends to have higher time and spatial resolution.
Fig. 1 is a schematic structural diagram of a multi-beam array multi-photon rescan microscopic imaging apparatus according to an embodiment of the present invention, as shown in fig. 1, including:
the light source module 100 generates laser light that can be used for multi-photon excitation.
It should be noted that the light source module includes a laser that can be used for multi-photon excitation, and laser light is generated by the laser used for multi-photon excitation. In one particular embodiment, the laser may be generated using a femtosecond laser.
And a beam splitter 101 for converting the laser light into a plurality of two-dimensional laser arrays with equal angular intervals.
It should be noted that the beam expansion may be performed by a beam expander first. Optionally, a flat-top optical shaper may be used to change laser with gaussian intensity distribution into laser with uniform intensity distribution, thereby improving microscopic imaging quality. Then, a two-dimensional light shaping element is used for obtaining a two-dimensional focusing light beam array with a plurality of beams distributed on a plane and equal intervals. The two-dimensional light shaping element can be a two-dimensional microlens array distributed at equal intervals, or a spatial light modulator, a digital microlens, a diffractive optical element and the like. The two-dimensional focused light beam array passes through a scanning lens, so that each beam is overlapped to the focal point of the scanning lens, and a two-dimensional laser array with a plurality of beams at equal angular intervals is obtained.
And the two-dimensional scanning head 102 is used for receiving the two-dimensional laser array with the equal angular spacing of the multiple beams and scanning the two-dimensional laser array with the equal angular spacing of the multiple beams.
It should be noted that two scanning heads in the two-dimensional scanning head are respectively used for controlling two scanning directions of the light beam. The two-dimensional scanning head scans the incident two-dimensional laser array with a plurality of beams at equal angular intervals, and the emergent direction of the two-dimensional laser array with the plurality of beams at equal angular intervals is changed by controlling the scanning direction of the two-dimensional scanning head. By controlling the scanning mode of the two-dimensional scanning head, when the angle of the two-dimensional scanning head deflects, the emergent multiple beams of the two-dimensional laser array with equal angular intervals also deflect, and then each part of the sample is scanned. The two-dimensional scanning head can be a resonance-galvanometer scanning head, a galvanometer-galvanometer scanning head or a piezoelectric-piezoelectric scanning head, and the like, and the two scanning heads respectively scan the light beams in different directions. It should be clear to those skilled in the art that any two scanning heads can be combined into a two-dimensional scanning head to achieve the above-mentioned effects, and the two-dimensional scanning head is within the protection scope of the present invention.
And the focusing module 103 is used for converting the scanned multi-beam equiangular-spaced two-dimensional laser array into a multi-beam equiangular-spaced two-dimensional focusing light spot array and focusing the multi-beam equiangular-spaced two-dimensional focusing light spot array to a sample so as to excite fluorescence or generate a multi-photon high-order harmonic signal.
It should be noted that, a plurality of equiangular two-dimensional laser arrays emitted from the two-dimensional scanning head are subjected to light path adjustment, so that the conversion from equiangular two-dimensional laser arrays to equiangular two-dimensional focusing light spot arrays is realized, and the focusing light spots focused to the sample are ensured to be equiangular; the optical path adjustment may be a combination of at least one imaging lens and one scanning lens, and the embodiments of the present invention are not limited in particular. Therefore, when the scanning direction of the two-dimensional scanning head is changed, the two-dimensional condensed light spot array with the equal intervals can just scan the area to be measured of the sample without repetition. When a sample is scanned by the multi-beam two-dimensional focusing light spot array with equal spacing, the sample absorbs energy, so that fluorescence or multi-photon high-order harmonic signals are excited. The sample can be stained or marked by fluorescent substances such as probes, fluorescent proteins, quantum dots, nano fluorescent particles and the like, and the sample comprises a living body sample, a fixed biological sample, other non-living system samples and multi-photon non-marked high-order harmonic imaging.
A rescanning module 104 for collecting the fluorescence or multiphoton high-order harmonic signal and making the fluorescence or multiphoton high-order harmonic signal incident to the two-dimensional scanning head for rescanning; wherein, the rescanning angle of the fluorescence or multiphoton high-order harmonic signal is proportional to the scanning angle of the two-dimensional laser array with the equiangular spacing of the multiple beams.
It should be noted that, after being excited by multiple photons, the sample emits fluorescence or multiple photon higher harmonic signals; the fluorescence or multiphoton high-order harmonic signal changes direction through reflection and enters the same two-dimensional scanning head, so that rescanning is realized. Wherein, the scanning angle of the fluorescence or multiphoton high-order harmonic signal is proportional to the scanning angle of the multi-beam equiangular spacing two-dimensional laser array. Specifically, the scanning angle of the fluorescence or multiphoton high-order harmonic signal is 2 times of the scanning angle of the two-dimensional laser array with the equal angular spacing of the multiple beams.
For example, when the two-dimensional scanning head is deflected by an angle α, the outgoing two-dimensional array of multiple beams is correspondingly deflected by the same angle 2 α. The deflection angle of the fluorescence or multiphoton high-order harmonic signal before returning to the two-dimensional scanning head is-2 alpha relative to the incident angle of the incident multiple two-dimensional laser array; wherein the negative sign indicates and the direction of deflection is opposite to the direction of rotation of the two-dimensional scanning head. After the fluorescence or multi-photon high-order harmonic signal passes through the two-dimensional scanning head, the deflection angle is 4 alpha, namely 2 times of the deflection scanning angle of the multi-beam two-dimensional laser array, and therefore an ultrahigh resolution microscopic image with the spatial resolution 2 times of the diffraction limit of far-field imaging resolution can be obtained in the camera. The scanning angle of the fluorescence or multiphoton high-order harmonic signal can be any multiple of the scanning angle of the two-dimensional laser array with the equal angular spacing, and when the scanning angle of the fluorescence or multiphoton high-order harmonic signal is 2 times of the scanning angle of the two-dimensional laser array with the equal angular spacing, the resolution of the obtained microscopic image is 2 times of the diffraction limit of the far-field imaging resolution.
The imaging module 105 is used for collecting and imaging the rescanned fluorescence or multi-photon high-order harmonic signals;
after the fluorescence or multiphoton higher harmonic signal is emitted from the two-dimensional scanning head, the fluorescence or multiphoton higher harmonic signal is incident on the camera through a series of optical path adjustments. The camera converts an optical signal of the fluorescence or multiphoton higher-order harmonic signal into an electrical signal, thereby generating a sample image. Furthermore, images can be reconstructed through deconvolution algorithms such as wiener filtering, and the spatial resolution can be further improved.
The optical path adjustment may be to separate the fluorescence or multiphoton high-order harmonic signal from the multi-beam equiangular-spaced two-dimensional laser array by using at least one dichroic mirror, or to reflect the fluorescence or multiphoton high-order harmonic signal by using at least one reflecting mirror. The aforementioned optical path adjustment is for changing the direction of the fluorescence or multiphoton higher harmonic signal, and therefore, the present invention is not limited to the specific implementation manner of the optical path adjustment; the dichroic mirror and the reflecting mirror are provided for illustration only and do not limit the scope of the embodiments of the present invention.
The multi-beam array multi-photon rescanning microscopic imaging device provided by the invention scans by adopting a plurality of beams of light, and enables fluorescence or multi-photon high-order harmonic signals excited by a sample to be incident to the same two-dimensional scanning head, thereby realizing rescanning and improving the spatial resolution of microscopic imaging.
Based on the content of the foregoing embodiments, as an alternative embodiment, in an embodiment of the present invention, the beam splitter includes a beam expander, a two-dimensional light shaping element, and a scanning lens.
It should be noted that the beam expander is used to expand one laser beam. Optionally, a flat-top optical shaper may be used to change laser with gaussian intensity distribution into laser with uniform intensity distribution, thereby improving microscopic imaging quality. Then, a two-dimensional light shaping element is used for obtaining a two-dimensional focusing light beam array with a plurality of beams distributed on a plane and equal intervals. The two-dimensional light shaping element can be a two-dimensional microlens array distributed at equal intervals, or a spatial light modulator, a digital microlens, a diffractive optical element and the like. The two-dimensional focused light beam array passes through a scanning lens, so that each beam is overlapped to the focal point of the scanning lens, and multiple beams of two-dimensional laser with equal angular spacing are obtained.
According to the multi-beam array multi-photon rescanning microscopic imaging device provided by the embodiment of the invention, laser of a single beam is split, and multiple beams of light are adopted for scanning, so that the scanning speed is increased compared with single beam scanning; in addition, by adopting the light beams of the two-dimensional array, each laser only needs to scan a part of the area of the sample, thereby realizing parallel scanning and obviously improving the time resolution of microscopic imaging.
Based on the content of the foregoing embodiment, as an optional embodiment, in the embodiment of the present invention, the focusing module includes: the device comprises a first scanning lens, a first imaging lens, a first dichroic mirror and an objective lens.
The two-dimensional focusing light beam array with a plurality of equiangular intervals is converted into a two-dimensional laser array with a plurality of equiangular intervals and then is incident to the two-dimensional scanning head; and the field of view is scanned by the multiple beams just without repetition by controlling the two-dimensional scanning head. A plurality of two-dimensional laser arrays with equal angular intervals are emitted from a two-dimensional scanning head, pass through a first scanning lens and then are converted into a plurality of two-dimensional focusing light beam arrays with equal intervals, and then the two-dimensional focusing light beam arrays are focused on a sample through a first imaging lens and an objective lens to excite the fluorescence or multi-photon high-order harmonic signals of the sample. The first dichroic mirror is positioned between the first imaging lens and the objective lens, and is used for transmitting the incident multiple equiangular-interval two-dimensional laser arrays and reflecting fluorescence or multi-photon high-order harmonic signals of the sample. The dichroic mirror may be a long-pass dichroic mirror or a short-pass dichroic mirror, and the type, model, and the like of the dichroic mirror are not limited in the present invention only to explain the effect of the dichroic mirror on changing the optical path direction.
Based on the content of the foregoing embodiment, as an optional embodiment, in the embodiment of the present invention, the rescanning module includes: the device comprises an objective lens, a reflecting unit, a second imaging lens, a second scanning lens, a second dichroic mirror and a two-dimensional scanning head.
Based on the content of the foregoing embodiment, as an optional embodiment, in the embodiment of the present invention, the imaging module includes: the two-dimensional scanning head, the third dichroic mirror, the third imaging lens and the camera.
It should be noted that, after receiving the incident multiple equidistant two-dimensional laser arrays, the sample excites the fluorescence or multiphoton high-order harmonic signal, and the fluorescence or multiphoton high-order harmonic signal sequentially passes through the objective lens, the reflection unit, the second imaging lens and the second scanning lens along the detection optical path direction, is reflected by the second dichroic mirror, and is incident to the two-dimensional scanning head. After rescanning the fluorescence or multiphoton high-order harmonic signals by the two-dimensional scanning head, the fluorescence or multiphoton high-order harmonic signals are incident to the camera and imaged after passing through the third dichroic mirror and the third imaging lens.
The reflecting unit can be at least one dichroic mirror which separates the fluorescent light or multi-photon high-order harmonic signal from the multi-beam equiangular-spacing two-dimensional laser array. It will be clear to those skilled in the art that the transmission of two-dimensional laser arrays, and the reflection of fluorescence or multiphoton higher harmonic signals can be achieved by using the characteristics of dichroic mirrors. The reflection unit may also be at least one mirror to reflect the fluorescence or multiphoton higher order harmonic signals. Alternatively, the reflecting unit may be a combination of a dichroic mirror and a reflecting mirror, which have various combinations, and therefore, are not expanded here. It should be clear that the dichroic mirror and the reflecting mirror are both provided for the purpose of changing the direction of the fluorescence or multiphoton higher harmonic signal, and the embodiments of the present invention are only illustrative and do not limit the scope of protection.
Based on the content of the foregoing embodiment, as an alternative embodiment, the physical size of a single pixel of the camera in the embodiment of the present invention satisfies shannon sampling theorem, and the physical size of the single pixel is not greater than 1/2 of the spatial resolution.
It should be noted that by controlling the scanning mode of the two-dimensional scanning head, the spatial sampling rate of the scanning satisfies shannon's sampling theorem, and the scanning angle of the fluorescence or multiphoton high-order harmonic signal is 2 times of the scanning angle of the multiple beams of laser light, thereby realizing optical rescanning and improving the spatial resolution of the imaging. The spatial resolution depends on the equivalent point spread function of the system, and the point spread function of the ordinary multi-photon microscope is the point spread function of the laser of the excitation light path, namely, the effective point spread function of the image scanning microscope in the optical rescanning mode, and is the product of the point spread function of the multi-beam laser of the excitation light path and the point spread function of the fluorescence or multi-photon higher-order harmonic signal on the detection light path, so that the full width at half maximum of the point spread function is reduced by 1/2, and the spatial resolution is correspondingly improved by 2 times.
The multi-beam array multi-photon rescanning microscopic imaging device provided by the embodiment of the invention adopts a multi-beam two-dimensional laser array with equal angular spacing and is matched with a two-dimensional scanning head to realize rescanning; meanwhile, a high-speed camera is adopted for imaging, the scanning spatial sampling rate meets the Shannon sampling theorem, and multi-photon microscopic imaging with ultrahigh spatial resolution is realized.
Based on the content of the foregoing embodiments, as an alternative embodiment, in an embodiment of the present invention, the two-dimensional scanning head includes a resonant galvanometer scanning head, a galvanometer scanning head, or a piezoelectric scanning head.
It should be noted that the two-dimensional scanning heads are used for scanning the light beams in different directions respectively through the two scanning heads, so that the imaging speed can be greatly increased while the spatial resolution is increased.
Fig. 2 is a schematic structural diagram of a beam splitter according to an embodiment of the present invention, as shown in fig. 2, for example, the beam splitter includes a lens 201, a lens 202, a light shaping element 203, and a second scanning lens 204, which are sequentially disposed along an optical path direction of a laser, where:
one beam of laser light passes through the lens 201 and the lens 202, and the beam of laser light is expanded. The laser beam passes through the light shaping element 203 to obtain a two-dimensional focused beam array of a plurality of beams at equal intervals. Wherein the light shaping element 203 may be a microlens array, a spatial light modulator, a digital microlens, a diffractive optical element, or the like, the multiple light beams are a two-dimensional array, for example, 2x5, 4x8, or 10x10, and the like, and the number of the corresponding laser lights may be 10 beams, 32 beams, or 100 beams, and the like. It should be noted that the array specification mentioned herein is only an example, and may be any value, and the scope is not limited herein. The two-dimensional focused light beam array with a plurality of equiangular intervals passes through the second scanning lens 204, and each laser beam is overlapped to the focal point of the second scanning lens 204 by the second scanning lens 204, so that the two-dimensional laser array with a plurality of equiangular intervals is obtained.
According to the multi-beam multi-photon rescanning microscopic imaging device provided by the embodiment of the invention, the laser of a single beam is split, and the parallel scanning of a sample is realized by using multiple beams of light, so that the scanning speed is improved. It will be appreciated that the beam splitter of embodiments of the present invention is provided as an example of one particular embodiment only, and does not limit the scope of the beam splitter.
Fig. 3 is a schematic view of a two-dimensional multi-beam laser array scanning according to an embodiment of the present invention, as shown in fig. 3, for example, in the embodiment of the present invention, M laser beams are arranged in a transverse direction, N laser beams are arranged in a vertical direction, and each laser beam scans an area of a sample. This region is 1/(N × M) of the single laser scan region when scanning with a single laser. The sample is scanned N x M times faster than the single laser scan. In this scanning system, the laser light is always emitted, which is called a bidirectional scanning system.
Fig. 4 is a schematic diagram of two-dimensional multi-beam laser array scanning according to another embodiment of the present invention, and as shown in fig. 4, a dashed line indicates that the laser is in an off state, which is called a unidirectional scanning mode. The rest of the drawings correspond to fig. 3, and the principle of the unidirectional scanning mode is similar to that of the bidirectional scanning mode, and for the specific principle, the above description is referred to, and the details are not repeated here.
It can be understood that the two-dimensional scanning head formed by any two scanning heads can achieve the above effects. For example, when the two-dimensional scanning head employs a galvanometer-galvanometer scanning head, the scanning direction X is the scanning direction of one galvanometer scanning head and the scanning direction Y is the scanning direction of the other galvanometer scanning head. The scanning speed is faster than that of the single-beam laser by N times by adopting a galvanometer-galvanometer scanning head. Similarly, when the two-dimensional scanning heads such as the piezoelectric-piezoelectric scanning head, the galvanometer-piezoelectric scanning head and the like are adopted for scanning, the scanning speed is faster by N × M times than that of the single-beam laser scanning.
Therefore, according to the multi-beam multi-photon rescanning device provided by the embodiment of the invention, the two-dimensional laser array is scanned by the two-dimensional scanning head, and compared with the single-beam scanning in the prior art, the scanning speed is obviously improved, so that the imaging time resolution is improved.
Based on the content of the foregoing embodiment, as an optional embodiment, the embodiment of the present invention further includes at least one relay module in the collimated light path, where the relay module is configured to adjust a beam diameter of the multi-beam equiangular-spaced two-dimensional laser array.
It should be noted that, in the embodiment of the present invention, a relay optical path may be added at any position of the collimation optical path, for example, between the resonant scanning head and the galvanometer scanning head, or between the objective lens and the imaging lens. The relay optical path can be a beam expander or a multi-reflector and is used for diameter adjustment of the light beam.
In order to facilitate understanding of the technical solutions of the present invention, the embodiments of the present invention provide a specific implementation manner for implementing the multi-beam array multi-photon rescan microscopic imaging apparatus described in the above embodiments. It should be understood that the embodiment is only an illustrative example, and the technical solution of the present invention is not meant to be realized only by the embodiment, and the protection scope of the present invention is not limited.
Fig. 5 is a schematic structural diagram of a multi-beam array multi-photon rescanning microscopic imaging apparatus according to another embodiment of the present invention, as shown in fig. 5, including a light source module 500, a beam splitter 501, a second dichroic mirror 502, a two-dimensional scanning head 503, a third dichroic mirror 504, a first scanning lens 505, a first imaging lens 506, a first dichroic mirror 507, and an objective lens 508, which are sequentially arranged along a laser optical path direction, the laser sequentially passes through the above-mentioned devices along the laser optical path direction, is focused on a sample, and simultaneously excites a fluorescence or multi-photon high-order harmonic signal of the sample, the fluorescence or multi-photon high-order harmonic signal passes through the objective lens 508, the reflection unit 509, the second imaging lens 510, the second scanning lens 511, the second dichroic mirror 502, the two-dimensional scanning head 503, the third dichroic mirror 504, and the third imaging lens 512, which are sequentially arranged along a detection optical path direction, and the fluorescence or multi-, and converting the fluorescence or multiphoton higher harmonic signals into electrical signals and generating an image. Wherein: a second dichroic mirror 502 is located between the beam splitter 501 and the two-dimensional scan head 503.
On the basis of the above-described embodiment, the pulse laser light for multiphoton excitation is passed through the optical modulator to control the intensity of the multiphoton laser light. Wherein the light modulator may be a pockels cell. And rapidly modulating the multi-photon laser through the Pockels cell to ensure that the intensity of the laser is matched with the two-dimensional scanning head. The multi-photon laser is converted into a multi-beam equiangular-spaced two-dimensional laser array (hereinafter referred to as a two-dimensional laser array for simplicity) by a beam splitter. Since the dichroic mirror has the characteristics of transmitting light with a certain wavelength and reflecting light with other wavelengths, the two-dimensional laser array passes through the second dichroic mirror 502 and enters the two-dimensional scanning head 503.
After passing through the two-dimensional scanning head 503, the two-dimensional laser array passes through the third dichroic mirror 504, then passes through the first scanning lens 505 and the first imaging lens 506, passes through the first dichroic mirror 507, is incident on the objective lens 508, and is focused on the sample, thereby scanning the sample.
When the two-dimensional laser array scans a sample, the sample simultaneously excites fluorescence or multi-photon higher-order harmonic signals at multiple points and reflects the fluorescence or multi-photon higher-order harmonic signals. The reflected fluorescence or multiphoton higher-order harmonic signals pass through the same objective lens 508 along the detection optical path direction, and then pass through a reflection unit 509; among them, the reflection unit 509 includes a first dichroic mirror 507 and a reflection mirror 514 on the laser light path. According to the characteristics of the dichroic mirror, the first dichroic mirror 507 and the reflecting mirror 514 reflect the fluorescence or multiphoton higher order harmonic signal on the detection optical path, thereby changing the direction of the fluorescence or multiphoton higher order harmonic signal. The fluorescence or multiphoton higher-order harmonic signal after the direction change is incident to the two-dimensional scanning head 503 through the second imaging lens 510, the second scanning lens 511, and the second dichroic mirror 502 on the laser optical path.
Wherein, the ratio of the focal lengths of the second imaging lens 510 and the second scanning lens 511 in the detection optical path is equal to the ratio of the focal lengths of the first imaging lens 506 and the first scanning lens 505 in the excitation optical path; and the fluorescence or multi-photon high-order harmonic signals are reflected by the second dichroic mirror 502 and then returned to the two-dimensional scanning head 503, so that rescanning is realized; the fluorescence or multiphoton higher order harmonic signals are then separated from the two-dimensional laser array by reflection from the third dichroic mirror 504, and the fluorescence or multiphoton higher order harmonic signals are redirected again and focused through a third imaging lens 512 to a camera 513. The camera 513 converts the optical signal of the fluorescence or multiphoton higher-order harmonic signal into an electric signal, thereby generating a sample image. Furthermore, images can be reconstructed through deconvolution algorithms such as wiener filtering, and the spatial resolution can be further improved.
Fig. 6 is a schematic diagram of a scanning dynamic optical path according to an embodiment of the present invention, in which only one laser beam of the multiple light beams is taken as an example for clarity. Corresponding to fig. 5, comprising: a light source module 600, a beam splitter 601, a second dichroic mirror 602, a two-dimensional scanning head 603, a third dichroic mirror 604, a first scanning lens 605, a first imaging lens 606, a first dichroic mirror 607, an objective lens 608, a reflection unit 609, a second imaging lens 610, a second scanning lens 611, a third imaging lens 612, a camera 613, and a reflection mirror 614. On the laser light path, one beam of light represents laser light before deflection, and the other beam of light represents laser light after deflection; on the detection optical path, one beam of light represents the fluorescence or multiphoton higher order harmonic signal before deflection, and the other beam of light represents the fluorescence or multiphoton higher order harmonic signal after deflection.
As shown in fig. 6, when the two-dimensional scanning head 603 deflects by an angle α, the emitted laser beam correspondingly deflects by an angle 2 α; the angle before the fluorescent or multiphoton higher order harmonic signal returns to the two-dimensional scan head 603 is correspondingly deflected by-2 α, with a negative sign indicating that the direction of deflection is opposite to the direction of rotation of the two-dimensional scan head 603. After the fluorescence or multiphoton high-order harmonic signal passes through the two-dimensional scanning head 603, the deflection angle is 4 alpha, which is 2 times of the deflection angle of the laser; thus, an ultra-high resolution microscopic image with a spatial resolution 2 times the diffraction limit of the far-field imaging resolution can be obtained in the camera 613. The spatial resolution depends on an equivalent point spread function of the system, that is, an effective point spread function of the image scanning microscope in the optical rescanning mode, and is a product of a point spread function of a plurality of laser beams on the excitation optical path and a point spread function of a fluorescence or multiphoton higher-order harmonic signal on the detection optical path.
In a specific embodiment, the ratio of the scan angle of the fluorescent or multiphoton higher harmonic signal to the scan angle of the two-dimensional multi-beam laser array satisfies: 1+ (f)a/fb)*(fd/fc)＝2；
Wherein f isaIs the focal length, f, of the first imaging lens 606bIs the focal length of the first scanning lens 605, fcIs the focal length, f, of the second imaging lens 610dIs the focal length of the second scan lens 611; and has fa/fb＝fc/fd。
According to the multi-beam array multi-photon rescanning microscopic imaging device provided by the embodiment of the invention, the rescanning of the fluorescence or multi-photon high-order harmonic signals is realized by returning the fluorescence or multi-photon high-order harmonic signals to the same two-dimensional scanning head; and the scanning angle of the fluorescence or multiphoton higher-order harmonic signal returned to the two-dimensional scanning head is just 2 times of the scanning angle of the incident two-dimensional laser array, so that a microscopic image with the spatial resolution 2 times of the diffraction limit of far-field imaging resolution is obtained, the resolution of the image is obviously improved, and a better imaging effect is achieved.
It should be clear that the above described embodiments of the apparatus are merely illustrative, wherein the units described as separate parts may or may not be physically separate, and the parts shown as units may or may not be physical units, i.e. may be located in one place, or may also be distributed over a plurality of network units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the solution of the present embodiment. One of ordinary skill in the art can understand and implement it without inventive effort.
In addition, it should be understood by those skilled in the art that the terms "comprises," "comprising," or any other variation thereof, in the specification of the present invention, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
In the description of the present invention, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In some instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description. Similarly, it should be appreciated that in the foregoing description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.
However, the disclosed method should not be interpreted as reflecting an intention that: that the invention as claimed requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A multi-beam array multi-photon rescan microscopic imaging apparatus, comprising:
a light source module for generating laser light usable for multi-photon excitation;
the beam splitter is used for converting the laser into a plurality of two-dimensional laser arrays with equal angular intervals;
the two-dimensional scanning head is used for receiving the two-dimensional laser arrays with the equiangular intervals and scanning the two-dimensional laser arrays with the equiangular intervals;
the focusing module is used for converting the scanned two-dimensional laser array with the equiangular intervals into a two-dimensional focusing light spot array with the equiangular intervals and focusing the two-dimensional focusing light spot array to a sample so as to excite fluorescence or generate a multi-photon high-order harmonic signal;
the rescanning module is used for collecting the fluorescence or multiphoton high-order harmonic signals and enabling the fluorescence or multiphoton high-order harmonic signals to be incident to the two-dimensional scanning head so as to perform rescanning;
the imaging module is used for collecting and imaging the rescanned fluorescence or multi-photon high-order harmonic signal;
wherein a rescanning angle of the fluorescent or multiphoton high-order harmonic signal is proportional to a scanning angle of the multi-beam equiangular-spaced two-dimensional laser array to achieve rescanning;
the method specifically comprises the following steps: the scanning angle of the fluorescence or multiphoton high-order harmonic signal is any multiple of the scanning angle of the two-dimensional laser array with the equal angular spacing of the multiple beams.
2. The multi-beam array multi-photon rescan microscopy imaging apparatus according to claim 1, wherein the scanning angle of the fluorescence or multi-photon higher order harmonic signal is proportional to the scanning angle of the multi-beam equiangularly spaced two-dimensional laser array, in particular:
the scanning angle of the fluorescence or multiphoton high-order harmonic signal is 2 times of that of the two-dimensional laser array with the equiangular spacing.
3. The multi-beam array multi-photon rescan microimaging device of claim 1, wherein the beam splitter comprises a beam expander, a two-dimensional light shaping element, and a scan lens.
4. The multi-beam array multi-photon rescan microscopic imaging apparatus according to claim 3, wherein the two-dimensional light shaping element comprises: a microlens array, a spatial light modulator, a digital microlens, or a diffractive optical element.
5. The multi-beam array multi-photon rescan microscopic imaging apparatus according to claim 1, wherein the focusing module comprises, arranged in order along the laser light path direction: the device comprises a first scanning lens, a first imaging lens, a first dichroic mirror and an objective lens.
6. The multi-beam array multi-photon rescan microscopic imaging apparatus according to claim 1, wherein the rescan module comprises, arranged in sequence along the detection optical path direction: the device comprises an objective lens, a reflecting unit, a second imaging lens, a second scanning lens, a second dichroic mirror and a two-dimensional scanning head.
7. The multi-beam array multi-photon rescan microscopic imaging apparatus according to claim 1, wherein the imaging module comprises, arranged in sequence along the detection optical path direction: the two-dimensional scanning head, the third dichroic mirror, the third imaging lens and the camera.
8. The multi-beam array multi-photon rescan microscopic imaging apparatus according to claim 7, wherein a physical size of a single pixel of the camera satisfies shannon's sampling theorem, the physical size of the single pixel being not greater than 1/2 of the spatial resolution.
9. The multi-beam array multi-photon rescan microscopic imaging apparatus according to claim 6 or 7, wherein the two-dimensional scanning head comprises a resonance-galvanometer scanning head, a galvanometer-galvanometer scanning head, or a piezo-electric scanning head.
10. The multi-beam array multi-photon rescan microscopic imaging apparatus according to claim 1, further comprising at least one relay module in the optical path for adjusting the beam diameter of the plurality of angularly equispaced two-dimensional laser arrays.
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