CN107028590A - One kind miniaturization adaptive optics two-photon fluorescence imaging system and method - Google Patents
One kind miniaturization adaptive optics two-photon fluorescence imaging system and method Download PDFInfo
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Abstract
Adaptive optics two-photon fluorescence imaging system and method is miniaturized the invention discloses one kind, the imaging system includes：Laser light-source device, it is used to export exciting light；Miniature probe device, it is used for the exciting light for receiving laser light-source device output, and produces fluorescence signal；And be distributed for the average wavefront distortion of each isoplanatic region according to the tissue plane imaging viewing field inside biopsy sample, wavefront correction is carried out to each isoplanatic region；Wave-front measurement device, it is connected with miniature probe device, for detecting average wavefront distortion distribution；Go to scan component, it is used for before the average wavefront distortion distribution of Wave-front measurement device detection, the isoplanatic region off-axis effect that real-Time Compensation miniature probe device is produced；Fluoroscopic imaging device, it is used for the fluorescence signal for gathering the output of miniature probe device, completes the imaging of biopsy sample interior tissue plane.The present invention, which can be realized, to be had and can carry out big visual field, high-spatial and temporal resolution, deep layer imaging in biological tissues in the freely animal of activity.
Description
Technical field
The present invention relates to two-photon fluorescence imaging technical field, more particularly to a kind of miniaturization adaptive optics two-photon
Fluoroscopic imaging systems and method.
Background technology
Directly record neuron activity is to study the pass between animal behavior and nervous function in the freely animal of activity
One of most directly effective method of system.In a very long time before, this task is all by electrophysiology approach
(electrophysiological approach) is come what is performed.In the last few years, optical imagery particularly fluorescence microscopy existed
Serve in this task more and more important.Compared to electro physiology method, it is non-that the maximum advantage of optical imagery is that it has
Invasive and bigger visual field and more observable targets.And compared with common single photon fluorescence imaging technique,
Two Photon Fluorescence has more preferable optical section ability and deeper penetration depth.This causes Two Photon Fluorescence to turn into utilization
Most important and most widely used instrument during fluorescence imaging is observed cerebral neuron.Meanwhile, in order to observe sobering animal in work
Nervous activity under dynamic state, researcher would generally be transformed large-scale desk-top Two Photon Fluorescence, add some simulations
The device of motion such as treadmill or runner etc.；But experiment when the head of mouse must be fixed on micro- lens head it
Under, the activity of trunk is not only limited, simulates real motion in this way.With going deep into for research, scientist has found
The drawbacks of this simulation exposes many.First, it is believed that this simulation can not reflect real active state.Because animal
The many processes participated in for example in mouse authentic activity required for, the twisting of such as body, the clue of surrounding environment, turn of gravity
Change etc. and not possess in head-fixed.Secondly, many classical praxiology researchs, such as frightened, social and exploration etc.
It can not be realized when head is fixed.
So, since 2001, people begin to attempt manufacture it is miniature, may be mounted at animal head such as rat or
Mouse head, the miniature Two Photon Fluorescence system of fluorescence imaging can be carried out in its freely activity completely.From 2001
U.S. Denk professors seminar attempts the 5g that the small-sized Two Photon Fluorescences of 25g made to Kerr professors seminar in 2011 made
Miniature Two Photon Fluorescence.Scientist is attempted for several times, but is all not reaching to highly desirable effect.Generally speaking, by
All optical mirror slip sizes are all greatly reduced in miniature Two Photon Fluorescence so that overall optical quality is very
Hardly possible control.Along with the assembling between small camera lens and coupling are difficult with good large-scale eyeglass.Importantly, small-sized
Object lens are more sensitive to the distortion that is introduced by sample due to the limitation of number of lenses.All these problems cause double light are miniaturized
Sub- microscope is unable to reach with the high-resolution imaging large-scale desk-top Two Photon Fluorescence.This significantly limit miniaturization
The promotion and application of Two Photon Fluorescence.
On the other hand, adaptive optics is all utilized well in astronomy, funduscopy and micro-imaging field,
With correct by system and template band Lai distortion, and then improve as matter.Particularly in large-scale desk-top Two Photon Fluorescence,
Have tried to use adaptive optics to correct aberration, improve image quality.However, because conventional adaptive optics system is needed
Wave-front measurement device and wavefront correction device that will be complicated, overall volume be huge, are that can not apply micro- in miniaturization two-photon
Among mirror.So, only propose new adaptive optics scheme, design new adaptive optics system, using new miniature
Adaptive optics, could be combined adaptive optics with miniature Two Photon Fluorescence, and then it is aobvious to improve miniaturization two-photon
The image quality of micro mirror.
Thus, it is desirable to have a kind of technical scheme come overcome or at least mitigate prior art drawbacks described above at least one
It is individual.
The content of the invention
It is an object of the invention to provide one kind miniaturization adaptive optics two photon imaging system and method overcoming or
At least mitigate at least one in the drawbacks described above of prior art.
To achieve the above object, the present invention provides a kind of miniaturization adaptive optics two-photon fluorescence imaging system, described
Miniaturization adaptive optics two-photon fluorescence imaging system includes：Laser light-source device, it is used to export exciting light；Miniature probe
Device, it is used for the exciting light for receiving the laser light-source device output, and using inside the excitation biopsy sample
Tissue plane, to produce fluorescence signal；And in the case of the biopsy sample is released, according to the live body sample
The average wavefront distortion distribution of each isoplanatic region of the tissue plane imaging viewing field of this inside, wavefront school is carried out to each isoplanatic region
Just；Wave-front measurement device, signal output port of its signal input port operably with the miniature probe device is connected,
For in the case of the biopsy sample is fixed, receiving the fluorescence signal of the miniature probe device output, and detect institute
State average wavefront distortion distribution；Go to scan component, it is arranged on the light between the miniature probe device and Wave-front measurement device
Lu Shang, for before the Wave-front measurement device detection average wavefront distortion distribution, miniature probe described in real-Time Compensation
The isoplanatic region off-axis effect that device is produced；And fluoroscopic imaging device, its signal input port operably with the miniature spy
The signal output port connection of head device, for gathering believing via the fluorescence after wavefront correction for the miniature probe device output
Number, complete the imaging of the tissue plane inside the biopsy sample.
Further, the miniature probe device includes：Laser input module, it is defeated for receiving the laser light-source device
The exciting light gone out；Wavefront correction module, in the case of the biopsy sample is released, according to the group inside biopsy sample
The average wavefront distortion distribution for knitting each isoplanatic region of planar imaging visual field carries out wavefront correction to each isoplanatic region；Be scanned into
As module, for receiving the exciting light after wavefront correction, the exciting light in the way of two dimensional motion to the biopsy sample inside
Tissue plane be scanned, to excite the biopsy sample to produce the fluorescence signal.
Further, the scanning imagery module includes：First twin shaft tilting mirror, it is used to enter by rotating change exciting light
The mode of firing angle angle by by the exciting light after the wavefront correction module wavefront correction to the tissue inside the biopsy sample
Plane carries out two-dimensional scan；Eyepiece, it is used to the exciting light from the first twin shaft tilting mirror converging to the biopsy sample
Inside, to excite the biopsy sample to produce the fluorescence signal；And for exporting the fluorescence signal；Scanning mirror, its cloth
Put in the light path between the first twin shaft tilting mirror and eyepiece, for by produced by the first twin shaft tilting mirror two-dimensional scan
The exciting light of angle change changes into the exciting light of change in location；And dichroic mirror, it is located between the scanning mirror and eyepiece, is used
In separating and export the fluorescence signal by exciting light and fluorescence signal.
Further, it is described to go scanning component to include：Second micro electronmechanical twin shaft tilting mirror, it is arranged on the miniature probe dress
In light path between the signal output port and Wave-front measurement device put；With the first lens, it is micro electronmechanical that it is arranged on described first
In light path between the signal output port of twin shaft tilting mirror and the miniature probe device, for receiving the miniature probe device
The fluorescence signal of output, and the fluorescence signal is transformed into wavefront and that the conjugate planes of the wavefront is projected into described second is micro-
On electromechanical twin shaft tilting mirror；The second micro electronmechanical twin shaft tilting mirror is with being capable of the first micro electronmechanical twin shaft rotating mirror scanning described in real-Time Compensation
The mode of the isoplanatic region off-axis effect of generation is engaged with the described first micro electronmechanical twin shaft tilting mirror, then by exciting after real-Time Compensation
Optical transport gives the Wave-front measurement device；Between the second micro electronmechanical twin shaft tilting mirror and the first micro electronmechanical twin shaft tilting mirror
Matching relationship needs to meet：In time, same frequency, same-phase；Spatially, scanning angle requirement be：Second microcomputer
The ratio for the angle that the angle that electric twin shaft tilting mirror is rotated is rotated with the described first micro electronmechanical twin shaft tilting mirror is Jiao of the scanning mirror
Away from the ratio with the focal length of first lens；Scanning direction is required：In the opposite direction.
Further, the Wave-front measurement device includes：Wavefront sensor, it is used to receive the first lens output
Wavefront；With the first relay facility, it is arranged in the light path between the described second micro electronmechanical twin shaft tilting mirror and Wavefront sensor, is used
In making the described second micro electronmechanical twin shaft tilting mirror be conjugated with Wavefront sensor, it is able to detect that the Wavefront sensor described average
Wavefront distortion is distributed.
Further, the wavefront correction module includes：Changeable type speculum, it is used to receive the laser input module
The exciting light of output, and be distributed according to the average wavefront distortion, the wavefront for the exciting light launched is varied and controlled；With second
Relay facility, it is used to be conjugated the changeable type speculum and scanning mirror, and will be by the changeable type speculum wavefront correction
Exciting light afterwards projects the reflecting surface of the first twin shaft tilting mirror.
Further, the wavefront correction module also includes the second lens, the first half-wave plate, polarization being arranged in light path
Dichroic cube, the second half-wave plate and quarter-wave plate, wherein：The exciting light of laser input module output successively via
Second lens and the first half-wave plate, then enter the polarization spectro cube from the cubical first side of the polarization spectro
Body, then from the cubical second side output of the polarization spectro, and passes sequentially through second half-wave plate and a quarter
The reflecting surface of the changeable type speculum is projected after wave plate；The exciting light that the changeable type speculum is reflected is successively logical again
The quarter-wave plate and the second half-wave plate are crossed, enters the polarization spectro from the cubical second side of the polarization spectro
Cube, is projected described via the cubical exciting light of the polarization spectro from cubical 3rd side of the polarization spectro
Second relay facility.
Further, the signal input port of the fluoroscopic imaging device is filled by flexible light shafts and the miniature probe
The signal output port connection put.
The present invention provides a kind of miniaturization adaptive optics two-photon fluorescence imaging method, the miniaturization adaptive optics
Two-photon fluorescence imaging method includes：Step 1, Wave-front measurement, it is specifically included：Step 11, fixed biopsy sample, and described
Miniature probe device is fixedly mounted in the predeterminated position of biopsy sample；Step 12, it is miniature described in scanning component real-Time Compensation by going
The isoplanatic region off-axis effect that probe apparatus is produced；With step 13, detected by Wave-front measurement device inside the biopsy sample
The average wavefront distortion distribution of each isoplanatic region of tissue plane imaging viewing field；Step 2, fluorescence imaging, it is specifically included：Step
21, discharge the biopsy sample；Step 22, the average wavefront distortion distribution detected according to the step 13, it is described micro-
Type probe apparatus carries out wavefront correction to each isoplanatic region and exports the fluorescence signal after wavefront correction；With step 23, pass through
Fluoroscopic imaging device gathers the tissue plane inside the fluorescence signal that the step 22 is exported, the completion biopsy sample
Imaging.
The present invention can export the wavefront of fluorescence signal from miniature probe device in the case of fixed biopsy sample,
And using going to scan the isoplanatic region off-axis effect that component real-Time Compensation miniature probe device is produced, recycle the inspection of Wave-front measurement device
The average wavefront distortion distribution of each isoplanatic region for the tissue plane imaging viewing field surveyed inside the biopsy sample, then lives in release
Under the precondition of body sample, miniature probe device is distributed according to the average wavefront distortion detected, and traveling wave is entered to each isoplanatic region
It is preceding to correct and export the fluorescence signal after wavefront correction, the imaging of the tissue plane inside biopsy sample is completed, therefore can be real
Big visual field, high-spatial and temporal resolution, deep layer imaging in biological tissues can be carried out in the freely animal of activity by now having.
Brief description of the drawings
Fig. 1 is the principle schematic diagram of miniaturization adaptive optics two photon imaging system provided by the present invention.
Fig. 2 is Wave-front measurement part is realized in Fig. 1 miniaturization adaptive optics two photon imaging system one preferred real
Apply the structural representation of mode.
Fig. 3 is that the matching relationship for going scanning component and scanning imagery module in Fig. 2 illustrates schematic diagram.
Fig. 4 is fluorescent imaging moiety is realized in Fig. 1 miniaturization adaptive optics two photon imaging system one preferred real
Apply the structural representation of mode.
Fig. 5 is the structural representation of Fig. 1 laser light-source device.
Embodiment
In the accompanying drawings, same or similar element is represented or with same or like function using same or similar label
Element.Embodiments of the invention are described in detail below in conjunction with the accompanying drawings.
In the description of the invention, term " " center ", " longitudinal direction ", " transverse direction ", "front", "rear", "left", "right", " vertical ",
The orientation or position relationship of the instruction such as " level ", " top ", " bottom " " interior ", " outer " are to be closed based on orientation shown in the drawings or position
System, is for only for ease of the description present invention and simplifies description, rather than indicate or imply that the device or element of meaning must have
Specific orientation, with specific azimuth configuration and operation, therefore it is not intended that limiting the scope of the invention.
The biopsy sample of the present invention is lived toy, for example：Mouselet etc..In view of the volume of biopsy sample compared with
Small, therefore, the invention belongs to microdevice, integrally-built volume is in 1~5cm3In the range of.
Scanning component in miniature probe device of the prior art in imaging/Wave-front measurement, i.e., laser scanning when pair
Exciting light generates a certain degree of deviation, and this causes the converged position of exciting light to deviate from the optical axis of whole optical system, enters
And make it that the isoplanatic region of the fluorescence signal produced has also offset from optical axis, this phenomenon is also referred to as " off-axis effect ".In the prior art
Wave-front measurement be nearly all to use based on the constant wavefront sensing mode of isoplanatic region, and there is " off-axis effect " in this mode
In the case of, it is impossible to the average wavefront distortion for detecting each isoplanatic region of the tissue plane imaging viewing field inside biopsy sample divides
Cloth.
In view of problem of the prior art, the present invention is intended to by the light path between miniature probe device and wavefront sensing apparatus
It is newly-increased it is a set of remove sweep mechanism, and go sweep mechanism to be linked with sweep mechanism in miniature probe this using control algolithm, will
The reverse off-axis isoplanatic region of the deviation of fluorescence signal is corrected on the optical axis of whole optical system in real time, so, is even adopted
With the wavefront sensing mode constant based on isoplanatic region of the prior art, the group inside biopsy sample can be also accurately detected
Knit the average wavefront distortion distribution of each isoplanatic region of planar imaging visual field.
According to the technical problem to be solved in the present invention, the miniaturization adaptive optics two-photon fluorescence that the present embodiment is provided
Imaging method includes：
Step 1, Wave-front measurement, it is specifically included：
Step 11, fixed biopsy sample, and miniature probe device is fixedly mounted in the predeterminated position of the biopsy sample.
" predeterminated position " is generally chosen at the windowing position on biopsy sample head (head of mouselet).The windowing position can be manifested
Cerebral tissue after fluorescence labeling.
Step 12, the isoplanatic region produced by the scanning component gone to scan in miniature probe device described in component real-Time Compensation
Off-axis effect.The step can be adopted and realized with the following method：
As shown in figures 1 and 3, scanning component 4 is gone to include the second micro electronmechanical lens 42 of twin shaft tilting mirror 41 and first, second is micro-
Electromechanical twin shaft tilting mirror 41 is arranged in the light path between the signal output port of miniature probe device 2 and Wave-front measurement device 3.The
One lens 42 are arranged in the light path between the signal output port of the first micro electronmechanical twin shaft tilting mirror 31 and miniature probe device 2,
It is transformed into wavefront and by the ripple for receiving the fluorescence signal that the miniature probe device 2 is exported, and by the fluorescence signal
Preceding conjugate planes is projected on the second micro electronmechanical twin shaft tilting mirror 41.Scanning component in miniature probe device 2 includes the first twin shaft
Tilting mirror 231 and scanning mirror 233, the first twin shaft tilting mirror 231 is used for will be by wavefront correction module 22 in the way of rotating and change angle
Exciting light after wavefront correction carries out two-dimensional scan to the tissue plane inside the biopsy sample.Scanning mirror 233 is used for the
The exciting light of angle change produced by the two-dimensional scan of one twin shaft tilting mirror 231 changes into the exciting light of change in location.Second microcomputer
The electric synchronous scanning of 41 and first twin shaft tilting mirror of twin shaft tilting mirror 231 needs to meet claimed below：
1. in time, frequency is identical, scope is in 100~300Hz；Phase is identical.
2. spatially, scanning angle requirement is：The angle and the first twin shaft that second micro electronmechanical twin shaft tilting mirror 41 is rotated turn
Mirror 231 rotate angle ratio for scanning mirror 233 focal length and the first lens 42 focal length ratio.Scanning direction is required
For：In the opposite direction.
By the above-mentioned means, isoplanatic region " off-axis effect " is gone to scan the synchronization of component just caused by scanning component scanning
Deflection compensated, so the fluorescence signal that miniature probe device is exported deviation reversely off-axis isoplanatic region be corrected in real time it is whole
On the axle of individual optical system, so that average wavefront distortion distribution detection is effectively carried out for step 13 provides advantage.
Step 13, detect that each of the tissue plane imaging viewing field inside the biopsy sample waits dizzy by Wave-front measurement device
The average wavefront distortion distribution in area.
Step 2, fluorescence imaging, it is specifically included：
Step 21, the biopsy sample is discharged, and the glimmering of the miniature probe device output is collected using flexible optical fibre beam
Optical signal, flexible optical fibre beam needs enough length, and such biopsy sample even crown the miniature probe device, also can
It is enough freely movable, without suffering restraints, and then be conducive to reflecting the real active state of biopsy sample, be conducive to obtaining live body
The real image data of the deep brain tissue of sample.
Step 22, the average wavefront distortion distribution detected according to the step 13, the miniature probe device pair
Each isoplanatic region carries out wavefront correction and exports the fluorescence signal after wavefront correction.
Step 23, the fluorescence signal that the step 22 is exported is gathered by fluoroscopic imaging device, completes the live body
The imaging of the tissue plane of sample interior.
Operating procedure 1 and step 2 repeatedly, that is to say, that every time it is upper once on the basis of continue to optimize, until converging to
One optimum value, i.e., remaining wavefront error is minimum, and big visual field, high space-time are carried out to whole deep layer biological tissue so as to realize
Resolution ratio, point by point scanning fluorescent microscopic imaging.
As shown in figure 1, the present embodiment provides realization is miniaturized adaptive optics two-photon fluorescence imaging method as described above
Adaptive optics two-photon fluorescence imaging system is miniaturized, the system includes laser light-source device 1, miniature probe device 2, wavefront
Detection means 3, scanning component 4 and fluoroscopic imaging device 5 is removed, wherein：
As shown in figure 5, laser light-source device 1 is used to export exciting light.Laser light-source device 1 is by a femto-second laser
11, the pulsewidth of femto-second laser 11 is between 80 to 250fs (femtoseconds), and repetition frequency range is 40 to 100MHz, peak power
In more than 500mW.
Miniature probe device 2 is fixedly mounted on the head windowing position of the predeterminated position of biopsy sample, i.e. biopsy sample.It is micro-
Type probe apparatus 2 is used for the exciting light for receiving the output of laser light-source device 1, and using inside the excitation biopsy sample
Tissue plane, to produce fluorescence signal；And in the case of the biopsy sample is released, according in biopsy sample
The average wavefront distortion distribution of each isoplanatic region of the tissue plane imaging viewing field in portion, wavefront correction is carried out to each isoplanatic region.
As shown in figure 1, in one embodiment, miniature probe device 2 includes laser input module 21, wavefront correction module
22 and scanning imagery module 23, wherein：
As shown in figure 5, laser input module 21 is used for the exciting light for receiving the output of laser light-source device 1.Laser inputs mould
Block 21 specifically includes intensity modulation module and fiber coupling module 215, wherein, the intensity modulation module includes electronic shutter
211st, half-wave plate 212 and electric light/acousto-optic modulator 213.Fiber coupling module 215 is by non-spherical lens 214 by collimation laser
Beam is coupled into photonic crystal fiber (hollow-core photon crystal fiber).
As shown in figure 1, wavefront correction module 22 is used in the case of the biopsy sample is released, according to biopsy sample
The average wavefront distortion distribution of each isoplanatic region of internal tissue plane imaging viewing field carries out wavefront correction to each isoplanatic region.
As shown in Fig. 2 in one embodiment, wavefront correction module 22 is relayed including changeable type speculum 221 and second
Mechanism 222, wherein：Changeable type speculum 221 is used for the exciting light for receiving the output of laser input module 21, and according to described average
Wavefront distortion is distributed, and the wavefront for the exciting light launched is varied and controlled.
Second relay facility 222 is used to be conjugated the twin shaft tilting mirror 231 of changeable type speculum 221 and first, and will be variable
Exciting light after the wavefront correction of type speculum 221 projects the reflecting surface of the first twin shaft tilting mirror 231.That is, setting this reality
The wavefront that example needs to detect with illustrate the #1 faces come in correction chart is applied, passes through the second relay facility 222, changeable type speculum
221 and the first one 4f system of formation of twin shaft tilting mirror 231, then, the conjugate planes in the #1 faces that changeable type speculum 221 is corrected is the
The surface of emission of one twin shaft tilting mirror 231.
During wavefront correction is carried out, between the control electric current of changeable type speculum 221 and the first twin shaft tilting mirror 231
Matching relationship can be represented using following formula：
Δ S (N)=- 2*T (IN)/λ
Wherein：Δ S (N) is the average wavefront distortion (phase between plane wave front in n-th sub-aperture on conjugate planes #1
Potential difference)；INFor the electric current being applied on the n-th independent deformation unit of changeable type speculum 221；T (I) is changeable type speculum
221 driving function, i.e., when applying the electric current that numerical value is I, the space displacement amount on a certain independent deformation unit；λ is incidence
Optical maser wavelength.
In one embodiment, wavefront correction module 22 also includes the second lens 223, the first half-wave being arranged in light path
Piece 224, polarization spectro cube 225, the second half-wave plate 226 and quarter-wave plate 227, wherein：Laser input module 21 is defeated
The exciting light that goes out is successively via the half-wave plate 224 of the second lens 223 and first, then from the polarization spectro cube 225
First side enters the polarization spectro cube 225, is then exported from the second side of the polarization spectro cube 225,
And pass sequentially through the reflecting surface that changeable type speculum 221 is projected after second half-wave plate 226 and quarter-wave plate 227.
The exciting light that changeable type speculum 221 is reflected is again successively by the half-wave plate 226 of quarter-wave plate 227 and second, from inclined
Shake dichroic cube 225 second side enter polarization spectro cube 225, via the exciting light of polarization spectro cube 225
The second relay facility 222 is projected from the 3rd side of polarization spectro cube 225, is thrown finally by the second relay facility 222
On the reflecting surface for being mapped to the first twin shaft tilting mirror 231.In the present embodiment, the second lens 223 are used to collimate the laser sent from light.
First half-wave plate 224 is used to change laser polarization direction.Polarization spectro cube 225 is used to separate incident light and reflected light.
Second half-wave plate 226 and quarter-wave plate 227 are provided commonly for make it that incident light is just vertical with the polarization direction for launching light, this
Sample can just separate incident light and reflected light using 225.
Scanning imagery module 23 is used to receive the exciting light after wavefront correction, and the exciting light is in the way of two dimensional motion to institute
State the tissue plane inside biopsy sample to be scanned, to produce the fluorescence signal.
In one embodiment, scanning imagery module 23 includes the first twin shaft tilting mirror 231, eyepiece 232, the and of scanning mirror 233
Dichroic mirror 234, wherein：
First twin shaft tilting mirror 231 is used for after the wavefront correction of wavefront correction module 22 in the way of rotating and change angle
Exciting light carries out two-dimensional scan to the tissue plane inside the biopsy sample.First twin shaft tilting mirror 231 often converts an angle,
The wavefront correction of wavefront correction module 22 updates once, to compensate the different wavefront distortions of different isoplanatic regions.First twin shaft tilting mirror
231 use micro electronmechanical twin shaft tilting mirror, and its parameter area includes：Minute surface size：0.8~1.0mm；Scanning angle：± 5~±
7°；First resonant frequency：More than 2000Hz.
Eyepiece 232 is used to converge to the exciting light from the first twin shaft tilting mirror 231 inside the biopsy sample, to excite
The fluorescence signal is produced inside the biopsy sample, and for exporting the fluorescence signal.The #2 faces shown in Fig. 2 are into
The imaging surface of tissue plane inside image planes, the biopsy sample, with #1 faces Fourier transformation each other.
Scanning mirror 233 is arranged in the light path between the first twin shaft tilting mirror 231 and eyepiece 232, for the first twin shaft to be turned
The exciting light of angle change produced by the two-dimensional scan of mirror 231 changes into the exciting light of change in location.Dichroic mirror 234 is located at scanning
Between mirror 233 and eyepiece 232, for exciting light and fluorescence signal to be separated.
As shown in figure 1, the signal of the signal input port of Wave-front measurement device 3 operably with miniature probe device 2
Output port is connected, the fluorescence letter in the case of the biopsy sample is fixed, receiving the output of miniature probe device 2
Number, and detect the average wavefront distortion distribution.Wave-front measurement device 3 is in Wave-front measurement, and coordinating with miniature probe device 2 makes
With.In Wave-front measurement, it is necessary to the fixed biopsy sample.
As shown in Fig. 2 in one embodiment, Wave-front measurement device 3 includes the relay facility of Wavefront sensor 31 and first,
Wherein：Wavefront sensor 31 is used for the wavefront for receiving the output of the first lens 42.It is micro electronmechanical double that first relay facility is arranged on second
In light path between axle tilting mirror 41 and Wavefront sensor 31, for making the described second micro electronmechanical twin shaft tilting mirror 41 and Wavefront sensor
31 conjugation, make Wavefront sensor 31 be able to detect that the average wavefront distortion distribution.First relay facility is by lens 33 and thoroughly
Mirror 34 is constituted so that second one 4f system of micro electronmechanical twin shaft tilting mirror 41 and Wavefront sensor 31 formation, then, the conjugation in #1 faces
Face is the detection faces of Wavefront sensor 31.The 4f relations formation that #2 faces are formed by the lens 42 of lens 33 and first is in speculum 35
Reflecting surface on.
That is, in the case where not needing optical fiber 6 that fluorescence signal is introduced into system 5, detector 37 can also be utilized
To be imaged.So it is easy to evaluate the improvement of image quality in Wave-front measurement and during continuing to optimize wavefront correction.
As depicted in figs. 1 and 2, in one embodiment, go scanning component 4 to be arranged on miniature probe device 2 to examine with wavefront
Survey in the light path between device 3, for before Wave-front measurement device 3 detects the average wavefront distortion distribution, real-Time Compensation
The isoplanatic region off-axis effect that miniature probe device 2 is produced.
Specifically, scanning component 4 is gone to include the second micro electronmechanical lens 42 of twin shaft tilting mirror 41 and first, wherein：
Second micro electronmechanical twin shaft tilting mirror 41 is arranged on the signal output port and Wave-front measurement device 3 of miniature probe device 2
Between light path on.Second micro electronmechanical twin shaft tilting mirror 41 uses the micro electronmechanical twin shaft tilting mirror of heavy caliber, and its parameter area includes：
Minute surface size：3-5mm；Scanning angle：± 1~± 3 °；First resonant frequency：More than 200Hz.
First lens 42 are arranged between the signal output port of the first micro electronmechanical twin shaft tilting mirror 31 and miniature probe device 2
Light path on, for receiving the fluorescence signal that the miniature probe device 2 is exported, and by the fluorescence signal be transformed into wavefront with
And project conjugate planes #1 on the second micro electronmechanical twin shaft tilting mirror 41.
The ratio for the angle that the angle that second micro electronmechanical twin shaft tilting mirror 41 is rotated is rotated with the first micro electronmechanical twin shaft tilting mirror 231
The ratio of the focal length of focal length and the first lens 42 for scanning mirror 233, is scanned with the micro electronmechanical twin shaft tilting mirror 231 of real-Time Compensation first
The isoplanatic region off-axis effect of generation, then exciting light after real-Time Compensation are transferred to the Wave-front measurement device 3.
With reference to Fig. 3, in order to realize the isoplanatic region off-axis effect of scanning component 4 generation of real-Time Compensation miniature probe device 2,
The second micro electronmechanical synchronous scanning of 41 and first twin shaft tilting mirror of twin shaft tilting mirror 231 needs to meet claimed below：
1. in time, frequency is identical, scope is in 100~300Hz；Phase is identical.
2. spatially, scanning angle requirement is：The angle and the first twin shaft that second micro electronmechanical twin shaft tilting mirror 41 is rotated turn
The ratio for the angle that mirror 231 is rotated for scanning mirror 233 focal length and the first lens 42 focal length ratio, i.e. illustrate in Fig. 3
Comeβ1The angle rotated for the second micro electronmechanical twin shaft tilting mirror 41, α1The angle rotated for the first twin shaft tilting mirror 231, F1
For the focal length of scanning mirror 233, F2For the focal length of the first lens 42.Scanning direction is required：In the opposite direction.
By the above-mentioned means, isoplanatic region " off-axis effect " is gone to scan the synchronization of component just caused by scanning component scanning
Deflection compensated, so the fluorescence signal that miniature probe device is exported deviation reversely off-axis isoplanatic region be corrected in real time it is whole
On the axle of individual optical system, so that average wavefront distortion distribution detection is effectively carried out for Wave-front measurement device 3 provides favourable bar
Part.
As shown in figure 4, signal of the signal input port of fluoroscopic imaging device 5 operably with miniature probe device 2 is defeated
Exit port is connected, for gather the output of miniature probe device 2 via the fluorescence signal after wavefront correction, complete the live body sample
The imaging of the tissue plane of this inside.The composition of fluoroscopic imaging device 5 is prior art not reinflated description herein.Fluorescence imaging
Device 5 is used cooperatively in fluorescence imaging with miniature probe device 2.The signal input port of fluoroscopic imaging device 5 passes through flexibility
Light shafts 6 are connected with the signal output port of the miniature probe device 2.In fluorescence imaging, it is necessary to discharge the live body sample
This, and the fluorescence signal that the miniature probe device is exported is collected using flexible optical fibre beam 6, flexible optical fibre beam needs enough length
Degree, such biopsy sample even crown the miniature probe device, also can be freely movable, without suffering restraints, enters
And be conducive to reflecting the real active state of biopsy sample, be conducive to obtaining the true picture of the deep brain tissue of biopsy sample
Data.
The course of work for carrying out Wave-front measurement using the present invention is as follows：
Miniature probe device 2 and Wave-front measurement device 3 are combined, as shown in Figure 2.The femtosecond that femto-second laser 11 is exported is excited
Light is reached in miniature probe device 2 by Hollow-Core Photonic Crystal Fibers.First, collimated by the second lens 223, then pass through
Half of wave plate 224 adjusts polarization direction, and is reflected by polarization spectro cube 225, passes through the second half-wave plate 226 for the first time respectively
With quarter-wave plate 227, the surface of changeable type speculum 221 is reached.The collimated light second reflected by changeable type speculum 221
It is secondary to pass through the second half-wave plate 226 and quarter-wave plate 227 and pass through polarization spectro cube 225.Then, the second relaying is utilized
Mechanism 222, exciting light light beam is projected onto the surface of the first twin shaft tilting mirror 231.First twin shaft tilting mirror 231 passes through two-way high-pressure electrostatic
Signal is controlled, and laser scanning can be carried out in the two directions.Pass through the anti-of the converging action of scanning mirror 233 and dichroic mirror 234
The effect of penetrating, scanning light beam is focused in the imaging surface of eyepiece 232, then focuses on biopsy sample by eyepiece 232, so as to carry out two dimension
Spot scan excite.The fluorescence signal inspired is collected with falling to penetrate form by eyepiece 232 again, through dichroic mirror 234, from miniature spy
Head device 2 is projected, then is collimated by the first lens 42.Light beam after collimation is irradiated on the second micro electronmechanical twin shaft tilting mirror 41, then by
Speculum reflects, and the first relay facility being made up of lens 33 and lens 4, projects Wavefront sensor 31 or photoelectricity is visited
On the receiver for surveying device 37.
In above process, all rear emergent pupil faces have imaging conjugate relation (#1)；All image planes have imaging altogether
Yoke relation (#2).Distance 1 is equal with the operating distance of the first lens 42；Distance 2 and distance 3 and the operating distance with lens 3
It is identical, and equal to distance 4；Distance 5 and distance 6 are identical, equal to the operating distance of lens 44.In wavefront measurement, it is desirable to first
The micro electronmechanical twin shaft tilting mirror 41 of twin shaft tilting mirror 231 and second synchronizes scanning, i.e. scan frequency, and phase, waveform is identical.Amplitude
Ratio requirement reaches " go scanning " effect, i.e., due to caused by miniature micro electronmechanical twin shaft rotating mirror scanning isoplanatic region off-axis effect it is lucky
By the synchronous deflection compensated of the micro electronmechanical twin shaft tilting mirror of heavy caliber, the constant effect of isoplanatic region is reached.And then to larger visual field all
Accurate wavefront sensing can be accomplished.
The course of work for carrying out fluorescence imaging using the present invention is as follows：
Miniature probe device 2 and fluoroscopic imaging device 5 are combined, as shown in Figure 4.The fluorescence letter that eyepiece 232 is received and sent
Number collected, and then collected by fluoroscopic imaging device 5 by flexible optical fibre beam 6.During this, according to each isoplanatic region calculated rear
After average wavefront distortion distribution on the #2 faces of emergent pupil face, the changeable type speculum 221 in driving miniature probe device 2 produces minute surface
Deformation, completes wavefront correction.The operation of two steps more than repeatedly, you can realize and big visual field, height are carried out to whole deep layer biological tissue
Spatial and temporal resolution, point by point scanning fluorescent microscopic imaging.
It is last it is to be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations.This
The those of ordinary skill in field should be understood：Technical scheme described in foregoing embodiments can be modified, or it is right
Which part technical characteristic carries out equivalent substitution；These modifications are replaced, and the essence of appropriate technical solution is departed from this
Invent the spirit and scope of each embodiment technical scheme.
Claims (9)
1. one kind miniaturization adaptive optics two-photon fluorescence imaging system, it is characterised in that including：
Laser light-source device (1), it is used to export exciting light；
Miniature probe device (2), it is used for the exciting light for receiving the laser light-source device (1) output, and is excited described in
Light excites the tissue plane inside biopsy sample, to produce fluorescence signal；And the feelings for being released in the biopsy sample
Under shape, it is distributed according to the average wavefront distortion of each isoplanatic region of the tissue plane imaging viewing field inside the biopsy sample, to each
The isoplanatic region carries out wavefront correction；
Wave-front measurement device (3), the signal output part of its signal input port operably with the miniature probe device (2)
Mouth connection, the fluorescence letter in the case of the biopsy sample is fixed, receiving miniature probe device (2) output
Number, and detect the average wavefront distortion distribution；
Scanning component (4) is removed, it is arranged in the light path between the miniature probe device (2) and Wave-front measurement device (3), used
In before the Wave-front measurement device (3) the detection average wavefront distortion distribution, miniature probe device described in real-Time Compensation
(2) the isoplanatic region off-axis effect produced；With
Fluoroscopic imaging device (5), the signal output part of its signal input port operably with the miniature probe device (2)
Mouthful connection, for gather the miniature probe device (2) output via the fluorescence signal after wavefront correction, complete the live body
The imaging of the tissue plane of sample interior.
2. adaptive optics two-photon fluorescence imaging system is miniaturized as claimed in claim 1, it is characterised in that described miniature
Probe apparatus (2) includes：
Laser input module (21), the exciting light for receiving the laser light-source device (1) output；
Wavefront correction module (22), in the case of the biopsy sample is released, according to the tissue inside biopsy sample
The average wavefront distortion distribution of each isoplanatic region of planar imaging visual field carries out wavefront correction to each isoplanatic region；With
Scanning imagery module (23), for receiving the exciting light after wavefront correction, the exciting light is in the way of two dimensional motion to institute
State the tissue plane inside biopsy sample to be scanned, to excite the biopsy sample to produce the fluorescence signal.
3. adaptive optics two-photon fluorescence imaging system is miniaturized as claimed in claim 2, it is characterised in that the scanning
Image-forming module (23) includes：
First twin shaft tilting mirror (231), it is used for will be by the wavefront school by way of rotating and changing Exciting incidence angle
Exciting light after positive module (22) wavefront correction carries out two-dimensional scan to the tissue plane inside the biopsy sample；
Eyepiece (232), it is used to converge to the exciting light from the first twin shaft tilting mirror (231) in the biopsy sample
Portion, to excite the biopsy sample to produce the fluorescence signal；And for exporting the fluorescence signal；
Scanning mirror (233), it is arranged in the light path between the first twin shaft tilting mirror (231) and eyepiece (232), for by institute
The exciting light for stating the angle change produced by first twin shaft tilting mirror (231) two-dimensional scan changes into the exciting light of change in location；With
Dichroic mirror (234), it is located between the scanning mirror (233) and eyepiece (232), for exciting light and fluorescence signal to be divided
Open and export the fluorescence signal.
4. adaptive optics two-photon fluorescence imaging system is miniaturized as claimed in claim 3, it is characterised in that described to go to sweep
Retouching component (4) includes：
Second micro electronmechanical twin shaft tilting mirror (41), its signal output port for being arranged on the miniature probe device (2) and wavefront inspection
Survey in the light path between device (3)；With
First lens (42), it is arranged on the letter of the described first micro electronmechanical twin shaft tilting mirror (31) and the miniature probe device (2)
In light path between number output port, the fluorescence signal for receiving miniature probe device (2) output, and by the fluorescence
Signal is transformed into wavefront and projects the conjugate planes of the wavefront on the described second micro electronmechanical twin shaft tilting mirror (41)；
The second micro electronmechanical twin shaft tilting mirror (41) can produce described in real-Time Compensation in the first micro electronmechanical twin shaft tilting mirror (231) scanning
The mode of raw isoplanatic region off-axis effect is engaged with the described first micro electronmechanical twin shaft tilting mirror (231), then by after real-Time Compensation
Exciting light is transferred to the Wave-front measurement device (3)；
Matching relationship between the second micro electronmechanical twin shaft tilting mirror (41) and the first micro electronmechanical twin shaft tilting mirror (231) needs
Meet：
In time, same frequency, same-phase；
Spatially, scanning angle requirement be：The angle and described first that the second micro electronmechanical twin shaft tilting mirror (41) rotates are micro-
Focal length and first lens (42) of the ratio for the angle that electromechanical twin shaft tilting mirror (231) rotates for the scanning mirror (233)
The ratio of focal length；Scanning direction is required：In the opposite direction.
5. adaptive optics two-photon fluorescence imaging system is miniaturized as claimed in claim 4, it is characterised in that the wavefront
Detection means (3) includes：
Wavefront sensor (31), it is used for the wavefront for receiving the first lens (42) output；With
First relay facility, it is arranged on the light path between the described second micro electronmechanical twin shaft tilting mirror (41) and Wavefront sensor (31)
On, for making the described second micro electronmechanical twin shaft tilting mirror (41) be conjugated with Wavefront sensor (31), make the Wavefront sensor (31)
It is able to detect that the average wavefront distortion distribution.
6. the miniaturization adaptive optics two-photon fluorescence imaging system as any one of claim 3 to 5, its feature exists
In the wavefront correction module (22) includes：
Changeable type speculum (221), it is used for the exciting light for receiving laser input module (21) output, and according to described flat
Equal wavefront distortion distribution, is varied and controlled the wavefront for the exciting light launched；With
Second relay facility (222), it is used to be conjugated the changeable type speculum (221) and scanning mirror (233), and will be by institute
State the reflecting surface that the exciting light after changeable type speculum (221) wavefront correction projects the first twin shaft tilting mirror (231).
7. adaptive optics two-photon fluorescence imaging system is miniaturized as claimed in claim 6, it is characterised in that the wavefront
Correction module (22) also includes the second lens (223), the first half-wave plate (224), the polarization spectro cube being arranged in light path
(225), the second half-wave plate (226) and quarter-wave plate (227), wherein：What the laser input module (21) exported excites
Light is successively via second lens (223) and the first half-wave plate (224), then from the of the polarization spectro cube (225)
One side enters the polarization spectro cube (225), and then the second side from the polarization spectro cube (225) is defeated
Go out, and project the changeable type speculum after passing sequentially through second half-wave plate (226) and quarter-wave plate (227)
(221) reflecting surface；The exciting light that the changeable type speculum (221) reflects is again successively by the quarter-wave plate
(227) it is and the second half-wave plate (226), vertical from the second side of the polarization spectro cube (225) into the polarization spectro
Cube (225), via the exciting light of the polarization spectro cube (225) from the 3rd of the polarization spectro cube (225)
Side projects second relay facility (222).
8. adaptive optics two-photon fluorescence imaging system is miniaturized as claimed in claim 7, it is characterised in that the fluorescence
The signal input port of imaging device (5) passes through flexible light shafts (6) and the signal output port of the miniature probe device (2)
Connection.
9. one kind miniaturization adaptive optics two-photon fluorescence imaging method, it is characterised in that including：
Step 1, Wave-front measurement, it is specifically included：
Step 11, fixed biopsy sample, and miniature probe device is fixedly mounted in the predeterminated position of the biopsy sample；
Step 12, by going to scan the isoplanatic region off-axis effect that miniature probe device described in component real-Time Compensation is produced；With
Step 13, each isoplanatic region of the tissue plane imaging viewing field inside the biopsy sample is detected by Wave-front measurement device
Average wavefront distortion distribution；
Step 2, fluorescence imaging, it is specifically included：
Step 21, the biopsy sample is discharged；
Step 22, the average wavefront distortion distribution detected according to the step 13, the miniature probe device is to each institute
Isoplanatic region is stated to carry out wavefront correction and export the fluorescence signal after wavefront correction；With
Step 23, the fluorescence signal that the step 22 is exported is gathered by fluoroscopic imaging device, completes the biopsy sample
The imaging of internal tissue plane.
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