CN207459392U - Femtosecond pulse modulator and with its miniature two-photon microscopic imaging device - Google Patents

Abstract

The utility model discloses a kind of femtosecond pulse modulator and there is its miniature two-photon microscopic imaging device, the femtosecond pulse modulator has negative dispersion light path and positive dispersion light path, wherein：The negative dispersion light path includes laser input optical fibre, and the laser input optical fibre is used to the laser that pulse broadening prechirp has compensated being transferred to the scanning imagery portion；The positive dispersion light path, between the femtosecond pulse laser and the negative dispersion light path, for compensating by the laser input optical fibre caused negative dispersion in laser process is transmitted.The femtosecond pulse modulator can carry out stable observation to the freely dendron of the animal of activity and dendritic spines activity in natural physiological environment for the miniature two-photon microscopic imaging device and provide advantage.

Description

Femtosecond pulse modulator and with its miniature two-photon microscopic imaging device

Technical field

The utility model is related to optical image technology fields, more particularly to a kind of femtosecond pulse modulator and have Its miniature two-photon microscopic imaging device.

Background technology

One of final goal of Neuscience is that subcellular fraction, cell, loop and higher are understood on the freely animal of activity The basic principle of the neuronal messages processing of level.With reference to fluorescence indicator, light microscope has become in this task Basic research instrument, because it allows directly to observe neuron activity on multiple times and space scale.Single cynapse is letter Breath transfers, the base unit of processing and storage, for understanding that brain function and pathogenic mechanism are most important.Post-synaptic structures -- dendron Spine is submicrometer structure, buried in intracerebral, and with the speed activity of Millisecond.Due to its intrinsic optical section and deep tissues Penetration capacity, multiphoton microscope are always the one preferred technique of internal non-invasive optical Brian Imaging in 20 years in the past.Using desk-top Two Photon Fluorescence (English full name is " Two-Photon Microscopy ", is hereafter referred to as " TPM "), can be in work The metamorphosis of dendritic spines, such as the activity of the neuron of learning and memory are observed in vivo.Observation live body under waking state The dendritic spines activity of sample header complexity can by gathered equipped with rapid image (frequency acquisition be more than 15Hz), high excitation and The state-of-the-art desk-top multiphoton microscope of photodetection efficiency is realized.However, the head of biopsy sample is fixed always, entirely During the experiment is under physical constraint and emotional stress, and no priori evidence shows neuron to extraneous response in void It is of equal value to intend reality and freely explore down.Importantly, many social actions, such as parent-offspring's nursing, mating and fight, all It cannot be studied with the fixed experiment in head.

In order to tackle these challenges, a preferable solution is that exploitation can observe biopsy sample in freedom for a long time Microscope is dynamically miniaturized in the 26S Proteasome Structure and Function of dendritic spines in active procedure.Denk and his colleague established in 2001 (English full name is " micro Two-Photon to first miniature Two Photon Fluorescence based on fiber optic tip scanning Microscopy " is hereafter referred to as " mTPM "), then in next ten years, other many seminars take distinct methods It continues to attempt to.However, these mTPM neither ones are used for subsequent biologic applications, mainly due to two main limitations： First, no one can the widest fluorescence probe of imaging applications, such as GCaMPs, reason be a lack of appropriate optical fiber be used for transmit 920 nanometers of femto-second laser pulses, to ensure that pulse does not have distortion to sample.Second, mTPMs are often showed in testing in vivo Go out less than their theoretical resolution, reason may be that sample rate is low, and (English full name is " Graded- to miniature graded index Index Lenses " are hereafter referred to as " GRIN ") aberrations of lens, the defects of minitype optical device and caused by movement into As noise etc..At present, the activity of the neuronal structure of dendritic spines resolution ratio cannot well be solved by mTPMs, although it is managed It it is about 1 μm by lateral resolution.

On the other hand, mini single-photon wide-field microscope, such as by Ghosh and its microscope of colleague's exploitation, it is real Quick Acquisition and big visual field (English full name is " Field of Vision ", is hereafter referred to as " FOV ") are showed and have solved Noise problem caused by movement (in neuron resolution ratio).However, current noninvasive miniature wide-field microscope is only capable of obtaining cell Resolution ratio, and picture contrast is influenced be subject to the afocal background signal accumulated.Up to the present, can provide well into As ability and experimental program, and the new mTPM to the routine experimentation that can meet neuroscientist powerful enough still needs It completes.

Thus, it is desirable to have a kind of technical solution is come at least one in the drawbacks described above that overcomes or at least mitigate the prior art It is a.

Utility model content

Utility model be designed to provide a kind of femtosecond pulse modulator and with its miniature two-photon it is micro- Imaging device is come at least one in the drawbacks described above that overcomes or at least mitigate the prior art.

To achieve the above object, the utility model provides a kind of femtosecond pulse modulator, the femtosecond pulse Modulator has negative dispersion light path and positive dispersion light path, wherein：The negative dispersion light path includes laser input optical fibre, the laser Input optical fibre is used to the laser that pulse broadening prechirp has compensated being transferred to the scanning imagery portion；The positive dispersion light path, It is being transmitted between the femtosecond pulse laser and the negative dispersion light path for compensating by the laser input optical fibre Caused negative dispersion in laser process.

The utility model also provides a kind of miniature two-photon microscopic imaging device, the miniature two-photon microscopic imaging device Including：Femtosecond pulse laser is used for the laser that generation wavelength is 920 nanometers；Femtosecond pulse modulator is right It is required that femtosecond pulse modulator as described above, for receiving the laser of the femtosecond pulse laser output, and pre- Zhou The pulse broadening for compensating laser sing to preset value, and exports；And miniature probe, the miniature probe include：Scanning imagery portion Point, for receiving the laser of the femtosecond pulse modulator output, which sweeps the tissue inside biopsy sample It retouches, the biopsy sample to be excited to generate fluorescence signal；With fluorescence output optical fibre, it is used to receiving the scanning imagery part defeated The fluorescence signal gone out, and exported.

Miniature two-photon microscopic imaging device provided by the utility model (hereinafter referred to as " FIRM-TPM ") is tested and should Speed is fast, high resolution, can be used in solving a whole set of experiment of the imaging problem of single dendritic spines in free moving animals Method.In the behavior example of irregular, frequent body and head movement is related to (for example, tail is hung, diving tower and society Bank of Communications is), the mini microscope of the utility model can be to the body cell of the GCaMP6f cortical neurons marked, dendron It is observed with dendritic spines.It integrates, FIRM-TPM represents next-generation mini microscope, it meets in free moving animals The middle demand for carrying out high-resolution Brian Imaging.

Description of the drawings

Fig. 1 is the structure diagram of mono- preferred embodiments of FIRM-TPM provided by the utility model.

Fig. 2 is the view that the miniature probe in Fig. 1 is mounted on mouselet.

Fig. 3 is the light path principle schematic diagram of the miniature probe in Fig. 1.

Fig. 4 is the structure diagram of one preferred embodiment of femtosecond pulse modulator provided by the utility model.

Fig. 5 a are biopsy sample behavior imaging system of the utility model using the miniature two-photon microscopic imaging device of Fig. 1 Structure diagram.

Fig. 5 b are the decomposition diagrams of the data collecting assembly and circuit mounting assembly in Fig. 5 a.

Fig. 6 is the integrated platform of desk-top TPM, miniature wide field fluorescence microscope and FIRM-TPM properties in desk-top double light Light path schematic diagram under subpattern, the figure illustrate the structure diagram of desk-top TPM.

Fig. 7 is light path schematic diagram of the integrated platform of Fig. 6 under the imaging pattern of wide field.

Fig. 8 is light path schematic diagram of the integrated platform of Fig. 6 under FIRM-TPM imaging patterns, which illustrates in Fig. 6 Desk-top TPM in object lens and object plane (biopsy sample or biopsy sample) between light path on be provided with the FIRM-TPM of Fig. 1.

Fig. 9 a-9f are the two-photon excitation efficiencies for comparing GCaMP-6f under 800nm, 920nm and 1030nm excitation.

Figure 10 a are the transmission loss and dispersion parameters of the HC-920 optical fiber of custom design, wherein, the illustration of upper left is shown The cross-section photograph of HC-920 is shown, the illustration of upper right, which is shown, to be projected and by HC-920 by the pairing of the focal length with 3mm The image of 920nm laser after collimated.

Figure 10 b are the auto-correlations of 1 meter of HC-920 exits 920-nm femtosecond laser pulse width under different laser powers Distribution.

In Figure 10 c, left hand view：The frequency response of the x (light gray) and y (black) axis of MEMS scanners.Right plate：Machinery Function of the inclination angle as the DC driving voltages of the x (black) and y (light gray) axis of MEMS scanners.

Figure 11 a-11d be Fig. 4 femtosecond pulse modulator in dispersion compensation explanation.

Figure 12 a-12e be compare fix and move freely on head under the conditions of under dark surrounds biopsy sample V-1 cortex In dendron activity.

Figure 13 a-13c are to utilize the biopsy sample in different behavior examples of the biopsy sample behavior imaging system in Fig. 5 Imaging vision cortical activity.

Figure 14 a-14d are FIRM-TPM compared with desk-top TPM, the microscopical performance of miniature wide visual field.

Specific embodiment

In the accompanying drawings, represent same or similar element using same or similar label or have the function of same or like Element.The embodiment of the utility model is described in detail below in conjunction with the accompanying drawings.

In the description of the utility model, term " " center ", " longitudinal direction ", " transverse direction ", "front", "rear", "left", "right", " perpendicular Directly ", the orientation of the instructions such as " level ", " top ", " bottom " " interior ", " outer " or position relationship are based on orientation shown in the drawings or position Relation is for only for ease of description the utility model and simplifies description rather than instruction or imply that signified device or element must There must be specific orientation, with specific azimuth configuration and operation, therefore it is not intended that scope of protection of the utility model Limitation.

As shown in Figure 1, (English full name is " Fast to a kind of miniature two-photon microscopic imaging device that the present embodiment is provided high resolution miniature two photon microscope”；Hereafter it is referred to as " FIRM-TPM ") include Femtosecond pulse laser, femtosecond pulse modulator 1 and miniature probe 2, wherein：

The femtosecond pulse laser is used for the laser that generation wavelength is 920 nanometers.The femtosecond pulse laser is being schemed In do not illustrate, can use the company's chameleon solid state laser that is concerned with as light source, centre wavelength 920nm is defeated Go out 100 ± 1mW of power.Compared to the laser that existing wavelength is 1030nm and 800nm, wavelength is that 920 nanometers of laser can Effectively exciting common biological indicator, (such as Thy1-GFP and GCaMP-6f, GCaMP therein are most generally used Calcium ion indicator) generate fluorescence.Here compares GCaMP-6f respectively under 800nm, 920nm and 1030nm laser excitation Two-photon excitation efficiency utilizes tunable ti∶sapphire laser femto-second laser (Coherent, USA, Vision-S, 680- 1080nm, 85-110fs) in selection different wave length excited.

As shown in figs. 9 a-9f, Fig. 9 a are the AAV transfections for expressing GCaMP-6f in V-1 cortex under different excitation wavelengths Identical group pericaryon cell1, cell2 and cell3 are imaged in biopsy sample, is sequentially to use respectively from left to right in figure The fluoroscopic image that 800nm, 920nm and 1030nm laser obtain.Fig. 9 b are the Ca of 10 spongiocytes²⁺The pericaryon of signal Frequency.C in Fig. 9 c is the Ca of 10 spongiocytes²⁺The amplitude of the pericaryon of signal, the d in Fig. 9 c is 10 colloids The Ca of cell²⁺The average time-histories of the pericaryon of signal.Fig. 9 d are as in fig. 9 a, in different focal plane imaging neuron trees Prominent dendrite1, dendrite2 and dendrite3, be respectively sequentially from left to right in figure using 800nm, 920nm and The fluoroscopic image that 1030nm laser obtains.Fig. 9 e are from ten dendron Ca shown in Fig. 9 d²⁺The frequency of signal.In Fig. 9 f G is from ten dendron Ca shown in Fig. 9 d²⁺The amplitude of signal, the h in Fig. 9 f is from ten dendrons shown in Fig. 9 d Ca²⁺The average time-histories of signal.It can be seen that from these experimental datas：Femto-second laser pulse centered on 800nm completely cannot The GCaMP6 signals from pericaryon or dendron are excited, 1030nm excitations compared with 920nm is excited are only capable of that dendron is recorded The 1/10 of calcium ion activity, while the amplitude of the fluorescence signal recorded significantly reduces.Experimental data further proves to use ripple The laser of a length of 920nm can more efficiently excite GCaMP-6f to generate fluorescence.

Referring again to Fig. 1, femtosecond pulse modulator 1 is used to receive the laser of the femtosecond pulse laser output, And prechirp compensates the pulse broadening of laser to its preset value, and export.Laser pulse centered on 85fs, 920nm, via After the compensation of 1 prechirp of femtosecond pulse modulator, the about laser pulse of 100fs is modulated into, transimission power can be from 5mW to 200mW (data are to measure to obtain in the exit of laser input optical fibre 11 by experiment).

Miniature probe 2 includes scanning imagery part and fluorescence output optical fibre 21, wherein：The scanning imagery part is used to connect The laser of 1 output of femtosecond pulse modulator is received, which is scanned the tissue inside biopsy sample, with described in excitation Biopsy sample generates fluorescence signal.Fluorescence output optical fibre 21 is used to receive the fluorescence letter of the scanning imagery part output Number, and exported.

As depicted in figs. 1 and 2, what fluorescence output optical fibre 21 selected is a kind of new flexible optical fibre beam (SFB), this reality The fluorescence output optical fibre 21 applied in mode is formed by 700-900 root glass optical fiber weldings, meanwhile, keep each glass optical fiber loosely simultaneously It separates therebetween, the lightweight microscope for being carried by the biopsy sample of freely activity and being reduced motion artifacts is so convenient for visit Head, and then torque and tension caused by animal movement are minimized, without reducing photon collection efficiency.All glass optical fiber weldings Two ends be in 1.5mm diameters cylinder, to be easily installed on miniature probe 2.The following table 1 is the fluorescence in the present embodiment The performance parameter of output optical fibre 21 has higher collection efficiency compared with multimode fibre, and compares the plastics used before Optical fiber and traditional pattern of fusion fiber optic bundle are more flexible, can carry out multi-wavelength emission detection.Fluorescence output optical fibre 21 will come from micro- The fluorescence signal of object lens in type probe 2 is transferred in the GaAsP PMT (10770P-40, Hamamatsu, Japan) of distal end, Its total collection efficiency is about 80% at 532nm.

Table 1

Number of fibers	800
		Single fibre diameter (μm)	30
Numerical aperture (NA)	0.65
		Collection angle (degree)	80
Efficiency of transmission (%/m)	90
		Duty cycle (%)	90
Overall transfer efficiency (%)	80

As shown in Fig. 2, it is integrated in miniature probe 2 on fixing bracket 28.By fixing bracket 28, miniature probe 2 is with can The mode (screw connection) of dismounting is mounted on the head of biopsy sample (biopsy samples such as mouselet as shown in Figure 2).It is miniature The total weight about position 2.15g of probe 2 and fixing bracket 28, volume are no more than 1cm³, system is sufficiently small and compact and light enough So that biopsy sample carries.Fixing bracket 28 is made of aluminum material, such as aluminium chassis.The shape of fixing bracket 28 can be set A helmet shape is counted into, is so easily installed and dismantles, mainly can miniature probe 2 be firmly mounted on freely activity Biopsy sample head, therefore be conducive to carry out a few hours imaging in same animal, and keep miniature two-photon micro- The visual field of imaging device (hereinafter referred to as " FOV ") does not generate drift in the case of there are strong body and head movement. In addition, before being imaged using the utility model, the advance training living body sample of meeting adapts to be mounted on micro- on its skull Type probe 2, and be added dropwise 1.5% low melting-point agarose make its be filled in the brain tissue of the object lens of miniature probe 2 and biopsy sample it Between, these operations all significantly reduce relative motion between miniature probe 2 and brain, these measures improve experiment in short term and Long-term stability allows to carry out high-resolution in the behavioral value for carrying out comprising the free activity of a large amount of bodies and head in animal Imaging.

In one embodiment, as shown in figure 4, femtosecond pulse modulator 1 has negative dispersion light path and countenance astigmatism Road, wherein：The negative dispersion light path includes laser input optical fibre 11, and laser input optical fibre 11 is used for pulse broadening prechirp The laser compensated is transferred to the scanning imagery part.The positive dispersion light path is located at the femtosecond pulse laser and described Between negative dispersion light path, for compensating by the laser input optical fibre 11 caused negative dispersion in laser process is transmitted.

Due to material dispersion and nonlinear effect, single mode optical fiber can be by femto-second laser pulse broadening.And make in the present embodiment Laser input optical fibre 11 is Hollow-Core Photonic Crystal Fibers (hereinafter referred to as " HC-PCF "), i.e., occurs in figure or in text HC-920, wherein most of laser propagations be by HC-920 among inflation core, this has been effectively minimized due to non-linear The pulse broadening that effect generates.The laser input optical fibre 11 of the present embodiment is used for transmission 920 nanometers of femtosecond lasers of hundreds of milliwatts Pulse, nonlinear pulse broadening can be neglected (Figure 10 a and table 2).

Table 2

The laser input optical fibre 11 of the present embodiment provides undistorted transmission for 920 nanometers of femto-second laser pulses, this to change Into allowing common biological indicator is effectively excited to be possibly realized, such as Thy1-GFP and GCaMP-6f.

In one embodiment, the positive dispersion light path includes dispersive compensation element 12 and acousto-optic modulator 13, wherein： Acousto-optic modulator 13 adjusts laser intensity for receiving the laser after being compensated via dispersive compensation element 12, is then output to The scanning imagery part.Existing product realization may be employed in acousto-optic modulator 13.

Dispersive compensation element 12 closes on the femtosecond pulse laser and sets, and is being passed for compensating by laser input optical fibre 11 Caused negative dispersion in defeated laser process.

Negative dispersion in laser input optical fibre 11 is relatively low, as long as dispersive compensation element 12 is used with specific length The commercial glass material of positive dispersion can be accurately compensated for by prechirp method.After prechirp compensation, 85fs, Laser pulse centered on 920nm becomes the about pulse of 100fs after the laser input optical fibre 11 of 1m long transmits, and transmits Power (data are to measure to obtain in the exit of laser input optical fibre 11 by experiment) (can scheme from 5mW to 200mW 10b)。

Preferably, dispersive compensation element 12 has using H-ZF62 glass tubes in the laser of 920nm wavelength The positive dispersion of 458ps/nm/km.Wherein：Figure 11 a are to compensate HC-920 dispersion schematic diagrames using H-ZF62 glass tubes；Figure 11 b are The dispersion parameters of glass H-ZF62；Figure 11 c are the 920nm femtosecond lasers of 1 meter of the HC-920 outputs of never dispersion compensation Pulse width；Figure 11 d are the 920nm femto-second lasers before and after the HC-920 with and without the 1 of dispersion compensation meter Pulse width.

Here is that femtosecond pulse modulator 1 is realized to compensate the laser pulse prechirp centered on 85fs, 920nm Afterwards, the about laser pulse of 100fs, the detailed description that transimission power can be from 5mW to 200mW are become.

During the transmission of ultrashort pulse, two cause pulse broadening because being known as：The dispersion of material and high laser The nonlinear effect of peak power.In laser input optical fibre 11, most of laser propagations are by air-core, therefore non-linear effect It should minimum.In the light path of miniature probe 2, the optical density at the center of the optical component of such as lens and speculum is relatively It is low, because beam sizes are larger (a diameter of to be no more than 2mm), therefore hardly cause non-linear.Due to microscopical forepiece It is relatively short with the length of the micro-component in sample, therefore also can be neglected.Therefore, main expansion effect originates from by desk-top Material dispersion caused by optical device and laser input optical fibre.By simplification, second order is only considered in the system of present embodiment Dispersion.Therefore, the pulse broadening of whole system is：

B_system=| ∑_n{L_n∫_λ,ΔλD_n(λ)dλ}| (1.1)

Wherein：The quantity of optical device in n expression systems；D_n(λ) color as caused by n-th of component (optical device) Dissipate parameter；L_nRepresent the optical path length of the n-th component (optical device)；λ and Δ λ is the centre wavelength and spectral width of laser；|| Represent the positive dispersion (negative D (λ)) from lens and the absolute value from laser input optical fibre anomalous dispersion (positive D (λ)) summation.Cause This, as Δ λ<<λ, in the system of the utility model (λ=920nm, Δ λ are no more than 15nm), equation simplification is：

B_system=| ∑_n{L_n·D_n(λ)·Δλ}| (1.2)

Final pulse width is expressed as：

P_output=P_input+|B_system| (1.3)

Wherein P_inputAnd P_outputRepresent laser outputs and inputs pulse width.

The length of the HC-920 used in system provided in this embodiment is 1m (L_hc-920), by manufacturer measure and It is shown in Figure 10 a.Therefore, the extension of HC-920 is：

L_hc-920·D_hc-92015 (nm)=1.125 (ps) of (920nm) Δ λ~1 (m) 75 (ps/nm/km)

The easily definite such as length of polarization beam apparatus, lens and acousto-optic modulator, and the eyeglass used by them Refractive index information calculates, and is expressed as：

With

Wherein, GVD represents group velocity dispersion in medium, and c is the light velocity, and n is the refractive index of medium.

It is added together, these components cause the broadening of about -200fs, therefore defeated from the HC-920 of 85fs Transform Limited Pulses The calculating pulse width gone out is about：

(0.085+1.125-0.2) (ps)=1.01 (ps)

It is matched with the pulse width (being no more than 1ps) (Figure 11 c) of experiment measurement, and for effective two-photon excitation It is too weak.By the way that H-ZF62 glass tubes (dispersive compensation element 12 in the present embodiment uses H-ZF62 glass tubes) insertion is had In the light path of positive dispersion, the present embodiment has pre-compensated for the negative dispersion as caused by HC-920.The present embodiment selects H-ZF62 glass tubes, Because it has big positive dispersion (dispersion in wavelength is 920nm is about 58ps/nm/km), provided by Scott companies Refractive index information and equation (1.4) calculate, as shown in figure 11b).The length computation of required H-ZF62 glass tubes is as follows：

(1.125-0.2) (ps)/458 (ps/nm/km)/15 (nm)=13.5 (cm)

In view of small positive dispersion as caused by microscope front end and sample, the present embodiment using 12 centimeter lengths, The H-ZF62 glass tubes of 12.7 mm dias, less than calculated value.In this " the pre- line of H-ZF62 glass tubes (Figure 11 b and Fig. 6) Property frequency modulation " after, the laser pulse width from HC-920 test lighting power gamut in be compressed to about 100fs (Figure 11 d and Figure 10 b).The FIRM-TPM of the present embodiment has and can neglect to transmit by using the HC-PCF of designed, designed 920 nanometers of femto-second laser pulses (entitled HC-920) of nonlinear pulse broadening slightly, can test to common microbial administration Agent is imaged, and is achieved and the comparable performances of desk-top TPM.

In one embodiment, the positive dispersion light path further includes laser azimuth adjustment component, the laser azimuth adjustment Component is arranged between dispersive compensation element 12 and acousto-optic modulator 13, and the laser azimuth adjustment component includes the first half-wave plate 14th, the first speculum 15 and the second speculum 16, wherein：

First half-wave plate 14 adjusts laser polarization side for receiving the laser after being compensated via dispersive compensation element 12 To so that the modulation efficiency highest of acousto-optic modulator 13.First speculum 15 swashing via the first half-wave plate 14 for reception Light, and reflection laser, to adjust the position that laser injects acousto-optic modulator 13.Second speculum 16 is anti-via first for receiving The laser of mirror 15, and reflection laser are penetrated, the angle of acousto-optic modulator 13 is injected to adjust laser, and is transferred to acousto-optic modulator 13。

In one embodiment, the positive dispersion light path further includes spectrum groupware, and the spectrum groupware closes on laser input Optical fiber 11 is set, and regulates the laser of intensity via acousto-optic modulator 13 for reception and the laser is divided at least two beams, And it is transferred to laser input optical fibre 11.

In one embodiment, the quantity of laser input optical fibre 11 is at least two, including first laser input optical fibre and Second laser input optical fibre.

The spectrum groupware includes polarization beam apparatus 17, the second half-wave plate 18, the 3rd speculum 19, the first collimation lens 110th, the 3rd half-wave plate 111, the 4th speculum 112, the second collimation lens 113 and the 4th half-wave plate 114, wherein：

Polarization beam apparatus 17 regulates the laser of intensity via acousto-optic modulator 13 and is divided into the laser for receiving At least two beams are transferred to the first laser input optical fibre and second laser input optical fibre respectively.Second half-wave plate 18 is arranged in Between polarization beam apparatus 17 and acousto-optic modulator 13, for changing the splitting ratio of polarization beam apparatus 17.3rd speculum 19 is arranged Between 17 and second half-wave plate 18 of polarization beam apparatus, for reflection laser, to adjust the position of laser and project described inclined It shakes on the plane of incidence of beam splitter 17.First collimation lens 110 is arranged in the first exit facet and first laser of polarization beam apparatus 17 Between input optical fibre 11, for the laser for receiving the output of polarization beam apparatus 17 and by laser coupled into first laser input light Fibre 11.3rd half-wave plate 111 is arranged between the first exit facet of polarization beam apparatus 17 and the first collimation lens 110, for connecing The laser that polarization beam apparatus 17 exports is received, and adjusts laser polarization direction so that laser coupled is into first laser input optical fibre 11 Efficiency highest.4th speculum 112 is arranged in the second exit facet of polarization beam apparatus 17 and the second laser input optical fibre Between, for receiving the laser of the output of polarization beam apparatus 17, for reflection laser, to adjust the position of laser and project described In second laser input optical fibre.Second collimation lens 113 is arranged between the 4th speculum 112 and second laser input optical fibre, For the laser for receiving the output of polarization beam apparatus 17 and by laser coupled into the second laser input optical fibre.4th half-wave plate 114 are arranged between the 4th speculum 112 and the second collimation lens 113, for receiving the laser of the 4th speculum 112 reflection, And adjust laser polarization direction so that laser coupled is into the efficiency highest of the second laser input optical fibre.

As shown in figures 1 and 3, in one embodiment, the scanning imagery part includes micro electromechanical scanning instrument 22, object lens 23rd, scanning lens 24, collimator 25, dichroic mirror 26 and collection len 27, wherein：

Micro electromechanical scanning instrument 22 (MEMS) is used for laser by way of rotating and changing laser light incident angle angle to described Tissue plane inside biopsy sample carries out two-dimensional scan.Preferably, a diameter of 0.8mm of micro electromechanical scanning instrument 22 encapsulates ruler Very little is 9 × 9mm², the first resonant frequency is no more than 6kHz, and greatest optical scanning angle is ± 10 degree, supports that frame sign is 256 × 256 and maximum field of view be 130 × 130 μm²40Hz imaging, with realize video rate image gather.With other scanning sides Formula is compared, such as is compared using the spiral of piezoelectric actuator or the optical fiber scanning of Lissajous patterns, provided in this embodiment micro- Electromechanical scanner 22 is scanned for its high speed in entire visual field, and uniformly excitation and wide angle scanning, the big visual field are all favourable. X and y control signals in 22 scanning process of micro electromechanical scanning instrument are raw by FPGA (field programmable gate array) cards (PXI-7853R) Into the card is also used for driving acousto-optic modulator to adjust laser intensity and triggering other devices (for example, infrared camera scan). Signal from photoelectric detector (H10770P-40, Hamamatsu Japan) passes through high speed preamplifier (DHPCA- 100, FEMTO Gmbh, Berlin, Germany) amplification, it is then attached to 120MS/s digital quantizer adaptor modules (NI5734, National Instruments Inc., Austin, TX, USA) and FlexRIO FPGA (PXIe-7961R, National Instruments Inc., Austin, TX, USA), for high-speed data acquisition.Whole system is by being based on The customized development software control of LabVIEW platform.

Object lens 23 are for the laser from micro electromechanical scanning instrument 22 to be converged to inside the biopsy sample, with described in excitation Biopsy sample generates the fluorescence signal and for exporting the fluorescence signal.Preferably, the numerical aperture of object lens 23 is (hereafter It is referred to as " NA ") for 0.8, NA higher, optical layer is cut relatively thin, this is advantageously implemented sub-micrometer imaging resolution ratio, and then can The 26S Proteasome Structure and Function variation of dendron and dendritic spines is told in the freely sample of activity.

In the present embodiment, object lens 23 use the high-resolution water logging of GRINTECH GmbH (Jena, Germany) small Type object lens (GT-MO-080-018-AC900-450), this be with 4.76 × magnifying power, the picture of 0.80 object space NA and 0.173 Square NA carries out finite correction.The length of object lens 23 is 6mm long, and diameter is 1.4mm, and with 200 μm of operating distance.Object lens 23 specifically include thin Fresnel lens 231, planoconvex spotlight 232, relay lens 233 and condenser lens 234, wherein, thin Fresnel Lens 231 and planoconvex spotlight 232 are all miniature graded index (GRIN) lens.Compared with the object lens used in the prior art, The diffraction components (Fresnel Lenses) that the present embodiment is incided into object lens 23 effectively correct during two photon imaging excitation and Big aberration between launch wavelength, this can cause better light beam focusing quality, and improve signal collection efficiency, so as to be conducive to Realize sub-micrometer imaging resolution ratio, and then structure and work(for dendron and dendritic spines can be told in the freely sample of activity It can variation offer advantage.

Scanning lens 24 is arranged in the light path between micro electromechanical scanning instrument 22 and object lens 23, for by micro electromechanical scanning instrument The laser of angle change changes into the laser of change in location caused by 22 two-dimensional scans.Scanning lens 24 uses non-spherical lens (#355160B, Lightpath Technologies, Orlando, FL, USA；Diameter：3mm；Equivalent focal length：2.7mm) conduct Scanning lens, to reduce spherical aberration.

Collimator 25 is arranged between laser input optical fibre 11 and micro electromechanical scanning instrument 22, is inputted for collimating from laser Aberration between laser and reduction different frequency laser that optical fiber 11 exports, to match object lens 23 jointly with scanning lens 24 Image NA.The collimator 25 of the present embodiment use achromatism collimation lens (#65-286, Edmund Optics Inc., Barrington, NJ, USA；Diameter：2mm, equivalent focal length：3mm, special near infrared light), output laser can be collimated and subtracted Aberration between the different frequency component of few femto-second laser is so conducive to improve efficiency of transmission (from lasing light emitter to sample height Up to 50%), light beam focuses on and launching efficiency.

Dichroic mirror 26 is located between scanning lens 24 and object lens 23, for separating and exporting institute by laser and fluorescence signal State fluorescence signal.The product that dichroic mirror 26 is manufactured using Fujian China Sunlight Co., Ltds, size 3 × 3 × 0.2mm³, reflection band is 750-1100nm, and transmission bands are 400-650nm, are reflected into the laser beam of self-scanning in future lens 24 Object lens 23.Collection len 27 is for effectively collection fluorescence signal.

The utility model is simulated using optical design software (ZEMAX) and is optimized all optical elements between them Distance, and carry out geometry designs using business CAD software (Solidworks).

The specific calculating process of the resolution ratio of miniature probe 2 provided by the utility model is as follows：

First, NA (NA are effectively excited_ex) determined by the entire light path excited.The diameter D of laser beam after collimator 25₁ For：

D₁=2NA_hc-920·EFL_c (2.1)

Wherein, NA_hc-920It is the NA (being about 0.15) and EFL of HC-920_cThe equivalent focal length (3mm) of collimator 25.

Because the diameter D of laser beam₁(0.9mm) be more than micro electromechanical scanning instrument 22 size (for x directions be 0.8mm, it is right In y directions for be about 0.6mm), it is contemplated that the gradient of micro electromechanical scanning instrument 22 be limited by its size and by following formula to Go out：

NA_ex=(1/2D_m/EFL_s)·NA_o/NA_i (2.2)

Wherein, EFL_sIt is the equivalent focal length (2.7mm) of scanning lens 24.NA_o,NA_iThe NA and image of object lens are represented respectively NA is respectively 0.8 and 0.173.

Then, the maximum excitation NA determined by the x directions of micro electromechanical scanning instrument 22 is：

NA_ex=(1/20.8/2.7) 0.8/0.173~0.685

Use the equation calculation two-photon excitation (I in bibliography 2_PSF ²) point spread function diffraction limit it is horizontal (ω_xy) and axial (ω_z) 1/e radiuses, the present embodiment has：

Wherein, the refractive index of immersing medium (physiological saline) is no more than 1.34, and the wavelength of laser is 920nm. aNA_exFor 0.685, ω_xy=0.304 μm and ω_z=1.840 μm.

Resolution ratio is normally defined PSF²Halfwidth (FWHM), the present embodiment has：

It is obtained using the image measurement of the 100nm fluorescent beads in embedded agarose：Miniature double light provided by the utility model The lateral resolution of sub- microscopic imaging device is 0.64 ± 0.02 μm, and axial resolution is 3.35 ± 0.36 μm.

In the above-described embodiments, the method for making experiment sample is when testing the resolution ratio of the utility model：First by 2 μ L TetraSpeck 100-nm fluorescent beads (T7270, Life Technologies, Oregon USA) be diluted to 100 μ L physiology In brine, then mixed with 900 μ L, 1.5% low melting-point agaroses.The agarose droplet of microsphere fluorescence pearl (4 μM) will be contained again It is added on coverslip, it is spare after ten minutes.Equivalent horizontal pixel size is 61nm, and axial scan is at intervals of 80nm, both greatly In 5 times or more of nyquist sampling theorem requirement, and then it is enough the true optical resolution of measuring microscope.

Table 3 is FIRM-TPM provided by the utility model and the contrast table of the resolution ratio of mTPM in the prior art, and the 2nd row are The resolution ratio and its parameter declaration of FIRM-TPM provided by the utility model, 3-6 row are the resolution ratio of mTPM in the prior art And its parameter declaration.It should be noted that：* in table 3:Data come from document.It is proved from existing experimental data：The prior art Middle mTPM although having obtained higher spatial resolution in the external of animal, most of mTPM are in the internal performance of animal It is bad, it fails to obtain the fluoroscopic image of high s/n ratio (SNR).It can be drawn from the comparative analysis in table 3：The utility model carries The resolution ratio of the FIRM-TPM of confession is about twice of the highest resolution of mTPM of the prior art, and this high-resolution is solution The imaging problem of single dendritic spines provides advantage in certainly free moving animals.

Table 3

For test and desk-top TPM more of the prior art and miniature wide-field microscope and FIRM- on same benchmark Performance difference between TPM, the present embodiment establish an integrated platform, allow to carry out desk-top TPM mode on same sample Switching between (Fig. 6), wide field imaging pattern (Fig. 7) and FIRM-TPM imaging patterns (Fig. 8).As shown in figs 6-8, this is integrated Platform equipped with miniature and conventional objective lens, multiple light sources (laser for TPM and the high power mercury lamp for wide field) and Detection device (GaAsP for TPM and the sCMOS cameras for wide field).Different imaging patterns has identical focal plane With concentric visual field, total frame acquisition time and imaging NA, and average illumination power and detector sensitivity are also carefully selected And matching, the composition of above-mentioned integrated platform is illustrated with reference to Fig. 6 to Fig. 8.

Above-mentioned integrated platform is respectively to same under desk-top TPM mode, wide field imaging pattern and FIRM-TPM imaging patterns The same focal plane of sample is imaged.Laser (Chameleon Vision-S, Coherent, USA；Centre wavelength： 920nm), by H-ZF62 glass tubes, then with acousto-optic modulator (MT110-B50A1.5-IR-Hk, AA Sa, Orsay Cede, France).Then the laser that centre wavelength is 920nm is divided into two beams, it is a branch of be coupled to used in FIRM-TPM A HC-920 in, another beam is coupled in another HC-920 used in desk-top TPM.

As shown in fig. 6, in desk-top TPM mode, the alignment laser from HC-920 is by a pair of of galvanometer meter scanning mirror A1 (6215H, Cambridge Technology, MA, USA) scan, then by scanning lens A2 (LMS05-BB, Thorlabs Inc, New Jersey), pipe lens A3 (Olympus Japan) and the first dichroscope A4 (DM1, DMLP650R, Thorlabs Inc, New Jersey, USA), then it is ultimately delivered to back focal plane (BFP) (CFI of object lens A5 Apo 40XW NIR, Nikon, Japan；40 × NA 0.8, operating distance：3.5mm).Fluorescent emission signals from object lens A5 It is reflected by the first dichroscope A4, passes through collecting lens A6 (LA1213-A, Thorlabs Inc, New Jersey, USA), the (DM2, #87-284, Edmund Optics Inc., Barrington, USA reflect band to two dichronic mirror A7：375-393,466- 490 and 546-565nm；Transmission bands：420-460,510-531,590-624 and 677-725nm), pass through collector lens A8 (ACL25416U-A, Thorlabs Inc, New Jersey, USA), last Photoelectric multiplier tube A9 (GaAsP PMT 7422P-40, Hamamatsu, Japan) detection.In order to realize the excitation NA identical with FIRM-TPM (0.685), by object lens A5 Back focal plane (BFP) at the beam sizes of excitation laser beam be arranged to：

(2 × 0.685 × 200) (mm)/40=6.85 (mm)

Wherein, 200mm is the EFL of the matching pipe lens A3 for object lens A5, and 40 be the magnifying power of object lens A5.

It as shown in Figure 7 and Figure 8, will be provided by the utility model in wide field imaging pattern and FIRM-TPM imaging patterns Miniature probe 2 is connected on the small manual micromanipulator of three axis (Sigma Koki, Tokyo, JAPAN) of customization, miniature spy Object lens image plane is air object lens (Plan Apo NIR 5X, Mitutoyo, Japan in first 2；5 ×, NA 0.14, working distance From for 37.5mm) focal plane.Using with high-precision two-way repeated (± 2.5 μm) 4 port rotating nosepieces (OT1, Thorlabs Inc, New Jersey, USA) switch between miniature probe 2 and 40 × Nikon object lens.By adjusting Nikon object The screw thread of lens barrel before mirror and miniature probe 2, the utility model can set miniature probe 2 and Nikon object lens to have phase Same focal plane.

As shown in fig. 7, in miniature wide visual field fluorescence structure, using with light filter of blue light (MF475-35, Thorlabs Inc, New Jersey, USA；Centre wavelength is 475nm, bandwidth 40nm) fluorescent luminaire (X-Cite 120Q, Excelitas Technologies, MA, USA)：35nm) it is used as light source B1.By desk-top TPM construct in the one or two to Look mirror A4 changes into dichronic mirror B2 (DM3, # 87-284, Edmund Optics Inc., Barrington, NJ, USA；Reflection Band：375-393,466-490 and 546-565nm；Transmission bands include：420-460,510-531,590-624 and 677- 725nm), illumination light is reflected into miniature probe 2, and the fluorescent emission signals from miniature probe 2 is transferred to science CMOS camera B3 (8050M-GE, Thorlabs Inc, New Jersey, USA).In desk-top TPM and FIRM-TPM configurations Be imaged on after target and use similar mean power, be usually 10-25mW.Due to the absorption of single photon and two-photon excitation The difference in section, the mean power used in single photon image are usually more much lower than the mean power in two photon imaging.Therefore, The utility model is illuminated using 100-500 μ W (after miniature object lens) in the configuration of wide field fluorescence microscope, similar to before The 170-600 μ W illumination used in report.

The left-side images of Figure 14 a are the neuron dendron of Thy1-GFP transgenosis biopsy sample brains and the 3D forms of dendritic spines Imaging, 1s total exposure (average value of 8 frames of FIRM-TPM and the microscopical 8Hz of miniature wide visual field and desk-top TPM's The average value of 2 frames of 2Hz) under obtain the image of roughly the same focal plane, the right side graph of Figure 14 a is shown in left-side images Clipping region in two pairs of adjacent dendritic spines cross-sectional profiles.Figure 14 b are in the PFC of the biopsy sample of expression GCaMP6f The image of plane (- 130 μm) rich in pericaryon, top picture：30 seconds the average images of identical ROI.Miniature wide field figure As being shown as normalized Δ F/F (referring in line method)；Lower wave line：Ca in the neuron of three selections²⁺Time-histories become Change (the duration：100 seconds) (numeral mark is used in upper figure).The left-side images of Figure 14 c come from identical work in (Figure 14 b) The Ca of the dendron and spinous process (- 120 μm of focal plane) of body sample²⁺Due to lacking recognizable dendrite signal, use is not shown in signal The image of miniature wide visual field microscope record, the right upper portion image of Figure 14 c captured with desk-top TPM one is with dendron axis (D1) Imaging and Ca with dendritic spines (S1, S2, S3)²⁺Signal (the duration：100s), the lower right side image of Figure 12 c is to use FIRM One band dendron axis (D1) of capture and imaging and the Ca of dendritic spines (S1, S2, S3) (left side)²⁺Signal (the duration：100s).Figure 12d is the average frequency and amplitude for the Ca2+ signals being imaged in different configurations, from identical group of pericaryon (n=4), Statistics：The data of dendron (n=8) and dendritic spines (n=6).

It is imaged for morphology, V1 region of the utility model in the fixation brain of Thy1-GFP transgenosis biopsy samples (130×130μm², from surface to less than 60 μm) and it is imaged, obtain the image data (Figure 14 a) of 3D.FIRM-TPM and desk-top TPM shows identical contrast and resolution ratio (Figure 14 a) in dendron imaging.However, under miniature wide field imaging pattern, base Resolution technology details is unable on this, is primarily due to the strong background signal from afocal tissue.In the comparison of functional imaging, this Utility model represents Ca using the mode of adeno-associated virus infection in biopsy sample prefrontal cortex neuron²⁺Index agent GCaMP 6f, and calcium imaging (Figure 14 b- Figure 14 d) is carried out in the fixed clear-headed biopsy sample in head.Two focal planes of selection are come with showing From the activity of cell space (below surface 130 μm) or dendron and dendritic spines (below surface 120 μm).In image normalization and comparison After enhancing (Δ F/F), miniature wide field imaging is with similar frequency discrimination body cell Ca²⁺Signal, and their amplitude (Δ F/F For 5-10%) than the low about an order of magnitude of amplitude (figure with desk-top TPM or FIRM-TPM (Δ F/F is about 150%) measurements 14d).It is worth noting that, most of Ca for being derived from dendritic arbors²⁺Signal is not detected (figure in this imaging configuration 14b and Figure 14 d).In short, the test of these benchmark proves that the FIRM-TPM of the utility model reaches the property identical with desk-top TPM Can, while it is better than miniature wide-field microscope in terms of the 26S Proteasome Structure and Function activity of dendron and dendritic spines in sobering animal is differentiated.

The quick micro electromechanical scanning instrument 22 used in FIRM-TPM is than the resonance scanner that is used in high speed desktop TPM With more linear voltage-tilt response and controllability.Therefore it assigns it and is imaged greater flexibility so that in single system The random scanning imaging in region (ROI) and the scanning of ultrafast line that system kind can realize quick grid type scanning imagery, be arbitrarily designated Imaging.The random scanning imaging of particularly 128Hz helps to differentiate Ca²⁺Ultra microstructure on signal time, and can assign MTPM carries out the ability of accurate light genetic manipulation in the freely animal of activity.10kHz line scanning imagery abilities are for utilizing It is most important that the voltage indicator of gene code carrys out rapid scan action potential.

Dendritic spines activity is the elementary event of neuronal messages processing, utilizes desk-top TPMs grinding on fixed animal Study carefully and show that the different dendritic spines of single nerve cell can be pierced by the sound of the visual stimulus of different directions or varying strength frequency It is activated.Although the mass activity of the hundreds of neurons obtained by miniature wide-field microscope can help to detect in difference Neuroid encoding characteristics under the conditions of behavior, the FIRM-TPM of the utility model provide one and alternative more strengthen Big instrument, to the space-time characterisation of more basic neural coding unit in irrealizable behavior normal form on head fixation animal It is observed.

In short, FIRM-TPM newly developed has been realized in high-spatial and temporal resolution, good mechanical stability and correspondence With effective excitation of indicator widest in area.By using the mTPM of a new generation, the utility model is realized in free activity Animal in the long-times of single synaptic levels be imaged the target that this scientist dreams of.It is contemplated that future has more It improves and extends.Using the miniature high NA object lens correctly designed, FOV and penetration depth can be further expanded.Deep Brian Imaging can By the way that directly miniature object lens are inserted into brain or are realized by being embedded in grin lens in the brain, to be reported similar to former Method.For larger animal, such as rat or marmoset, multiple FIRM-TPM can be installed on skull, to explore brain The relevant issues of medium-long range 26S Proteasome Structure and Function connection.The utility model, it is expected that FIRM-TPM will be proved to be neuroscientist with And biomedical science man explores the important tool of health and disease mechanisms in animal body.

As shown in Figure 5 a, the utility model also provides a kind of biopsy sample behavior imaging system, the biopsy sample behavior Imaging system includes babinet 3, femtosecond pulse laser, femtosecond pulse modulator 1, miniature probe 2, data collecting assembly 7 With circuit mounting assembly, wherein：

Femtosecond pulse laser is used for the laser that generation wavelength is 920 nanometers.Femtosecond pulse modulator 1 is used to connect Receive the laser of femtosecond pulse laser output, and the pulse broadening of prechirp compensation laser, to preset value, the preset value is excellent Choosing is no more than 100fs, and exports.Miniature probe 2 is used to be mounted on biopsy sample, and input terminal passes through the femtosecond pulse Laser input optical fibre in laser modulator connects the output terminal of the femtosecond pulse laser, for receiving the femtosecond pulse The laser of laser modulator output, the laser are scanned the tissue inside biopsy sample, the biopsy sample to be excited to produce Raw fluorescence signal；And the fluorescence signal for receiving the scanning imagery part output, and exported.Femtosecond pulse Laser, femtosecond pulse modulator 1 and miniature probe 2 have been discussed in detail above, and details are not described herein again.

Babinet 3 provides restriction space for moving freely for live body biopsy sample, and the restriction space is compared to biopsy sample Build is much bigger, can provide sufficiently large space for the free activity of biopsy sample.Data collecting assembly 7 is mounted on babinet On 3, the input terminal of data collecting assembly 7 connects the output terminal of miniature probe 2 by fluorescence output optical fibre 21, micro- for collecting The fluorescence signal of 2 output of type probe.Laser input optical fibre 11 and fluorescence output optical fibre 21 pass through the circuit mounting assembly So as to the mode arbitrarily rotated compared with 3 babinets is mounted on babinet 3.By circuit mounting assembly, it can prevent that fluorescence is defeated Go out the mutually winding each other of optical fiber 21, laser input optical fibre 11 and other supply lines 8, provided for the free activity of biopsy sample Advantage.

In one embodiment, data collecting assembly 7 provides detection light path, includes coaxially arranged light in the detection light path Electric multiplier tube 71, concentrator 72, transmitting optical filter 73, short pass filter 74 and collecting lens 75, wherein：Photomultiplier 71 is used It is exported after electric signal is converted optical signal into.The fluorescence signal that concentrator 72 is transmitted for convergence by fluorescence output optical fibre 21. Transmitting optical filter 73 is used to filter the laser that wavelength is 920 nanometers.Short pass filter 74 is used to filter out in addition to flashlight Stray light.Fluorescence output optical fibre 21 is coaxial with collecting lens 75, and fluorescence output optical fibre 21 is located at the focal plane of collecting lens 75 On, collecting lens 75 is used to the fluorescence signal being more fully collected into photomultiplier 71.

Preferably, the circuit mounting assembly includes electronic rotation first 6 and stent 10, wherein：Electronic rotation first 6, which runs through, to be set It puts at the top of babinet 3, also, one end connection external power supply, other end connection supply lines 8 makes the external power supply and power supply Line 8 can be electrically connected.Stent 10 is located at outside the electronic rotation first 6 at 3 top of babinet, and laser input optical fibre 11 and fluorescence are defeated Go out the shell that optical fiber 21 passes through electronic rotation first 6, fluorescence output optical fibre 21 and 75 light of collecting lens in data collecting assembly 7 Signal connects.Fluorescence output optical fibre 21 is connected to data collecting assembly 7 by bearing 9, in this way, electronic rotation first 6 and bearing 9 are permitted Perhaps fluorescence output optical fibre 21 and supply lines 8 are independently rotated, at the same keep detection light path it is constant, this design prevents work The winding of connecting line caused by when body sample freely explores its environment drastically increases the stability of miniature probe 2, even if living Body sample carries out strong body movement and there will not be influence, so that animal is minimum in the distortion and winding of free probe phase top-stitching Change.

In one embodiment, the biopsy sample behavior imaging device that moves freely further includes supply lines 8, is micro electronmechanical Scanner 22 is powered.Supply lines 8 is pacified in a manner that the circuit mounting assembly compared with the babinet so as to arbitrarily rotate On the babinet, so as to ensure supply lines 8 can normal power supply, can also avoid supply lines 8 and laser input optical fibre 11 It is wound each other with fluorescence output optical fibre 21, so that animal minimizes in the distortion and winding of free probe phase top-stitching.

In one embodiment, the biopsy sample behavior imaging device that moves freely further includes multiple cameras 4, camera 4 All on the inner wall of babinet 3, than the side and the top surface that are mounted on babinet 3 as shown in Figure 2, respectively with different angles Degree shooting and the behavior for moving freely process of record biopsy sample.

In one embodiment, the biopsy sample behavior imaging device that moves freely further includes headlamp 5, headlamp 5 On the inner wall of babinet 3, for the inner cavity of illumination box 3.

As shown in Figure 12 a-12e, Figure 12 a are that (this practicality is newly by 50 of V-1 cortex L2/3 neurons average FIRM-TPM The miniature two-photon microscopic imaging device of type) image, 1,2,3 dendron has been irised out in image.Figure 12 b are that biopsy sample is being raced Two-dimentional track (the duration in：100 seconds), track is calculated from the videograph of the camera capture at wherein top, and is moved Dynamic speed color coding.Figure 12 c and Figure 12 d are that (12c) is fixed on head and is moved freely under condition (12d) from three respectively The Ca of a dendron (being irised out in Figure 12 a)²⁺Time-histories (the duration of signal：100s), the time correlation of biopsy sample speed becomes Change is also shown in bottom.Figure 12 e are all dendron shape Ca under the conditions of fixing and move freely on head²⁺The average frequency of transition； Data are expressed as average value ± s.e.m., n=15 dendron.***p<0.001, paired t-test.It, can be with by more than experimental data It was found that when biopsy sample freely exploration in the dark (track is shown in Figure 12 a-12e), the neuron of visual cortex Dendron activity has significant increase (Figure 12 a-12e) compared with the fixed situation in biopsy sample head.

As shown in Figure 13 a-13c, Figure 13 a are to be imaged neuron in the visual cortex in three kinds of different behavior examples to live It is dynamic：Qutstanding tail test, diving tower, Social behaviors.Leftmost side first row image：Pass through two phases of the side on behavior device side face The biopsy sample of machine shooting participates in the snapshot of different behaviors；Intermediate secondary series image：The pericaryon of GCaMP6f marks, tree Prominent and dendritic spines FIRM-TPM are imaged；The 3rd row image of right side：The corresponding calcium activity distribution figure (duration：100s).Most The 4th row image of right side：The Ca of three ROI indicated in FIRM-TPM images²⁺Signal (the duration：50 seconds).Figure 13 b are The short-term and long-time stability of FIRM-TPM imagings, Figure 13 b leftmost curves are based between the frame and frame in each behavior example Cross-correlation analysis transverse shakiness distribution, in curve, FM：It moves freely；TS：Experiment；SD：Diving tower；SI：Social behaviors；It is flat Transverse shakiness between frame and frame is：In FM, 0.11 ± 0.06 μm (75% in 0.14 μm)；In TS for 0.10 ± 0.07 μm (75% in 0.13 μm)；It is 0.19 ± 0.09 μm (75% in 0.24 μm) in SD；In SI, 0.06 ± 0.03 μm (75% in 0.07 μm)；Figure 13 b right side graphs are the whole drifts of the visual field of the during the experiment when 4 is small.Figure 13c is dendron and the activity of the global and local calcium of dendritic spines, top in qutstanding tail test：Dendron with multiple dendritic spines branches 10s the average images, illustration are shown in the Ca of its root dendron axis of 3 pairs of dendritic spines²⁺Signal, global calcium activity are defined as simultaneously Activate dendritic spines and its root dendron axis.

This result and multidigit point electricity probe records to movement can enhance animal vision cortical neuron activity one It causes.Next, in order to prove the robustness of FIRM-TPM, the utility model in the primary visual cortex of biopsy sample 130 ×130μm²FOV on carry out the imaging of 40Hz to observe the activity of its neuron, the biopsy sample is in three behaviors example It tests successively, including tail-suspention test, diving tower and Social behaviors.These examples all cannot be real in the fixed experimental strategy in head It is existing, entire test process last about 4 it is small when.It is worth noting that, even if when biopsy sample is struggled strongly in qutstanding tail test, When jumping off from plateau or being exchanged with its compatriot, the activity from different cell spaces, dendron and dendritic spines still can be by stable sight It surveys.Different behavior normal forms seems associated (Figure 13 a) with the nervous activity of the different zones of same V1 cortexes.

In order to quantify the short-term and long-time stability of miniature Two Photon Fluorescence provided by the utility model, the utility model Lateral displacement (Figure 13 b) is analyzed to the drift of another a period of time by a period of time of cross-correlation frame by frame and visual field.This practicality New discovery, in 2000 successive frames, the average displacement between frame and frame is (0.19 ± 0.09 μm in diving tower behavior example； 75% below 0.24 μm) it is the largest, but still less than the half of pixel size.In entire 4h tests, FIRM-TPM's The whole drift of FOV<10 μm, most of drifts are happened at first hour of strenuous exercise in tail-suspention test.It is noticeable It is that higher Image Acquisition speed reduces the dislocation in frame, the motion artifacts of this level do not interfere the utility model pair The imaging of dendritic spines 26S Proteasome Structure and Function.In order to demonstrate miniature Two Photon Fluorescence provided by the utility model to single synaptic activity Imaging capability, by tracking the Ca on dendritic spines and his father's dendron axis²⁺Activity, the utility model further analyze whole drawn game Relation between portion's activity.To sum up, miniature Two Photon Fluorescence provided by the utility model can be in natural physiological environment In stable observation is carried out to the freely dendron of the animal of activity and dendritic spines activity.

The utility model also provides a kind of miniature two-photon micro imaging method, the miniature two-photon micro imaging method Including：

Step 1, region to be imaged is chosen：Fixed biopsy sample, and chosen and treated in the biopsy sample using desk-top TPM Imaging region.In step 1, it is necessary to using imaging platform as shown in Figure 6, and under desk-top TPM mode, that is to say, that utilize Desk-top TMP chooses region to be imaged in biopsy sample, and concrete operations are：Biopsy sample is fixed using body fixator, with logical Desk-top TPM is crossed to confirm the region of the virus infection on biopsy sample head.

Step 2, fluorescence signal is gathered：The miniature probe for being connected with laser input optical fibre is mounted on the biopsy sample On, biopsy sample is discharged, with the fluorescence signal that the region to be imaged chosen in detecting step 1 exports, completes the biopsy sample The imaging of internal tissue plane.Step 2 concrete operations are as follows：

First, miniature probe 2 provided by the utility model is adhered on the stent of biopsy sample head skull, the stent It is already fixed on the skull on biopsy sample head during operation of opening cranium prepares, then is reinforced miniature probe 2 with dental cement Onto the stent of biopsy sample head skull.

Then, 1.5% low melting-point agarose is covered to exposed brain regions.This method significantly inhibits probe Relative motion between brain.In order to realize the dyeing in arbitrary access to a large amount of neurons, the process of labeled neurons is： The utility model manually cerebrospinal fluid (Sigma-Aldrich, Chinese Shanghai；MM, 125NaCl, 4.5KCl, 26NaHCO₃, 1.25NaH₂PO₄, 2CaCl₂, 1MgCl₂, 20 glucose, when with 95%O₂And 5%CO₂PH exposes brain 7.4) to cover when full Area.50 microgram Cal-520AM (AAT Bioquest.CA, USA) are dissolved in 4 μ lDMSO (F-127 containing 20%Pluronic) In, and manually dilution (mM, 150NaCl, 2.5KCl, 10Hepes, pH 7.432) is diluted to 500 μM.Use 2-3M Ω electricity The borosilicate pipette of resistance, by applying pressure pulse (1 minute, 400 millibars) by Ca²⁺Dye Injections are to primary vision skin In the layer 2/3 of layer.1 it is small when after, SR-101 is added drop-wise to cortex surface to identify astroglia.Biopsy sample head is consolidated It is fixed, and be imaged in random access mode using FIRM-TPM.

Finally, biopsy sample from fixator is discharged, and is put into the babinet 3 of the small animal imaging system of above-described embodiment In, tail-suspention test test is carried out to biopsy sample using miniature probe 2 or slides experiment test (cylindrical flat, 5cm², 5cm It is high) and Social behaviors (30 × 30cm²Square space for activities).

Step 3, the fluorescence signal is handled, obtains the 3-D view of the free activity of biopsy sample.

Before above-mentioned steps, following steps 4 are further included, biopsy sample is made, specifically includes：

Step 41, biopsy sample is chosen:C57BL/6 wild types and Thy-1-GFP transgenosis live body samples are used in an experiment This (after birth 8-16 weeks).All programs meet Peking University's animal use and the nursing committee and experimental animal Nursing evaluation With the standard of Certification Association, including animal breeding and experimental implementation.Thy-1-GFP transgenosis biopsy samples are anaesthetized and are used in combination Then 0.9% perfusion of saline is perfused with 4% paraformaldehyde, and takes out brain.It was fixed in the PBS of 4 DEG C of 4% paraformaldehyde Night.

Step 42, for the osteotomy of live body Cerebral cortex imaging：C57BL/6 wild type live body samples are used in these experiments This.In short, by sucking pure O₂In 1-1.5% isofluranes biopsy sample is anaesthetized, be placed in three-dimensional locating frame On (68025, RWD, China Shenzhen).Meanwhile use the normal temperature of (37.5-38 DEG C) holding biopsy sample of heating plate.In office After portion applies xylocaine and removes skin and muscle, at the middle part of target cortex, high speed cranium bores (tip diameter 0.5mm) Brill opens small square head window (0.5 × 0.5mm²).GCaMP-6f is opened with restructuring AAV in Calmodulin-Dependent kinases II (CaMK II) Mover (serotype 2/9；> 2×10¹³A genome copies/ml, by University of Pennsylvania's gene therapy procedure carrier core production It is raw) under express.Using syringe (Nanoliter 2010, World Precision Instruments, Sarasota, USA) and glass microelectrode, the AAV that total amount is 0.5-0.8 μ l was slowly injected into the targeting cortex of biopsy sample in 20 minutes 2/3 layer (depth be 130-400 μm).It, will be certainly with cyanoacrylate glue with isoflurane anesthesia biopsy sample after expression 3 weeks The stent of system is connected on skull and with tooth cement stabilization.Be drilled in cranium drilled out in target cortex small square head window (2.5 × 2.5mm²).Endocranium is carefully removed, by small-size glass coverslip (3.5 × 2.5mm²) be placed on out on cranium.Recover after a week, so Daily training living body sample adapts to head and fixes 30 minutes afterwards, continues 3 days.After clear-headed biopsy sample adapts to, operation step Rapid 1 and step 2.

It is last it is to be noted that：Above example is only to illustrate the technical solution of the utility model rather than it is limited System.It will be understood by those of ordinary skill in the art that：Can modify to the technical solution recorded in foregoing embodiments or Person carries out equivalent substitution to which part technical characteristic；These modifications are replaced, and take off the essence of appropriate technical solution Spirit and scope from various embodiments of the utility model technical solution.

Claims (8)

1. a kind of femtosecond pulse modulator, which is characterized in that there is negative dispersion light path and positive dispersion light path, wherein：

The negative dispersion light path includes laser input optical fibre, and the laser input optical fibre is used for transmission 920 nanometers of hundreds of milliwatts Femto-second laser pulse, the laser that pulse broadening prechirp has been compensated export；

The positive dispersion light path, between the femtosecond pulse laser and the negative dispersion light path, for compensating by institute State the caused negative dispersion in laser process is transmitted of laser input optical fibre.

2. femtosecond pulse modulator as described in claim 1, which is characterized in that the positive dispersion light path includes：

Dispersive compensation element closes on the femtosecond pulse laser and sets, passed for compensating by the laser input optical fibre Caused negative dispersion in defeated laser process；With

Acousto-optic modulator is used to receive the laser after compensating via the compensating element, and adjusts laser intensity, then exports To the scanning imagery part.

3. femtosecond pulse modulator as claimed in claim 2, which is characterized in that the positive dispersion light path further includes：

Laser azimuth adjusts component, is arranged between the dispersive compensation element and the acousto-optic modulator, the laser side Position adjustment component includes：

First half-wave plate for receiving the laser after being compensated via the compensating element, and adjusts laser polarization direction, so that The modulation efficiency highest of the acousto-optic modulator；

First speculum, for receiving the laser via first half-wave plate, and reflection laser, to adjust described in laser injection The position of acousto-optic modulator；With

Second speculum, for receiving the laser via first speculum, and reflection laser, to adjust described in laser injection The angle of acousto-optic modulator, and it is transferred to the acousto-optic modulator.

4. the femtosecond pulse modulator as any one of claim 2 to 3, which is characterized in that the countenance astigmatism Road further includes：

Spectrum groupware, closes on the laser input optical fibre and sets, and intensity is regulated via the acousto-optic modulator for receiving Laser and the laser is divided at least two beams, and be transferred to the laser input optical fibre.

5. femtosecond pulse modulator as claimed in claim 4, which is characterized in that the quantity of the laser input optical fibre is extremely It is less two, is respectively first laser input optical fibre and second laser input optical fibre；

The spectrum groupware includes：

Polarization beam apparatus regulates the laser of intensity via the acousto-optic modulator for reception and is divided into the laser at least Two beams are transferred to the first laser input optical fibre and second laser input optical fibre respectively；

Second half-wave plate is arranged between the polarization beam apparatus and acousto-optic modulator, for changing the polarization beam apparatus Splitting ratio；

3rd speculum is arranged between the polarization beam apparatus and the second half-wave plate, for reflection laser, to adjust laser Position and project on the plane of incidence of the polarization beam apparatus；

First collimation lens, be arranged in the first exit facet of the polarization beam apparatus and the first laser input optical fibre it Between, for the laser that receives polarization beam apparatus output and by laser coupled into the first laser input optical fibre；

3rd half-wave plate is arranged between the first exit facet of the polarization beam apparatus and first collimation lens, is used for The laser of the polarization beam apparatus output is received, and adjusts laser polarization direction so that laser coupled is defeated into the first laser Enter the efficiency highest of optical fiber；

4th speculum is arranged between the second exit facet of the polarization beam apparatus and the second laser input optical fibre, For receiving the laser of polarization beam apparatus output, for reflection laser, to adjust the position of laser and project described the In dual-laser input optical fibre；

Second collimation lens is arranged between the 4th speculum and the second laser input optical fibre, for receiving State the laser of polarization beam apparatus output and by laser coupled into the second laser input optical fibre；

4th half-wave plate is arranged between the 4th speculum and second collimation lens, for receiving the described 4th The laser of speculum reflection, and adjusts laser polarization direction so that laser coupled is into the efficiency of the second laser input optical fibre Highest.

6. a kind of miniature two-photon microscopic imaging device, which is characterized in that including：

Femtosecond pulse laser is used for the laser that generation wavelength is 920 nanometers；

Femtosecond pulse modulator is the femtosecond pulse modulator any one of claim 1 to 5, is used for The laser of the femtosecond pulse laser output is received, and prechirp compensates the pulse broadening of laser to preset value, and export；With And

Miniature probe, the miniature probe include：

Scanning imagery part, for receiving the laser of the femtosecond pulse modulator output, the laser is in biopsy sample The tissue in portion is scanned, the biopsy sample to be excited to generate fluorescence signal；With

Fluorescence output optical fibre, is used to receive the fluorescence signal of the scanning imagery part output, and is exported.

7. miniature two-photon microscopic imaging device as claimed in claim 6, which is characterized in that the scanning imagery part includes Micro electromechanical scanning instrument, object lens, scanning lens, collimator and dichroic mirror, wherein：

The micro electromechanical scanning instrument is used for will be by the laser of the collimator by way of rotating and changing laser light incident angle angle Two-dimensional scan is carried out to the tissue plane inside the biopsy sample；

The object lens are for the laser from the micro electromechanical scanning instrument to be converged to inside the biopsy sample, with described in excitation Biopsy sample generates the fluorescence signal；And for exporting the fluorescence signal；

The scanning lens is arranged in the light path between the micro electromechanical scanning instrument and eyepiece, for by the micro electromechanical scanning The laser of angle change changes into the laser of change in location caused by instrument two-dimensional scan；

The collimator arrangement is defeated for collimating the laser between the laser input optical fibre and the micro electromechanical scanning instrument Enter the aberration between that part exports and luminous and reduction different frequency laser, described in being matched jointly with the scanning lens The image values aperture of object lens；With

The dichroic mirror is located between the scanning lens and eyepiece, described in laser and fluorescence signal are separated and exported Fluorescence signal.

8. miniature two-photon microscopic imaging device as claimed in claim 7, which is characterized in that the fluorescence output optical fibre by 700-900 root glass optical fiber weldings form, and two ends of welding are in the cylinder of 1.5mm diameters.