CN109211854B - Multi-beam multi-photon rescanning microscopic imaging device - Google Patents
Multi-beam multi-photon rescanning microscopic imaging device Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention provides a multi-beam multi-photon rescanning microscopic imaging device, comprising: a light source module for generating laser light usable for multi-photon excitation; the beam splitter is used for converting the single laser beam into a plurality of laser beams distributed on a straight line and at equal angular intervals; the two-dimensional scanning head is used for scanning a plurality of beams of laser with equal angular intervals; the focusing module is used for converting the scanned multiple beams of laser with equal angular intervals into multiple beams of focusing light spots with equal intervals so as to excite the fluorescence of the sample or generate multi-photon high-order harmonic signals; 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 for imaging. The microscopic imaging device provided by the invention realizes multi-beam optical scanning through the two-dimensional scanning head, so that fluorescence or multi-photon high-order harmonic signals are incident to the same two-dimensional scanning head, thus realizing rescanning and improving spatial resolution.
Description
Technical Field
The invention relates to the technical field of optics, in particular to a multi-beam 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 temporal resolution and the spatial resolution of the 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 multi-beam, multi-photon rescanning microimaging apparatus.
The invention provides a multi-beam multi-photon rescanning microscopic imaging device, 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 laser beams distributed on a straight line and at equal angular intervals;
the two-dimensional scanning head is used for receiving the laser beams with the equal angular spacing and scanning the laser beams with the equal angular spacing;
the focusing module is used for converting the scanned multiple laser beams with equal angular intervals into multiple focusing light spots with equal intervals and focusing the light spots 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 laser lights at the equiangular intervals.
Preferably, the scanning angle of the fluorescence or multiphoton higher-order harmonic signal is proportional to the scanning angle of the plurality of laser beams at the equiangular intervals, specifically:
the scanning angle of the fluorescence or multiphoton high-order harmonic signal is 2 times of that of the laser beams with the equal angular interval.
Preferably, the beam splitter comprises a lens element, a light shaping element and a scanning lens.
Preferably, the 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 objective lens, the reflecting unit, the second imaging lens, the second scanning lens, the second dichroic mirror and the 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 collimated light path, and the relay module is configured to adjust the beam diameter of the plurality of laser beams with equal angular spacing.
According to the multi-beam multi-photon rescanning microscopic imaging device, multi-beam light scanning is realized through the two-dimensional scanning head, and fluorescence or multi-photon high-order harmonic signals excited by a sample are incident to the same two-dimensional scanning head, so that rescanning is realized, the spatial resolution is improved, and the time resolution is improved.
Drawings
FIG. 1 is a schematic structural diagram of a multi-beam 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 multi-beam laser scanning according to one embodiment of the present invention;
FIG. 4 is a schematic view of a multi-beam laser scan according to another embodiment of the present invention;
FIG. 5 is a schematic diagram of a multi-beam multi-photon rescan microscopy 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.
Based on the development trend that the current optical microscopic imaging technology tends to have higher time and spatial resolution, the invention provides a multi-beam and multi-photon rescanning microscopic imaging device.
Fig. 1 is a schematic structural diagram of a multi-beam multi-photon rescanning 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.
The light source module includes a laser that can be used for multi-photon excitation, and laser light is generated by the laser that can be 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 laser lights at equal angular intervals.
First, a laser beam having a gaussian intensity distribution can be converted into a laser beam having a linear intensity distribution by a combination of a cylindrical lens and a lens. The laser beam can be converted into a plurality of focusing beams with equal intervals distributed on a straight line through a light shaping element. Wherein the light shaping element may be a microlens array, a spatial light modulator, a digital microlens, a diffractive optical element, or the like. A plurality of equally spaced focused beams pass through a scanning lens and are converted into a plurality of equally spaced laser beams.
And a two-dimensional scanning head 102 for receiving the laser beams at the equal angular intervals and scanning the laser beams at the equal angular intervals.
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 a plurality of incident laser beams with equal angular intervals, and the emitting direction of the laser beams with 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, a plurality of emergent laser beams with equal angular intervals also deflect along with the angle, and then each part of the sample is scanned.
And the focusing module 103 is used for converting the scanned multiple laser beams with equal angular intervals into multiple focusing light spots with equal intervals, and focusing the light spots onto the sample so as to excite fluorescence or generate a multi-photon high-order harmonic signal.
The laser beams emitted from the two-dimensional scanning head and having equal angular intervals are adjusted through an optical path, so that the conversion of the laser beams having equal angular intervals to focusing light spots having equal intervals is realized, and the light spots focused to a sample are ensured to be at equal intervals; the optical path adjustment may be a combination of at least one scanning lens and one imaging lens, and the embodiment of the present invention is not particularly limited. Therefore, when the scanning direction of the two-dimensional scanning head is changed, the plurality of equally-spaced focusing light spots can just scan the area to be measured of the sample without repetition. While the sample is scanned by the plurality of equally spaced focused spots, the sample absorbs energy, thereby exciting fluorescence or a multi-photon higher harmonic signal. 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 laser beams with equal angular spacing.
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 multiple laser beams with equal angular spacing. Specifically, the scanning angle of the fluorescence or multiphoton higher harmonic signal is 2 times the scanning angle of the laser beams having the equal angular interval.
For example, when the two-dimensional scanning head is deflected by an angle α, the outgoing laser beams are correspondingly deflected by the same angle 2 α. The angle before the fluorescence or multiphoton higher-order harmonic signal returns to the two-dimensional scanning head is-2 alpha in deflection angle relative to the incident angle of the incident multiple laser beams; 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 laser, so that an ultrahigh resolution microscopic image with the spatial resolution 2 times of the diffraction limit of far-field imaging 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 laser beams 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 laser beams 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.
And the imaging module 105 is used for collecting the rescanned fluorescence or multi-photon high-order harmonic signals and imaging.
After the fluorescence or multiphoton higher harmonic signal is emitted from the two-dimensional scanning head 102, 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 plurality of laser beams at the equal angular intervals 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 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 above description of the embodiments, as an alternative embodiment, the beam splitter in the embodiment of the present invention includes a lens element, a light shaping element, and a scanning lens.
It should be noted that the lens element may include a cylindrical lens and a lens which are arranged in this order in the laser light path direction. A laser beam with the intensity in Gaussian distribution passes through the cylindrical lens and the lens and is converted into a laser beam with the intensity in linear distribution. The laser beam can be converted into a plurality of focusing beams with equal intervals distributed on a straight line through a light shaping element. The light shaping element can be a micro-lens array, a spatial light modulator, a digital micro-lens or a diffractive optical element, and the like, and the generated light beams are a one-dimensional array. For example, 1x4, 1x16, or 1x64, etc., the corresponding number of lasers may be 4 beams, 16 beams, or 64 beams, etc. It should be noted that the array specifications mentioned herein are only examples and are not limiting in scope. A plurality of equally spaced focused beams pass through a scanning lens, and each of the focused beams is overlapped to a focal point of the scanning lens, so that a plurality of equally spaced laser beams are obtained.
Fig. 2 is a schematic structural diagram of a beam splitter apparatus according to an embodiment of the present invention, and as shown in fig. 2, the beam splitter apparatus includes a cylindrical lens 201, a lens 202, an optical shaping element 203, and a second scanning lens 204, which are sequentially disposed along a laser optical path direction, where:
one beam of laser light passes through the cylindrical lens 201 and the lens 202, and the intensity of the laser light is converted from gaussian distribution to linear distribution, so that the laser light is expanded in only one dimension. The laser beam passes through the light shaping element 203 to obtain a plurality of focused beams at equal intervals distributed on a straight line. 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 one-dimensional array, for example, 1x5, 1x16, or 1x50, and the number of the corresponding laser beams may be 5 beams, 16 beams, or 50 beams, and the like. It should be noted that the array specifications mentioned herein are only examples and are not limiting in scope. A plurality of equally spaced laser beams with focused beams pass through the second scanning lens 204, and each of the focused beams is overlapped by the second scanning lens 204 to the focal point of the second scanning lens 204, so that a plurality of laser beams with equal angular spacing are 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.
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.
It should be noted that, after a plurality of equally spaced focused beams are converted into a plurality of equally spaced lasers, the lasers are incident on 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 beams of equiangular interval laser emitted from the two-dimensional scanning head are converted into a plurality of beams of equiangular interval focused beams after passing through the first scanning lens, and then are focused on a sample through the first imaging lens and the 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 incident multiple beams of laser with equal angular spacing 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 focusing light spots, 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 light 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.
Wherein, the reflection unit can be at least one dichroic mirror for separating the fluorescence or multiphoton high-order harmonic signal from the plurality of laser beams at equal angular intervals. It will be clear to those skilled in the art that the effect of transmission of laser light, reflection of fluorescence or multiphoton higher harmonic signals can be achieved by using the features of the dichroic mirror. 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 multi-photon rescanning microscopic imaging device provided by the embodiment of the invention adopts a plurality of beams of lasers distributed on a straight line and in equal angular intervals, 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, as will be clear to those skilled in the art, the two-dimensional scanning head may be a resonant galvanometer scanning head, a galvanometer scanning head, or a piezoelectric scanning head, etc., and the two scanning heads respectively scan the light beam in different directions. The embodiment of the invention selects the resonance-galvanometer scanning head, and can greatly improve the imaging speed while realizing the improvement of the spatial resolution. When the resonant-galvanometer scanning head is selected, the scanning schematic diagrams of a plurality of laser beams are shown in fig. 3 and 4.
Fig. 3 is a schematic diagram of scanning of multiple laser beams according to an embodiment of the invention, as shown in fig. 3, the transverse direction is a scanning direction of the resonant scanning head, and the vertical direction is an arrangement direction of the multiple laser beams and a scanning direction of the galvanometer scanning head.
It is well known to those skilled in the art that resonant scan heads can only scan at a fixed frequency (i.e., the resonant frequency). In the embodiment of the invention, a plurality of laser beams are incident to the resonance scanning head, the arrangement direction of the laser beams is just vertical to the scanning direction of the resonance scanning head and is consistent with the scanning direction of the galvanometer scanning head, and the laser beams are overlapped at the geometric center position of the resonance-galvanometer scanning head. Therefore, the scanning frequency of the resonance scanning head does not need to be adjusted, and the rapid scanning imaging can be realized; and the field of view is scanned by the multi-beam without repetition by controlling the scanning mode of the galvanometer scanning head. The field scan speed is equal to the single beam scan speed multiplied by the number of beams of the multiple beams. Because the maximum scanning angle of the galvanometer scanning head in the scanning of the multiple beams of laser is 1/N of that of the galvanometer scanning head in the scanning of the single beam, the speed of the multiple beams of laser scanning is N times of that of the single beam of laser scanning for the same field of view; wherein N is the number of laser beams.
In this scanning system, the laser light is always emitted, which is called a bidirectional scanning system.
Fig. 4 is a schematic diagram of scanning by multiple lasers according to another embodiment of the present invention, and as shown in fig. 4, the dashed lines indicate that the lasers are 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.
According to the multi-beam multi-photon rescanning microscopic imaging device provided by the embodiment of the invention, through the scanning of a plurality of beams of laser, and the specific scanning mode is set for the plurality of beams of laser through the resonance-galvanometer scanning head, namely, the scanning directions of the resonance scanning head and the galvanometer scanning head are mutually vertical, and the arrangement direction of the plurality of beams of laser is consistent with the scanning direction of the galvanometer scanning head, compared with the single beam scanning in the prior art, the scanning speed is obviously improved, and the imaging time resolution is improved.
It can be understood that the two-dimensional scanning head formed by any two scanning heads can achieve the above effects. Therefore, according to the multi-beam multi-photon rescanning microscopic imaging device provided by the embodiment of the invention, the two-dimensional scanning head is adopted to scan a plurality of laser beams, so that compared with the single-beam scanning in the prior art, the scanning speed is obviously improved, and 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 multiple laser beams with equal angular intervals.
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 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 and 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, wherein laser sequentially passes through the above 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, a reflection unit 509, a second imaging lens 510, a second scanning lens 511, the second dichroic mirror 502, the two-dimensional scanning head 503, the third dichroic mirror 504, and a third imaging lens 512, which are sequentially arranged along a detection optical path direction, and the fluorescence or multi-photon, 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 beam is converted into a plurality of laser beams (hereinafter, referred to as one-dimensional multi-beam laser beam for simplicity) distributed on a straight line at equal angular intervals 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 one-dimensional multiple laser beams pass through the second dichroic mirror 502 and enter the two-dimensional scanning head 503.
After passing through the two-dimensional scanning head 503, the one-dimensional multiple beams of laser light pass through the third dichroic mirror 504, then pass through the first scanning lens 505 and the first imaging lens 506, pass through the first dichroic mirror 507, are incident on the objective lens 508, and are focused on the sample, thereby scanning the sample.
When the sample is scanned by the one-dimensional multi-beam laser, 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 return to the two-dimension 503, so that rescanning is realized; then, by reflection by the third dichroic mirror 504, the fluorescence or multiphoton higher-order harmonic signal and the one-dimensional plurality of laser beams are separated, and the fluorescence or multiphoton higher-order harmonic signal changes direction again and is focused to the camera 513 through the third imaging lens 512. 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 a laser light path, one beam of light represents laser before deflection, and the other beam of light represents laser after deflection; on the detection optical path, one beam of light represents the pre-deflection fluorescence or multi-photon higher order harmonic signal, and the other beam of light represents the post-deflection fluorescence or multi-photon higher order harmonic signal.
As shown in fig. 6, when the two-dimensional scanning head 603 deflects by an angle α, the emitted laser beams correspondingly deflect 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 multi-photon 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 multi-beam 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 the equivalent point spread function of the system, that is, the effective point spread function of the image scanning microscope in the optical rescanning mode is the product of the point spread function of a plurality of laser beams on the excitation light path and the point spread function of a fluorescence or multiphoton higher-order harmonic signal on the detection light path.
In a specific embodiment, the ratio of the scanning angle of the fluorescence or multiphoton higher harmonic signal to the scanning angle of the one-dimensional multi-beam laser 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 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 one-dimensional multi-beam laser, 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 (9)
1. A multi-beam 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 laser beams distributed on a straight line and at equal angular intervals;
the two-dimensional scanning head is used for receiving the laser beams with the equal angular spacing and scanning the laser beams with the equal angular spacing;
the focusing module is used for converting the scanned multiple laser beams with equal angular intervals into multiple focusing light spots with equal intervals and focusing the light spots 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 lasers;
the rescanning module comprises a plurality of modules which are sequentially arranged along the direction of the detection light path: 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.
2. The multi-beam multi-photon rescanning microimaging apparatus of claim 1, wherein a scan angle of the fluorescence or multi-photon higher order harmonic signal is proportional to a scan angle of the plurality of equally angularly spaced lasers, in particular:
the scanning angle of the fluorescence or multiphoton high-order harmonic signal is 2 times of that of the laser beams with the equal angular interval.
3. The multi-beam multi-photon rescan microimaging device of claim 1, wherein the beam splitter comprises a lens element, a light shaping element, and a scan lens.
4. The multi-beam multi-photon rescan microscopic imaging apparatus according to claim 3, wherein the light shaping element comprises: a microlens array, a spatial light modulator, a digital microlens, or a diffractive optical element.
5. The multi-beam multi-photon rescan microscopic imaging apparatus according to claim 1, wherein the focusing module comprises, arranged in sequence 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 multi-photon rescan microscopic imaging apparatus according to claim 1, wherein the imaging module comprises, arranged in sequence along the detection optical path: the two-dimensional scanning head, the third dichroic mirror, the third imaging lens and the camera.
7. The multi-beam multi-photon rescan microscopic imaging apparatus according to claim 6, 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.
8. The multi-beam multi-photon rescan microscopic imaging apparatus according to claim 6, wherein the two-dimensional scanning head comprises a resonance-galvanometer scanning head, a galvanometer-galvanometer scanning head, or a piezo-piezo scanning head.
9. The multi-beam multi-photon rescan microscopy imaging apparatus according to claim 1 further comprising at least one relay module in the collimated optical path for adjusting beam diameters of the plurality of equally angularly spaced lasers.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102096180A (en) * | 2009-12-11 | 2011-06-15 | 奥林巴斯株式会社 | Super-resolution microscope |
| WO2015164843A1 (en) * | 2014-04-24 | 2015-10-29 | Vutara, Inc. | Galvo scanning mirror for super-resolution microscopy |
| WO2018039361A1 (en) * | 2016-08-23 | 2018-03-01 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Systems and methods for instant total internal reflection fluorescence/structured illumination microscopy |
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| EP1953580B1 (en) * | 2000-09-18 | 2014-09-17 | Vincent Lauer | Confocal optical scanning device |
| US10247930B2 (en) * | 2014-09-24 | 2019-04-02 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Resolution enhancement for line scanning excitation microscopy systems and methods |
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| CN102096180A (en) * | 2009-12-11 | 2011-06-15 | 奥林巴斯株式会社 | Super-resolution microscope |
| WO2015164843A1 (en) * | 2014-04-24 | 2015-10-29 | Vutara, Inc. | Galvo scanning mirror for super-resolution microscopy |
| WO2018039361A1 (en) * | 2016-08-23 | 2018-03-01 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Systems and methods for instant total internal reflection fluorescence/structured illumination microscopy |
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