CN107028590B - Miniaturized self-adaptive optical two-photon fluorescence imaging system and method - Google Patents
Miniaturized self-adaptive optical two-photon fluorescence imaging system and method Download PDFInfo
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Abstract
The invention discloses a miniaturized self-adaptive optical two-photon fluorescence imaging system and a method, wherein the imaging system comprises the following components: a laser light source device for outputting excitation light; the miniature probe device is used for receiving the excitation light output by the laser light source device and generating a fluorescence signal; and the method is used for carrying out wavefront correction on each isochrone region according to the average wavefront distortion distribution of each isochrone region of the tissue plane imaging view field in the living body sample; the wavefront detection device is connected with the miniature probe device and is used for detecting average wavefront distortion distribution; the scanning removing component is used for compensating the corona area off-axis effect generated by the miniature probe device in real time before the wavefront detecting device detects the average wavefront distortion distribution; and the fluorescence imaging device is used for collecting fluorescence signals output by the micro probe device and completing imaging of the tissue plane in the living sample. The invention can realize imaging of deep biological tissues with large field of view, high space-time resolution and high depth in freely movable animals.
Description
Technical Field
The invention relates to the technical field of two-photon fluorescence imaging, in particular to a miniaturized self-adaptive optical two-photon fluorescence imaging system and method.
Background
Direct recording of neuronal activity in free-moving animals is one of the most directly effective methods to study the relationship between animal behavior and neurological function. This task has been performed for a long time previously by the electrophysiological route (electrophysiological approach). In recent years, optical imaging, in particular fluorescence microscopy, has played an increasingly important role in this task. The greatest advantage of optical imaging over electrophysiological methods is that it is non-invasive and has a larger imaging field of view and more observable targets. Compared with the common single photon fluorescence imaging technology, the two-photon microscope has better optical slicing capability and deeper penetration depth. This makes two-photon microscopy the most important and widely used tool in brain neuron observation using fluorescence imaging. Meanwhile, in order to observe the neural activity of awake animals in an active state, researchers often modify a large-sized desktop two-photon microscope, and add some devices simulating exercise such as a running machine or a rotating wheel, etc.; however, the head of the mouse must be fixed under the microscope lens during experiments, and the movement of the trunk is only not limited, so that the real movement is simulated. As research goes deep, scientists have found that such simulations expose a number of drawbacks. First, it is believed that such simulation does not reflect the actual activity state. Because many processes that the animal, such as mice, need to participate in the real activities, such as physical twisting, clues to the surrounding environment, gravitational conversion, etc., are not available in the head-mounted form. Second, many classical behavioral studies, such as fear, social and exploratory etc., cannot be realized while the head is fixed.
Therefore, since 2001, attempts have been made to fabricate miniature two-photon microscope systems that can be mounted on animal heads, such as rat or mouse heads, and that can perform fluorescence imaging when they are fully free to move. 25g small two-photon microscope made from the group of professor Denk topics in the United states of America in 2001 mirror 5g miniature two-photon microscope manufactured by Kerr professor task group in 2011. Scientists have tried several times, however none have achieved a very desirable result. In general, the optical quality of the whole is difficult to control because the micro two-photon microscope greatly reduces the size of all optical lenses. And the assembly and coupling between tiny lenses is difficult to achieve as good as large lenses. More importantly, small objectives are more sensitive to distortion introduced by the sample due to the limited number of lenses. All of these problems result in miniaturized two-photon microscopes that cannot achieve as high resolution imaging as large bench-top two-photon microscopes. This greatly limits the popularization and application of miniaturized two-photon microscopes.
On the other hand, the adaptive optics are well utilized in astronomy, fundus examination and microscopic imaging fields to correct distortion caused by the system and the template, thereby improving image quality. Particularly in large table-type two-photon microscopes, attempts have been made to correct aberrations using adaptive optics to improve imaging quality. However, since the conventional adaptive optics require a complicated wavefront sensing device and wavefront correction device, the overall volume is huge, and thus cannot be applied to a miniaturized two-photon microscope. Therefore, only a new self-adaptive optical scheme is provided, a new self-adaptive optical system is designed, and a new miniature self-adaptive optical device is used, so that the self-adaptive optics and the miniature two-photon microscope can be combined, and the imaging quality of the miniature two-photon microscope is improved.
It is therefore desirable to have a solution that overcomes or at least alleviates at least one of the above-mentioned drawbacks of the prior art.
Disclosure of Invention
It is an object of the present invention to provide a miniaturized adaptive optical two-photon imaging system and method that overcomes or at least alleviates at least one of the above-mentioned drawbacks of the prior art.
To achieve the above object, the present invention provides a miniaturized adaptive optical two-photon fluorescence imaging system including: a laser light source device for outputting excitation light; the miniature probe device is used for receiving the excitation light output by the laser light source device and exciting a tissue plane inside the living body sample by using the excitation light so as to generate a fluorescence signal; and performing wavefront correction on each of the isochrone regions according to an average wavefront distortion distribution of each of the isochrone regions of a tissue plane imaging field of view inside the living body sample in a state where the living body sample is released; a wavefront sensing device having a signal input port operatively connected to the signal output port of the microprobe device for receiving the fluorescent signal output by the microprobe device and sensing the average wavefront aberration distribution in the event that the living sample is immobilized; a descan component which is arranged on the light path between the miniature probe device and the wavefront detection device and is used for compensating the isochrone off-axis effect generated by the miniature probe device in real time before the wavefront detection device detects the average wavefront distortion distribution; and the signal input port of the fluorescence imaging device is operatively connected with the signal output port of the micro probe device and is used for collecting the fluorescence signal which is output by the micro probe device and subjected to wave front correction, and imaging the tissue plane inside the living body sample is completed.
Further, the micro probe device includes: the laser input module is used for receiving the excitation light output by the laser light source device; a wavefront correction module, configured to perform wavefront correction on each of the isochronal regions according to an average wavefront distortion distribution of each of the isochronal regions of a tissue plane imaging field of view inside the living body sample in a case where the living body sample is released; and a scanning imaging module for receiving the wave-front corrected excitation light, wherein the excitation light scans a tissue plane inside the living body sample in a two-dimensional motion mode so as to excite the living body sample to generate the fluorescent signal.
Further, the scanning imaging module includes: a first biaxial rotation mirror for two-dimensionally scanning the tissue plane inside the living body sample with the excitation light wavefront-corrected by the wavefront correction module by rotationally changing an angle of incidence of the excitation light; an eyepiece for converging excitation light from the first biaxial rotation mirror to an inside of the living sample to excite the living sample to generate the fluorescent signal; for outputting the fluorescent signal; a scanning mirror disposed on an optical path between the first biaxial turning mirror and the eyepiece for converting excitation light of an angle change generated by the two-dimensional scanning of the first biaxial turning mirror into excitation light of a position change; and a dichroic mirror provided between the scanning mirror and the eyepiece for separating the excitation light and the fluorescence signal and outputting the fluorescence signal.
Further, the descan means includes: the second micro-electromechanical double-shaft rotating mirror is arranged on a light path between a signal output port of the micro probe device and the wavefront detection device; the first lens is arranged on a light path between the first micro-electromechanical double-shaft rotating mirror and a signal output port of the micro probe device and is used for receiving a fluorescent signal output by the micro probe device, converting the fluorescent signal into a wave front and projecting a conjugate surface of the wave front onto the second micro-electromechanical double-shaft rotating mirror; the second micro-electromechanical double-shaft rotating mirror is matched with the first micro-electromechanical double-shaft rotating mirror in a mode of being capable of compensating an isochrone region off-axis effect generated by scanning of the first micro-electromechanical double-shaft rotating mirror in real time, and then excitation light after real-time compensation is transmitted to the wavefront detection device; the matching relation between the second micro-electromechanical double-shaft rotating mirror and the first micro-electromechanical double-shaft rotating mirror needs to satisfy: in time, the same frequency and the same phase; spatially, the scan angle requirements are: the ratio of the rotation angle of the second micro-electromechanical double-shaft rotating mirror to the rotation angle of the first micro-electromechanical double-shaft rotating mirror is the ratio of the focal length of the scanning mirror to the focal length of the first lens; the scanning direction requirements are: the opposite direction.
Further, the wavefront detection device includes: a wavefront sensor for receiving a wavefront output by the first lens; and the first relay mechanism is arranged on an optical path between the second micro-electromechanical double-shaft rotating mirror and the wavefront sensor and is used for enabling the second micro-electromechanical double-shaft rotating mirror to be conjugated with the wavefront sensor so that the wavefront sensor can detect the average wavefront distortion distribution.
Further, the wavefront correction module includes: a variable type reflecting mirror for receiving the excitation light output from the laser input module and changing and controlling the wavefront of the emitted excitation light according to the average wavefront distortion distribution; and a second relay mechanism for conjugating the variable mirror and the scanning mirror and projecting the excitation light wavefront-corrected by the variable mirror to the reflecting surface of the first biaxial rotation mirror.
Further, the wavefront correction module further comprises a second lens, a first half-wave plate, a polarization splitting cube, a second half-wave plate, and a quarter-wave plate disposed on the optical path, wherein: the excitation light output by the laser input module sequentially passes through the second lens and the first half-wave plate, enters the polarization beam-splitting cube from the first side surface of the polarization beam-splitting cube, is output from the second side surface of the polarization beam-splitting cube, sequentially passes through the second half-wave plate and the quarter-wave plate, and then is projected to the reflecting surface of the variable reflecting mirror; the excitation light reflected by the variable reflecting mirror passes through the quarter wave plate and the second half wave plate again, enters the polarization splitting cube from the second side surface of the polarization splitting cube, and is projected to the second relay mechanism from the third side surface of the polarization splitting cube through the excitation light of the polarization splitting cube.
Further, a signal input port of the fluorescence imaging device is connected with a signal output port of the micro probe device through a flexible light beam.
The invention provides a miniaturized self-adaptive optical two-photon fluorescence imaging method, which comprises the following steps: step 1, wavefront detection, which specifically includes: step 11, fixing a living body sample, and fixedly mounting a miniature probe device at a preset position of the living body sample; step 12, compensating the off-axis effect of the halation area generated by the miniature probe device in real time by removing the scanning component; step 13, detecting the average wavefront distortion distribution of each isochron region of the tissue plane imaging view field in the living body sample by a wavefront detection device; step 2, fluorescence imaging, which specifically comprises: step 21, releasing the living body sample; step 22, according to the average wavefront distortion distribution detected in the step 13, the micro probe device performs wavefront correction on each of the isochrone regions and outputs a fluorescence signal after the wavefront correction; and step 23, collecting the fluorescence signal output by the step 22 through a fluorescence imaging device, and completing the imaging of the tissue plane inside the living body sample.
Under the condition of fixing a living body sample, the invention can lead out the wave front of a fluorescent signal from the miniature probe device, uses the scanning component to compensate the off-axis effect of the isochron area generated by the miniature probe device in real time, then uses the wave front detection device to detect the average wave front distortion distribution of each isochron area of the tissue plane imaging view field in the living body sample, and then under the precondition of releasing the living body sample, the miniature probe device carries out wave front correction on each isochron area according to the detected average wave front distortion distribution and outputs the fluorescent signal after wave front correction, thereby completing the imaging of the tissue plane in the living body sample, and therefore, the invention can realize the imaging of the tissue plane with large view field, high space-time resolution and deep biological tissue in freely movable animals.
Drawings
Fig. 1 is a schematic diagram of a miniaturized adaptive optical two-photon imaging system provided by the present invention.
Fig. 2 is a schematic structural diagram of a preferred embodiment of implementing a wavefront sensing portion in the miniaturized adaptive optical two-photon imaging system of fig. 1.
FIG. 3 is a schematic diagram illustrating the mating relationship of the descan member and the scanning imaging module of FIG. 2.
Fig. 4 is a schematic structural diagram of a preferred embodiment of a miniaturized adaptive optical two-photon imaging system of fig. 1 for implementing a fluorescence imaging portion.
Fig. 5 is a schematic diagram of the laser light source device of fig. 1.
Detailed Description
In the drawings, the same or similar reference numerals are used to denote the same or similar elements or elements having the same or similar functions. Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
In the description of the present invention, the terms "center", "longitudinal", "lateral", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like indicate an orientation or a positional relationship based on that shown in the drawings, only for convenience of description and simplification of the description, and do not indicate or imply that the apparatus or element to be referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the scope of protection of the present invention.
The living sample of the present invention is a living small animal, for example: mice, etc. In view of the small volume of the living body sample, the invention belongs to a micro device, and the volume of the whole structure is 1-5 cm 3 Within the range.
The scanning element in the prior art microprobe device deflects the excitation light to a certain extent during imaging/wavefront detection, i.e. laser scanning, so that the converging position of the excitation light deviates from the optical axis of the whole optical system, and the isochrone region of the generated fluorescence signal also deviates from the optical axis, which phenomenon is also called "off-axis effect". In the prior art, a wavefront sensing mode based on constant vignetting areas is almost adopted, and in the case of the off-axis effect, the average wavefront distortion distribution of each vignetting area of a tissue plane imaging view field inside a living body sample cannot be detected.
In view of the problems in the prior art, a set of scanning removing mechanism is newly added on an optical path between the miniature probe device and the wavefront sensing device, and the scanning removing mechanism is linked with the scanning mechanism in the miniature probe by utilizing a control algorithm, so that the deflection reverse off-axis isochron area of a fluorescent signal is corrected to the optical axis of the whole optical system in real time, and even if a wavefront sensing mode based on the isochron area invariance in the prior art is adopted, the average wavefront distortion distribution of each isochron area of a tissue plane imaging view field in a living body sample can be accurately detected.
According to the technical problem to be solved, the miniaturized self-adaptive optical two-photon fluorescence imaging method provided by the embodiment comprises the following steps:
and step 11, fixing a living body sample, and fixedly mounting a miniature probe device at a preset position of the living body sample. The "preset position" is usually selected as a windowing position at the head of the living sample (the head of a small mouse). The fenestration site reveals fluorescently labeled brain tissue.
And step 12, compensating the off-axis effect of the halation area generated by the scanning component in the miniature probe device in real time by removing the scanning component. The method can be realized by the following steps:
as shown in fig. 1 and 3, the scanning means 4 includes a second micro-electro-mechanical biaxial rotation mirror 41 and a first lens 42, and the second micro-electro-mechanical biaxial rotation mirror 41 is disposed on an optical path between a signal output port of the micro probe device 2 and the wavefront detection device 3. The first lens 42 is disposed on the optical path between the first biaxial rotation mirror 231 and the signal output port of the micro probe device 2, and is configured to receive the fluorescent signal output by the micro probe device 2, convert the fluorescent signal into a wavefront, and project the conjugate plane of the wavefront onto the second microelectromechanical biaxial rotation mirror 41. The scanning means in the micro probe device 2 includes a first biaxial rotation mirror 231 for two-dimensionally scanning the tissue plane inside the living body sample with the excitation light wave-front corrected by the wave-front correction module 22 in a manner of changing the angle of rotation, and a scanning mirror 233. The scan mirror 233 is used to convert the angular-change excitation light generated by the two-dimensional scan of the first biaxial rotation mirror 231 into position-change excitation light. The synchronous scanning of the second micro-electromechanical biaxial rotation mirror 41 and the first biaxial rotation mirror 231 is required to satisfy the following requirements:
1. the frequency is the same in time and the range is 100-300 Hz; the phases are identical.
2. Spatially, the scan angle requirements are: the ratio of the angle at which the second micro electro mechanical dual axis turning mirror 41 is turned to the angle at which the first dual axis turning mirror 231 is turned is the ratio of the focal length of the scan mirror 233 to the focal length of the first lens 42. The scanning direction requirements are: the opposite direction.
By the mode, the off-axis effect of the isochron area caused by the scanning of the scanning component is exactly compensated by the synchronous deflection of the scanning component, and the off-axis isochron area of the deflection reverse direction of the fluorescent signal output by the micro probe device is corrected to the axis of the whole optical system in real time, so that the advantage is provided for effectively detecting the average wavefront distortion distribution in the step 13.
And step 13, detecting the average wavefront distortion distribution of each isochron area of the tissue plane imaging field of view inside the living body sample by a wavefront detection device.
And step 23, acquiring the fluorescence signals output in the step 22 through a fluorescence imaging device, and completing the imaging of the tissue plane inside the living body sample.
As shown in fig. 1, the present embodiment provides a miniaturized adaptive optical two-photon fluorescence imaging system that realizes the miniaturized adaptive optical two-photon fluorescence imaging method as described above, the system including a laser light source device 1, a microprobe device 2, a wavefront detection device 3, a descan member 4, and a fluorescence imaging device 5, wherein:
as shown in fig. 5, the laser light source device 1 is configured to output excitation light. The laser light source device 1 is composed of one femtosecond laser 11, the pulse width of the femtosecond laser 11 is between 80 and 250fs (femtosecond), the repetition frequency is 40 to 100MHz, and the highest power is more than 500 mW.
The micro probe device 2 is fixedly installed at a preset position of the living body sample, namely, a head windowing position of the living body sample. The miniature probe device 2 is used for receiving the excitation light output by the laser light source device 1 and exciting a tissue plane inside the living body sample by using the excitation light so as to generate a fluorescence signal; and performing wavefront correction on each of the isochrone regions according to an average wavefront distortion distribution of each of the isochrone regions of a tissue plane imaging field of view inside the living body sample in a state where the living body sample is released.
As shown in fig. 1, in one embodiment, the microprobe device 2 includes a laser input module 21, a wavefront correction module 22, and a scanning imaging module 23, wherein:
as shown in fig. 5, the laser input module 21 is configured to receive excitation light output from the laser light source device 1. The laser input module 21 specifically includes a light intensity modulation module and a fiber coupling module 215, where the light intensity modulation module includes an electrically operated shutter 211, a half-wave plate 212, and an electro-optic/acousto-optic modulator 213. The fiber coupling module 215 couples the collimated laser beam into a photonic crystal fiber through an aspheric lens 214 (hollow-core photon crystal fiber).
As shown in fig. 1, the wavefront correction module 22 is configured to perform wavefront correction on each of the isochrone regions of the tissue plane imaging field of view within the living sample according to an average wavefront distortion distribution of the isochrone regions in a state where the living sample is released.
As shown in fig. 2, in one embodiment, the wavefront correction module 22 includes a variable type mirror 221 and a second relay mechanism 222, wherein: the variable type mirror 221 is used for receiving the excitation light output from the laser input module 21 and changing and controlling the wavefront of the emitted excitation light according to the average wavefront distortion distribution.
The second relay mechanism 222 is configured to conjugate the variable mirror 221 and the first biaxial rotation mirror 231, and to project the excitation light wavefront-corrected by the variable mirror 221 onto the reflection surface of the first biaxial rotation mirror 231. That is, setting the wavefront of the #1 face, which is schematically illustrated in the drawing, to be detected and corrected in the present embodiment, the variable mirror 221 and the first biaxial mirror 231 form a 4f system by the second relay mechanism 222, then the conjugate plane of the #1 face corrected by the variable mirror 221 is the emission plane of the first biaxial mirror 231.
In performing the wavefront correction, the matching relationship between the control current of the variable type mirror 221 and the first biaxial rotation mirror 231 can be expressed by the following equation:
ΔS(N)＝-2*T(I N )/λ
wherein: Δs (N) is the average wavefront distortion (between the plane wavefront and the N-th sub-aperture on conjugate plane #1Phase difference); i N For the current applied to the nth independent deformation element of the variable mirror 221; t (I) is the driving function of the variable mirror 221, i.e., the amount of spatial displacement on a certain independent deformation unit when a current of value I is applied; lambda is the incident laser wavelength.
In one embodiment, the wavefront correction module 22 further comprises a second lens 223, a first half-wave plate 224, a polarization splitting cube 225, a second half-wave plate 226, and a quarter-wave plate 227 disposed in the optical path, wherein: the excitation light output by the laser input module 21 sequentially passes through the second lens 223 and the first half-wave plate 224, enters the polarization splitting cube 225 from the first side surface of the polarization splitting cube 225, is output from the second side surface of the polarization splitting cube 225, sequentially passes through the second half-wave plate 226 and the quarter-wave plate 227, and then is projected onto the reflecting surface of the variable reflecting mirror 221. The excitation light reflected by the variable type reflecting mirror 221 enters the polarization splitting cube 225 from the second side surface of the polarization splitting cube 225 again through the quarter wave plate 227 and the second half wave plate 226, is projected to the second relay mechanism 222 from the third side surface of the polarization splitting cube 225 through the excitation light of the polarization splitting cube 225, and is finally projected to the reflecting surface of the first biaxial turning mirror 231 through the second relay mechanism 222. In this embodiment, the second lens 223 is used to collimate the laser light emitted from the light. The first half wave plate 224 is used to change the polarization direction of the laser light. The polarization splitting cube 225 is used to separate the incident light from the reflected light. The second half-wave plate 226 and quarter-wave plate 227 together serve to make the polarization direction of the incident light exactly perpendicular to the polarization direction of the emitted light so that the incident light and the reflected light can be separated by 225.
The scanning imaging module 23 is configured to receive the wavefront-corrected excitation light, and scan a tissue plane inside the living body sample in a two-dimensional motion manner to generate the fluorescence signal.
In one embodiment, the scanning imaging module 23 includes a first dual axis turning mirror 231, an eyepiece 232, a scanning mirror 233, and a dichroic mirror 234, wherein:
the first biaxial rotation mirror 231 is used to scan the tissue plane inside the living body sample in two dimensions by the excitation light wave-front corrected by the wave-front correction module 22 in a rotation changing angle. The wavefront correction of the wavefront correction module 22 is updated once for each angular transformation of the first dual axis turning mirror 231 to compensate for the different wavefront distortions of the different halo regions. The first biaxial rotation mirror 231 is a microelectromechanical biaxial rotation mirror, and its parameter ranges include: mirror dimensions: 0.8-1.0 mm; scanning angle: 5 to 7 degrees; first resonant frequency: greater than 2000Hz.
The eyepiece 232 is used for converging excitation light from the first biaxial rotation mirror 231 to the inside of the living body sample to excite the inside of the living body sample to generate the fluorescence signal, and for outputting the fluorescence signal. The #2 plane shown in fig. 2 is an imaging plane, and the imaging plane of the tissue plane inside the living body sample and the #1 plane are fourier transforms of each other.
The scan mirror 233 is disposed on an optical path between the first biaxial rotation mirror 231 and the eyepiece 232 for converting the angle-changed excitation light generated by the two-dimensional scan of the first biaxial rotation mirror 231 into position-changed excitation light. A dichroic mirror 234 is provided between the scan mirror 233 and the eyepiece 232 for separating the excitation light and the fluorescent signal.
As shown in fig. 1, the signal input port of the wavefront sensing device 3 is operatively connected to the signal output port of the micro probe device 2 for receiving the fluorescent signal output by the micro probe device 2 and detecting the average wavefront distortion distribution in the case where the living sample is fixed. The wavefront sensing device 3 is used in conjunction with the microprobe device 2 during wavefront sensing. In wavefront detection, the living sample needs to be fixed.
As shown in fig. 2, in one embodiment, the wavefront sensing device 3 includes a wavefront sensor 31 and a first relay mechanism, wherein: the wavefront sensor 31 is configured to receive the wavefront output by the first lens 42. The first relay mechanism is disposed on the optical path between the second micro-electromechanical dual-axis turning mirror 41 and the wavefront sensor 31, and is used for conjugating the second micro-electromechanical dual-axis turning mirror 41 with the wavefront sensor 31, so that the wavefront sensor 31 can detect the average wavefront distortion distribution. The first relay mechanism is composed of a lens 33 and a lens 34, so that the second mems biaxial rotation mirror 41 and the wavefront sensor 31 form a 4f system, and then the conjugate plane of the #1 plane is the detection plane of the wavefront sensor 31. The #2 surface is formed on the reflecting surface of the reflecting mirror 35 by the 4f relationship formed by the lens 33 and the first lens 42.
That is, the detector 37 may also be used for imaging without the need for the optical fiber 6 to introduce the fluorescent signal into the system 5. This facilitates evaluation of the improvement in imaging quality during wavefront sensing and continuous optimization of wavefront correction.
As shown in fig. 1 and 2, in one embodiment, a descan means 4 is provided in the optical path between the micro-probe device 2 and the wavefront sensing device 3 for compensating in real time for the isochron off-axis effect produced by the micro-probe device 2 before the wavefront sensing device 3 detects the average wavefront distortion profile.
Specifically, the descan member 4 includes a second microelectromechanical biaxial rotating mirror 41 and a first lens 42, wherein:
the second micro-electromechanical biaxial turning mirror 41 is arranged on the optical path between the signal output port of the micro probe device 2 and the wavefront sensing device 3. The second micro-electromechanical double-shaft rotating mirror 41 adopts a large-caliber micro-electromechanical double-shaft rotating mirror, and the parameter ranges comprise: mirror dimensions: 3-5mm; scanning angle: (+ -1 to (+ -3); first resonant frequency: greater than 200Hz.
The first lens 42 is disposed on an optical path between the first biaxial rotation mirror 231 and a signal output port of the micro probe device 2, and is configured to receive a fluorescent signal output by the micro probe device 2, convert the fluorescent signal into a wavefront, and project the conjugate plane # 1 onto the second microelectromechanical biaxial rotation mirror 41.
The ratio of the rotation angle of the second micro-electro-mechanical dual-axis rotating mirror 41 to the rotation angle of the first dual-axis rotating mirror 231 is the ratio of the focal length of the scanning mirror 233 to the focal length of the first lens 42, so as to compensate the off-axis effect of the isochrone region generated by the scanning of the first dual-axis rotating mirror 231 in real time, and then the excitation light after the real-time compensation is transmitted to the wavefront detection device 3.
In connection with fig. 3, in order to realize real-time compensation of the isochrone off-axis effect generated by the micro probe device 2 by the scanning component 4, the synchronous scanning of the second micro electro-mechanical dual-axis turning mirror 41 and the first dual-axis turning mirror 231 needs to meet the following requirements:
1. the frequency is the same in time and the range is 100-300 Hz; the phases are identical.
2. Spatially, the scan angle requirements are: the ratio of the angle of rotation of the second micro-electromechanical biaxial rotation mirror 41 to the angle of rotation of the first biaxial rotation mirror 231 is the ratio of the focal length of the scan mirror 233 to the focal length of the first lens 42, i.e. as illustrated in fig. 3
β 1 Alpha is the rotation angle of the second micro-electromechanical double-shaft rotating mirror 41 1 F is the angle of rotation of the first biaxial rotation mirror 231 1 F is the focal length of the scan mirror 233 2 Is the focal length of the first lens 42. The scanning direction requirements are: the opposite direction. />
By the mode, the off-axis effect of the isochron area caused by the scanning of the scanning component is exactly compensated by the synchronous deflection of the scanning component, and the off-axis isochron area of the deflection reverse direction of the fluorescent signal output by the miniature probe device is corrected to the axis of the whole optical system in real time, so that the advantage is provided for effectively detecting the average wavefront distortion distribution of the wavefront detection device 3.
As shown in fig. 4, a signal input port of the fluorescence imaging device 5 is operatively connected to a signal output port of the micro probe device 2, and is used for acquiring the fluorescence signal output by the micro probe device 2 after wavefront correction, and completing imaging of a tissue plane inside the living body sample. The composition of the fluorescence imaging device 5 is prior art and will not be described here. The fluorescence imaging device 5 is used in cooperation with the microprobe device 2 at the time of fluorescence imaging. The signal input port of the fluorescence imaging device 5 is connected with the signal output port of the micro probe device 2 through a flexible light beam 6. In the fluorescence imaging process, the living body sample needs to be released, and the fluorescence signal output by the micro probe device is collected by using the flexible optical fiber bundle 6, and the flexible optical fiber bundle needs to be of a sufficient length, so that the living body sample can freely move even if the head is propped against the micro probe device without being restrained, thereby being beneficial to reflecting the real moving state of the living body sample and being beneficial to acquiring the real image data of the deep brain tissue of the living body sample.
The working process of the wavefront detection by using the invention is as follows:
the microprobe device 2 and the wavefront sensing device 3 are combined as shown in fig. 2. The femtosecond excitation light output by the femtosecond laser 11 is transmitted to the micro probe device 2 through the hollow photonic crystal fiber. First, the second lens 223 collimates the light, and the light passes through the first half-wave plate 224 to adjust the polarization direction, and is reflected by the polarization splitting cube 225, and passes through the second half-wave plate 226 and the quarter-wave plate 227 for the first time, respectively, to reach the surface of the variable mirror 221. The collimated light reflected by the variable mirror 221 passes through the second half-wave plate 226 and the quarter-wave plate 227 a second time and passes through the polarization splitting cube 225. Then, the excitation light beam is projected onto the surface of the first biaxial rotation mirror 231 using the second relay mechanism 222. The first biaxial rotation mirror 231 is controlled by two high-voltage electrostatic signals, and can perform laser scanning in two directions. The scanning beam is focused on the imaging surface of the eyepiece 232 by the converging action of the scanning mirror 233 and the reflecting action of the dichroic mirror 234, and is focused on the living sample by the eyepiece 232, thereby performing two-dimensional point scanning excitation. The excited fluorescent signal is collected by the eyepiece 232 in an epi-beam form, passes through the dichroic mirror 234, exits the micro probe device 2, and is collimated by the first lens 42. The collimated beam irradiates the second mems biaxial rotation mirror 41, is reflected by the reflecting mirror, and is projected onto the wavefront sensor 31 or the receiver of the photodetector 37 through the first relay mechanism composed of the lens 33 and the lens 4.
In the above procedure, all the rear exit pupil surfaces have an imaging conjugate relationship (# 1); all image planes have an imaging conjugate relationship (# 2). Distance 1 is equal to the working distance of the first lens 42; the sum of the distance 2 and the distance 3 is the same as the working distance of the lens 3 and is equal to the distance 4; distance 5 is the same as distance 6 and is equal to the working distance of lens 44. In wavefront measurement, the first biaxial rotation mirror 231 and the second microelectromechanical biaxial rotation mirror 41 are required to perform synchronous scanning, i.e., scanning frequency, phase, and waveform are the same. The ratio of the amplitude is required to achieve the effect of scanning, namely the off-axis effect of the isochron area caused by the scanning of the micro-electromechanical double-shaft rotating mirror is exactly compensated by the synchronous deflection of the large-caliber micro-electromechanical double-shaft rotating mirror, so that the effect of unchanged isochron area is achieved. And further, accurate wavefront sensing can be realized for a larger field of view.
The working process of fluorescence imaging by using the invention is as follows:
the microprobe device 2 and the fluorescence imaging device 5 are combined as shown in fig. 4. The fluorescent signals received and emitted by the eyepiece 232 are collected by the flexible optical fiber bundle 6 and further collected by the fluorescent imaging device 5. In this process, the variable mirror 221 in the microprobe device 2 is driven to generate mirror surface deformation according to the calculated average wavefront distortion distribution of each isochron area on the rear exit pupil plane # 2, and the wavefront correction is completed. The two steps are repeated, so that the whole deep biological tissue can be subjected to large-view-field high-space-time resolution point-by-point scanning fluorescence microscopic imaging.
Finally, it should be pointed out that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting. Those of ordinary skill in the art will appreciate that: the technical schemes described in the foregoing embodiments may be modified or some of the technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. A miniaturized adaptive optical two-photon fluorescence imaging system suitable for use with free-moving animals, comprising:
a laser light source device (1) for outputting excitation light;
the miniature probe device (2) is used for being fixed at a preset position of the living body sample, receiving excitation light output by the laser light source device (1), and exciting a tissue plane inside the living body sample by using the excitation light so as to generate a fluorescence signal; and performing wavefront correction on each of the isochrone regions according to an average wavefront distortion distribution of each of the isochrone regions of a tissue plane imaging field of view inside the living body sample collected by a flexible optical fiber bundle in a state where the living body sample is released;
a wavefront detection device (3) having a signal input port operatively connected to the signal output port of the microprobe device (2) for receiving a fluorescent signal output from the microprobe device (2) and detecting the average wavefront distortion distribution in the case where the living sample is immobilized;
a descan component (4) arranged on an optical path between the miniature probe device (2) and the wavefront detection device (3) and used for compensating an isochrone off-axis effect generated by the miniature probe device (2) in real time before the wavefront detection device (3) detects the average wavefront distortion distribution; and
a fluorescence imaging device (5), the signal input port of which is operatively connected with the signal output port of the micro probe device (2) and is used for collecting the fluorescence signal output by the micro probe device (2) after wave front correction, and completing the imaging of the tissue plane inside the living body sample;
the microprobe device (2) comprises:
a wavefront correction module (22) for performing wavefront correction on each of the isochronal regions of the tissue plane imaging field of view within the living sample according to an average wavefront distortion distribution of the isochronal regions in a state where the living sample is released;
a scanning imaging module (23) for receiving the wavefront-corrected excitation light, which scans a tissue plane inside the living sample in a two-dimensional motion to excite the living sample to generate the fluorescence signal; the scanning imaging module (23) comprises:
a first biaxial rotation mirror (231) for two-dimensionally scanning the tissue plane inside the living body sample with the excitation light wave-front-corrected by the wave-front correction module (22) by rotationally changing the angle of incidence of the excitation light;
an eyepiece (232) for converging excitation light from the first biaxial rotation mirror (231) into the inside of the living sample to excite the living sample to generate the fluorescent signal; for outputting the fluorescent signal;
a scanning mirror (233) disposed on an optical path between the first biaxial turning mirror (231) and the eyepiece (232) for converting excitation light of an angle change generated by the two-dimensional scanning of the first biaxial turning mirror (231) into excitation light of a position change;
the descan member (4) comprises:
a second micro-electromechanical biaxial rotating mirror (41) arranged on an optical path between a signal output port of the micro probe device (2) and the wavefront detection device (3); and
a first lens (42) arranged on an optical path between a second micro-electro-mechanical dual-axis turning mirror (41) and a signal output port of the micro probe device (2) and used for receiving a fluorescent signal output by the micro probe device (2), converting the fluorescent signal into a wave front and projecting a conjugate plane of the wave front onto the second micro-electro-mechanical dual-axis turning mirror (41);
the second micro-electromechanical double-shaft rotating mirror (41) is matched with the first double-shaft rotating mirror (231) in a mode of being capable of compensating an isochrone region off-axis effect generated by scanning of the first double-shaft rotating mirror (231) in real time, and then excitation light after real-time compensation is transmitted to the wavefront detection device (3);
the matching relation between the second micro-electromechanical double-shaft rotating mirror (41) and the first double-shaft rotating mirror (231) needs to be satisfied:
in time, the same frequency and the same phase;
spatially, the scan angle requirements are: the ratio of the rotation angle of the second micro-electromechanical double-shaft rotating mirror (41) to the rotation angle of the first double-shaft rotating mirror (231) is the ratio of the focal length of the scanning mirror (233) to the focal length of the first lens (42); the scanning direction requirements are: the opposite direction.
2. The miniaturized adaptive optical two-photon fluorescence imaging system according to claim 1, wherein the microprobe device (2) further comprises:
and the laser input module (21) is used for receiving the excitation light output by the laser light source device (1).
3. The miniaturized adaptive optical two-photon fluorescence imaging system of claim 2, wherein the scanning imaging module (23) further comprises:
a dichroic mirror (234) provided between the scanning mirror (233) and the eyepiece (232) for separating the excitation light and the fluorescence signal and outputting the fluorescence signal.
4. A miniaturized adaptive optical two-photon fluorescence imaging system according to claim 3, characterized in that the wavefront detection device (3) comprises:
a wavefront sensor (31) for receiving a wavefront output by the first lens (42); and
and the first relay mechanism is arranged on an optical path between the second micro-electromechanical double-shaft rotating mirror (41) and the wavefront sensor (31) and is used for enabling the second micro-electromechanical double-shaft rotating mirror (41) to be conjugated with the wavefront sensor (31) so that the wavefront sensor (31) can detect the average wavefront distortion distribution.
5. The miniaturized adaptive optical two-photon fluorescence imaging system of any of claims 3 to 4, wherein the wavefront correction module (22) comprises:
a variable type mirror (221) for receiving the excitation light output by the laser input module (21) and changing and controlling the wavefront of the emitted excitation light according to the average wavefront distortion distribution; and
and a second relay mechanism (222) for conjugating the variable mirror (221) and the scanning mirror (233) and projecting the excitation light wavefront-corrected by the variable mirror (221) to the reflecting surface of the first biaxial rotation mirror (231).
6. The miniaturized adaptive optical two-photon fluorescence imaging system of claim 5, wherein the wavefront correction module (22) further comprises a second lens (223), a first half-wave plate (224), a polarization splitting cube (225), a second half-wave plate (226), and a quarter-wave plate (227) disposed in the optical path, wherein: the excitation light output by the laser input module (21) sequentially passes through the second lens (223) and the first half-wave plate (224), enters the polarization splitting cube (225) from the first side surface of the polarization splitting cube (225), is output from the second side surface of the polarization splitting cube (225), sequentially passes through the second half-wave plate (226) and the quarter-wave plate (227), and then is projected to the reflecting surface of the variable reflecting mirror (221); the excitation light reflected by the variable reflecting mirror (221) passes through the quarter wave plate (227) and the second half wave plate (226) again, enters the polarization splitting cube (225) from the second side surface of the polarization splitting cube (225), and is projected to the second relay mechanism (222) from the third side surface of the polarization splitting cube (225) through the excitation light of the polarization splitting cube (225).
7. The miniaturized self-adaptive optical two-photon fluorescence imaging system according to claim 6, characterized in that the signal input port of the fluorescence imaging device (5) is connected with the signal output port of the microprobe device (2) through a flexible optical fiber bundle (6).
8. A miniaturized adaptive optical two-photon fluorescence imaging method using the miniaturized adaptive optical two-photon fluorescence imaging system of any of claims 1-7, comprising:
step 1, wavefront detection, which specifically includes:
step 11, fixing a living body sample, and fixedly mounting a miniature probe device at a preset position of the living body sample;
step 12, compensating the off-axis effect of the halation area generated by the miniature probe device in real time by removing the scanning component; and
step 13, detecting average wavefront distortion distribution of each isochron region of a tissue plane imaging view field in the living body sample by a wavefront detection device;
step 2, fluorescence imaging, which specifically comprises:
step 21, releasing the living body sample;
step 22, according to the average wavefront distortion distribution detected in the step 13, the micro probe device performs wavefront correction on each of the isochrone regions and outputs a fluorescence signal after the wavefront correction; and
and step 23, acquiring the fluorescence signals output in the step 22 through a fluorescence imaging device, and completing the imaging of the tissue plane inside the living body sample.
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| CN113100712A (en) * | 2021-04-08 | 2021-07-13 | 浙江大学 | Apparatus and method for long time in vivo imaging of awake small animals |
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