Background technique
Animal has the characteristics that high sensitivity, damage are small in body fluoroscopic image technology, has evolved into living animal imaging
A kind of common method, be widely used in the fields such as life science, biomedicine and medicament research and development [V.Wagner,
A.Dullaart,A.Bock,A.Zweck,The emerging nanomedicine landscape.Nature
Biotechnology.2006,24,1211-1217.].Whole body optical imaging system is ground in biomedical, Physiological Analysis and drug
Have broad application prospects in many researchs in fields such as hair.However, living animal Imaging-PAM is also faced with many difficulties
Topic have it is to be solved, for example, under the irradiation of visible light/burst of ultraviolel light source, the tissues such as skin, hair of living animal can generate compared with
Strong autofluorescence, the Overlapping of fluorescence spectra of they and common label probe, causes signal-to-noise ratio to be greatly reduced, and seriously affects inspection
The sensitivity and accuracy of survey;On the other hand, different types of cell and tissue to the absorption of light with scattering power different, living body
The problems such as animal tissue's structure and body fluid motion conditions are complicated, result in decaying, the distorted signals of specific fluorescence signal.In addition,
Some virose probes should not use in the living body, and small-molecule fluorescent probe luminous intensity is lower, and luminosity is by environment
Influence it is significant, the problems such as photostability and not high enough chemical stability be also restrict the development of living body fluorescent imaging technique it is important because
Element.How to overcome the interference of living animal autofluorescence, increase investigation depth, improves the accuracy of acquired Biochemical Information, be
Challenging scientific and technical problem in the development of living body fluorescent image technology.
Conventional fluorescent imaging technique is visited using the specificity fluorescent in continuous or modulated light source excitation living tissue or cell
Needle, which shines, realizes fluorescence imaging.Tissue, cell and body fluid send out the background fluorescences such as the scattering of exciting light and autofluorescence and probe
Light mixes, and generates biggish interference to specific target target image.Generally by with the matched narrow-band-filter of fluorescence probe
Piece compacting exciting light scattering improves the sensitivity of living body fluorescent image analysing computer with background fluorescence.But in living animal body
When needing under big sounding mark using higher excitation light intensity, the interference of background fluorescence is difficult to overcome.Background fluorescence interference leads to figure
As signal-to-noise ratio decline, specific fluorescence signal will be fallen into oblivion when serious, constrains the application of living animal Imaging-PAM significantly.
Using exciting light scattering and the background fluorescences such as archebiosis fluorescence and fluorescence probe shine between luminescent lifetime difference, when realization
Between resolved fluorometric imaging be a good solution.Since the duration of exciting light scattering and excitation light pulse width are big
It causes quite, can be avoided by the detection technique that is delayed.But background fluorescence lifetime and most fluorescent probe molecule longevity
Life is in nanosecond order, and e.g., protein, pigment and most of fluorescent target target fluorescence lifetimes are usually no more than 20ns, and the service life is poor
Different smaller, the corresponding detection technique that is delayed is appropriate only for micro- service life imaging, it is difficult to quick, the big visual field applied to living animal
Imaging.
In recent years, it is based on the unique fluorescence spectrum of dye molecule, it is dry by comparing standard fluorescence spectrum removal background fluorescence
Disturb, developed spectrum split bioluminescence imaging technology, improve to a certain extent imaging accuracy and sensitivity [X.H.Gao,
Y.Y.Cui,R.M.Levenson,L.W.K.Chung,S.M.Nie,In vivo cancer targeting and imaging
with semiconductor quantum dots,Nature Biotechnology,2004,22,969-976.].But due to
It seems that the Image Mosaic for containing spectral information by multiframe forms that light spectrum image-forming or spectrum, which are split into, is highly dependent on the calculating of image
Analytic technique, when bias light is stronger, it is difficult to guarantee the reliability that probe feature spectrum is split.Biomolecule and common fluorescent dye
The emission spectrum of material is all very wide, serious overlapping each other, still has suitable difficulty in terms of the characteristic spectrum for accurately obtaining object.By
Between animal species, Different Individual and the reasons such as the difference of different tissues, spectrum split technology and can also produce in practical applications
Raw some illusions.Therefore, detection sensitivity and reliability are further increased using new background Xanthophyll cycle philosophy and technique
Key point.
There is the Rayleigh scattering and other forms being inversely proportional with excitation wavelength biquadratic in animal tissue and cell
Scattering, strong scattering and absorption make exciting light be difficult to effectively reach deep layer target, are stranded greatly very much to realizing that its fluorescence imaging is brought
It is difficult.Biological tissue, cell and body fluid scatter the light of 610-900nm wave band and absorb relatively small, thus the wave band is referred to as
The optical window of biological tissue excitation and detection.Two-photon fluorescence excitation generally uses the femtosecond that wavelength is 750-1000nm closely red
As excitation light source, fluorescence labeling probe absorbs two photons simultaneously and reaches excitation state outer laser.Due to two-photon excitation probability
Directly proportional to two power of excitation light intensity, the probability that the luminescent substance other than focus is excited is greatly lowered.However, animal is intracorporal
Cucumber can also generate very strong background fluorescence because of two-photon effect, and signal-to-noise ratio is caused to decline, and the research of the present inventor
Show that animal cuticle can generate very strong background fluorescence or phosphorescence under femtosecond laser excitation, the service life is also longer than common fluorescent
The fluorescence lifetime of dyestuff is difficult to obtain the accurate information of internal luminous marker using common two-photon excitation imaging mode.
On the other hand, existing two-photon confocal imaging technology is in single photon confocal imaging technology and femtosecond laser light source base
On plinth, developed for the Two-photon fluorescent dye of visible range.The technology is imaged using point by point scanning, thus is not suitable for
Living animal imaging under big visual field.Simultaneously because the operating mode of point by point scanning, under the limitation of imaging time, every work
Making the time must be nanosecond order, this makes such technology be dfficult to apply to the efficient two-photon excitation imaging analysis of living animal.
Nano drug-carrying body has become a kind of effective novel medicine feeding mode, and positive effect has been played in oncotherapy.So
And the performance of nano drug-carrying body depends on its composition, size, surface targeting molecular structure, advanced optimizes the property of nano drug-carrying body
Can, need it is quantitative understand its cancer target kinetic property in living animal body and the distribution in major organs with it is above-mentioned
Relationship between structure feature.Efficiently and accurately to obtain above- mentioned information, the present inventor is proposed using two-photon excitation delay inspection
Survey the method for fluorescence imaging to it is in injection living animal body, there are long luminescence lifetime and two-photon excitation and fluorescence emission wavelengths
The nano drug-carrying body analogue probe for being in biological optical transmission window (600-1000nm) (calls fluorescence nano in the following text and carries medicine body simulation spy
Needle) distribution and kinetic property carry out quantitative analysis method.Due in animal cuticle blood vessel or in-vivo tissue away from light source more
Close nano drug-carrying body analogue probe will be excited first, and under general illumination mode, as the depth of detection increases, laser
Power density can be substantially reduced, the decline of two-photon excitation luminous intensity, therefore (such as one-dimensional having a size of 1mm-10 lis in big visual field
Rice) under will to the nano drug-carrying body analogue probe in tumour in living animal body or certain certain organs carry out it is lossless or non-intervention
Formula fluorescence analysis needs to reduce or eliminate the fluorescence interference that the above-mentioned probe around skin or test serum, organ generates.
Summary of the invention
To solve the above problems, the present invention provide a kind of two-photon excitation delay detection fluorescence animal imaging analytical technology,
Equipment and application increase fluorescence nano in animal body and carry medicine body analogue probe imaging analysis depth, disappear to realize under big visual field
Except the interference of autofluorescence and bias light, while reducing the fluorescence nano in animal cuticle and target acquisition position surrounding tissue
The interference for carrying medicine body analogue probe fluorescence, improves the reliability of the imaging analysis.
Technical scheme is as follows:
Distribution, fluorescence intensity or the concentration and probe concentration of fluorescent nano probe be at any time in a kind of living animal in-vivo tissue and organ
Between change kinetics property fluorescence imaging analysis method, which is characterized in that swashed with feux rouges or near infrared pulsed laser two-photon
It sends out fluorescent nano probe internal and shines, the fluorescence at the position that is stimulated in vivo is detected in a manner of being delayed detection, is imaged.
In above-mentioned fluorescence imaging analysis method, the fluorescent nano probe can be glimmering in the intracorporal tissue of animal or organ
Light nano drug-carrying body analogue probe.By this method can to the distribution of fluorescent nano probe in animal body, fluorescence intensity or
Concentration and probe concentration changes over time kinetic property and is tested and analyzed.
The imaging viewing field at least one dimension size of fluorescence imaging analysis method of the invention is at 1 millimeter to 10 cm ranges
It is interior.It drives detection device to carry out the controllable scanning of excitation hot spot to imaging position by motorized precision translation stage, realizes big imaging view
?.
In fluorescence imaging analysis method of the invention, it is preferred that excited by feux rouges or near-infrared femtosecond pulse glimmering
Two-photon in light nano-probe is sensitized rare earth luminous complex and shines, the wavelength of the feux rouges or near-infrared femtosecond pulse
In 610-1100 nanometer range, more preferably 700-1000 nanometer range.The output general power of the femtosecond pulse is
10-10000mW, preferably 300-1000mW, pulse excitation frequency are 10Hz-10000Hz, preferably 100Hz-400Hz.
In fluorescence imaging analysis method of the invention, after excitation light pulse excitation, it is delayed using fluorescent probe and detects body
It being inside stimulated and the fluorescence at position is imaged, the delay is the time difference before opening after excitation light pulse excites to fluorescent probe,
The time difference is 10ns to 500 μ s;The time window of delay detection fluorescence is 10ns to 100ms, preferably 100ns to 20ms.
Above-mentioned fluorescent nano probe can have two-photon to be sensitized rare earth luminous performance, and the rare earth luminous wavelength is located at red
Light or near infrared region, preferred wavelength range are 600-1050 nanometers.The illuminator of the fluorescent nano probe is substantially by matrix
Material and the rare earth compounding composition that rare earth luminous performance is sensitized with two-photon, it is hydrocarbon that the host material, which is by main chain,
Chain, side group are the high-molecular compound that carboxyl and hydrophobic grouping are constituted;The rare earth compounding is the lower transmitting of near infrared light excitation
The rare earth compounding of visible light or near infrared light.
Preferably, the rare earth compounding be selected from one of compound shown in Formulas I, Formula II and formula III general structure or
It is a variety of;
In Formulas I, Formula II and formula III, La represents europium, ytterbium or neodymium ion;R1And R2Respectively stand alone as C1~C4 alkyl;R3、
R4、R5、R6、R7And R8Respectively stand alone as methyl or H.
The mass ratio of the host material and the rare earth compounding is 1~10,000: 1;Form the host material
The number-average molecular weight of high-molecular compound is 1,500~150,000;In the high-molecular compound, carboxylic group accounts for the height
The 0.01%~40% of molecular compound gross mass.It is Illuminant nanometer grain by the illuminator that host material and rare earth compounding form
Son, partial size are 5~200 nanometers.
Excitation light pulse excitation, delay fluorescence signal acquisition in fluorescence imaging analysis method of the present invention, signal repeatedly tire out
Product, sample movement are carried out automatically under sequence controller control.
Fluorescence imaging analysis method of the invention can accomplish nondestructive analysis, as exciting swashing for fluorescent nano probe
Light emitting source is located in vitro, and fluorescent probe also is located at external human blood glucose.Another situation is that the fluorescent nano probe
Light excitation and phosphor collection be in by light pipe insertion body and close to carrying out in a manner of detection position, before the light pipe
The distance at end position and detection position is 0.1 to 3 centimetre;The light pipe can be hollow-core fiber, liquid-core optical fibre and overlay film light
Fibre etc..
It excites in many cases, detection target site is located at animal skin hereinafter, needing to make the focus of exciting light to be located at
Animal skin is hereinafter, make the optical power density of exciting light focal point much larger than the optical power density at other positions;Preferably, make skin
The optical power density of the following exciting light focal point of skin is 2 to 1000 times of the optical power density at skin, it is furthermore preferred that being skin
10 to 1000 times of the optical power density at place.
Fluorescence imaging analysis method of the invention can be with the linear femtosecond pulse beam pair of the uniform flat field of power density
Living animal (injection fluorescent nano probe) is scanned excitation fluorescence imaging analysis;Fluorescence detection is controlled using sequence controller
The fluorescence signal of the fluorescent nano probe excited in device (such as ICCD) delay acquisition animal body by above-mentioned mode of excitation.
A kind of method is subcutaneously to be gathered with two linear femtosecond pulse beams of the uniform flat field of power density in living animal
Coke excites fluorescent nano probe in a manner of this laser beam flying at a linear pulse laser beam of flat field, utilizes timing control
The fluorescent nano probe excited in device control fluorescent probe (such as ICCD) delay acquisition animal body processed by above-mentioned mode of excitation
Fluorescence signal.
It is also possible that by endless belt-shaped femtosecond pulse beam enter under Animal Skin excited in a manner of detected part focuses it is glimmering
Light nano-probe, it is interior by above-mentioned mode of excitation using sequence controller control fluorescent probe (such as ICCD) delay acquisition animal body
The fluorescence signal of the fluorescent nano probe of excitation.
It, can also be to being excited in addition to acquiring the fluorescence signal at position of being stimulated in fluorescence imaging analysis method of the invention
The luminescence generated by light for sending out position carries out spectra collection, and the wavelength of the spectra collection is located at 600 nanometers to 1000 nanometer ranges.
Further, under sequence controller, using the linear focusing laser beam of flat field to being scanned excitation in animal body,
The photoluminescence spectra for the position that is respectively stimulated is acquired in a manner of the detection that is delayed by spectrometer and ICCD;Alternatively, in timing control
Under device, it is focused excitation in animal body using endless belt-shaped laser beam, by spectrometer and ICCD in a manner of being delayed detection
Acquire the photoluminescence spectra for the position that is respectively stimulated.
Fluorescence imaging analysis method of the invention can be applied to research nano drug-carrying body and be distributed, transports or swell in animal body
In terms of tumor targets kinetic property, (the letter of medicine body analogue probe is carried in the intravenous injection fluorescence nano of tumor animal or intact animal
Claim fluorescent nano probe) front and back, respectively to detected part carry out two-photon excitation time delayed spectroscopy acquisition testing, with injection described in
Spectrum before fluorescent nano probe is background, using spectrum subtraction method, obtains animal after injecting the fluorescent nano probe
The kinetic property that the spectral signal and the signal of internal tested position fluorescent nano probe change over time.
The research to target-oriented drug can also be realized by following steps using fluorescence imaging analysis method of the invention:
1) medicine body analogue probe is carried to animals iv injection fluorescence nano;
2) it is shone with the probe in a normal non-internal organs position blood vessel in femtosecond pulse excitation animal body, with
The mode of time delayed signal acquisition records the position fluorescence intensity and changes with time;
3) having in medicine body targeting target site is contained with femtosecond pulse excitation animal body and carries medicine body targeting
The probe shines, and records the target site fluorescence intensity in a manner of time delayed signal acquisition and changes with time;
4) with equation Y=A1e-t/τ1+ C fit procedure 2) data that decay at any time of middle probe luminous intensity;
5) with equation Y=Aae-t/τ1+Abe-t/τ2+ B fit procedure 3) data that decay at any time of middle probe luminous intensity, with
Ab/(Aa+Ab) the load medicine body targeting of the probe is characterized, with 1/ τ 1 characterization because immune system removing or non-specific adsorption are led
It causes the probe in the concentration fall off rate at detection position, the probe is characterized in targeting moiety mean residence time with τ 2;Its
Middle C and B is constant or the function of time.
The present invention also provides a kind of for implementing the fluorescence imaging analysis equipment of above-mentioned fluorescence imaging analysis method, mainly
Component includes femto-second laser, fluorescent probe, sequence controller, light path control system and divides for space optical power density
The optical field distribution device of cloth regulation, wherein the tune of feux rouges or near infrared pulsed laser through optical field distribution device that femto-second laser issues
It after section and focusing, projects in the intracorporal detected part of animal, excites under the control of sequence controller and light path control system
The fluorescent nano probe of detected part shines, and then passes through fluorescence detection under the control of sequence controller and light path control system
Device carries out delay detection, and the excitation can be two-photon or multiphoton excitation, the delay when a length of 10 nanosecond to 500 micro-
Second.
Further, above-mentioned fluorescence imaging analysis equipment further includes for realizing the shifting of light source, sample and/or fluorescent probe
Dynamic motion platform.
The fluorescent probe generallys use ICCD detector, further can also include imaging spectrometer.
Fluorescence imaging analysis equipment utilization optical field distribution device of the invention can with spatial light field Energy distribution by femtosecond laser
Prosecutor formula will be put or linear excitation beam formula projects in the intracorporal detected part of animal.
The optical field distribution device is substantially made of three parts, to complete required optical field distribution regulatory function: to excitation
What light was expanded expands part, and the light field for carrying out beam shape control controls diaphragm, and the laser beam that will be adjusted
The focusing block of focusing.
Wherein, part is expanded to be extender lens group or expand reflection at least one of microscope group;The position of light field control diaphragm
Set can be located at extender lens group or reflect microscope group eyeglass between, or positioned at expand part and focusing block between, or be located at light
Side after beam focusing block;Focusing block is at least one in lens, lens group, reflection focus lamp or reflection focusing microscope group
Kind.
Preferably, the combination of the component of the optical field distribution device is as shown in Figure 1, wherein concavees lens 1-1 and convex lens
1-2 collectively constitutes extender lens group, and light field control diaphragm 1-3 is located between extender lens group two panels lens, and what is be adjusted swashs
Light light beam forms linear excitation hot spot after being focused by cylindrical convex lens 1-4.Optical field distribution device optical component aggregate (including expand
The cylindrical convex lens of beam lens group, light field control diaphragm and focusing) it is placed on electronic control translation stage 1-5, it can be described electronic
The controllable scanning of excitation hot spot is carried out under the driving of translation stage 1-5 to imaging section position to be measured.
The light field control diaphragm can be at least one of curved shape diaphragm, band-like diaphragm, the band-like diaphragm packet
Include strip-form diaphragm and annulus shape diaphragm.
The light field control mechanism of diaphragm schematic diagram is as shown in Figure 2, in which: a is curved shape diaphragm;B is strip-form diaphragm;
C is annulus shape diaphragm.The light of the curved shape diaphragm penetrates or reflection region is two shaped forms and two linear type frames
The shape in the region surrounded, the curve can be linear flat according to optical power density is changed in the femtosecond laser beam of Gaussian Profile
The needs of field femtosecond laser beam, which calculate, to be obtained.The strip-form diaphragm is saturating by prevention (or decrease) light with two straight flanges
Cross or light reflection region by above-mentioned curved shape diaphragm be separated into two with light penetrate or light reflective properties banded zones;Institute
The light for stating annulus shape diaphragm penetrates or reflection region is band-like in a ring, and the region in inner ring is to prevent or weaken light transmission or light
The region of reflection.The equipment can control excitation laser and form dotted or linear focusing in sample interior.In annulus shape light field
Under the action of controlling diaphragm (Fig. 2 c), laser is improved in focal spot and far from the power density difference of focal spot.In curve
It is under the action of shape light field controls diaphragm, energy density is equal at energy density in the laser beam transformation of Gaussian function formal distribution
The transmission region of even (flat field) linear focus on light beam, the diaphragm has curved shape and linear frame (Fig. 2 a), wherein curve
The shape of shape can carry out retrospectively calculate acquisition by the line focus optical field distribution strength relationship to Gaussian function.In strip-form light
Under the action of field control diaphragm, energy is formed to the linear focusing light of flat field in the laser beam transformation of Gaussian function formal distribution
Beam;And intermediate strap shape shelter to light beam block effect under, improve laser at focal line and far from focal line
Power density difference (Fig. 2 b).
Can by allow and obstruct light by realizing the control to light field, as transmission diaphragm;It can also pass through
Allow and obstructs the reflection mode of light to realize the control to light field, as reflection diaphragm.
Light field control diaphragm (transmission diaphragm) when working in the transmission mode, obstruct light part can be it is opaque
Material be made, exist in the form of independent self-supporting, the hollow form realization in the part of light transmission;The edge of light transmission and barrier light
Place is with a thickness of 500 nanometers to 1 millimeter;Opaque or translucency can also be plated or pasted by being coated on transparent substrate surface
The substance of difference block being formed to light.
The substance that the opaque or translucency of the light field control diaphragm is poor is metal, macromolecule and the high score containing dyestuff
At least one of son, the translucency matrix include one of quartz, glass, macromolecule resin.
When the light field control diaphragm works in this reflection mode (reflection diaphragm), allow the reflection of light can be by transparent
There is the coating of specific reflection ability to realize for plating in substrate, and the part for not plating reflecting layer can be prevented with plating anti-reflection film, blacking
Reflection or the modes such as additional blocking parts obstruct the reflection of light.
Fluorescence imaging analysis equipment of the invention, it is preferred that its respectively optically and mechanically connection type and knot between functional component
Structure schematic diagram is as shown in Fig. 3, Fig. 4 or Fig. 5;Connection type and structural schematic diagram be such as between its each mechanical, electronic control function component
Shown in Fig. 6.
Wherein, equipment shown in Fig. 3 includes the sample translation stage 3-3 for placing tested animal, light path control system
Including dichroscope 3-4, objective lens 3-5, steering reflection mirror 3-6, the first imaging lens group 3-7 and the second imaging lens group 3-
10, fluorescent probe includes imaging spectrometer 3-8, the first ICCD 3-9 and the 2nd ICCD 3-11, and optical field distribution device 3-2 contains
There is electronic control translation stage;Adjusting and focusing control of the laser 3-1 from femto-second laser through optical field distribution device 3-2 provided by the invention
The femtosecond excitation beam is formed after system after the reflection of dichroscope 3-4 and impinges upon tested animal on sample translation stage 3-3
Detected part on.The excitation beam scanning can excite portion to be measured under the driving of the electronic control translation stage of optical field distribution device 3-2
The different location of position;The position change shot between image field and sample drives sample by three-dimensional automatically controlled sample translation stage 3-3
Movement is to realize;The signal light transmission dichroscope 3-4 of generation is collected by objective lens 3-5.Steering reflection mirror 3-6 is for controlling letter
Number light leads to image analysing computer end (right side in figure) or spectral imaging analysis end (left side in figure).Image analysing computer end includes the second one-tenth
As lens group 3-10 and the 2nd ICCD detector 3-11;Spectral imaging analysis end then includes the first imaging lens group 3-7, imaging
Spectrometer 3-8 and the first ICCD detector 3-9.
Figure 4 and figure 5 respectively show equipment shown in Fig. 3, and image analysing computer end or spectral imaging analysis end Working mould is being used only
Optically and mechanically part and its connection structure under formula, in Fig. 4, the signal light transmission dichroscope 3-4 of generation is by objective lens
3-5 is collected, and is imaged in the picture plane of ICCD detector 3-11 after steering reflection mirror 3-6 and imaging lens group 3-10;?
In Fig. 5, the signal light transmission dichroscope 3-4 of generation is collected by objective lens 3-5, by steering reflection mirror 3-6 and imaging len
It is imaged at the entrance slit of imaging spectrometer 3-8 after group 3-7, by grating beam splitting formation spectral image by ICCD detector 3-
9 detections.
Fig. 6 describes the connection structure of the mechano-electronic control unit of present device (Fig. 3,4,5), femto-second laser output
Laser synchronization signal be delivered to delay time generator, when delay time generator controls femto-second laser pulse and ICCD acquisition fluorescence signal
Between between accurate delay relationship.The parameter set by computer to delay time generator and translation stage controller, can control
The specific location of the spatial position of imaging acquisition and femtosecond laser irradiation;By the parameter of computer settings, the on-off of shutter is determined
Be scheduled on equipment work in laser pulse before detector is acquired work proper time excitation sample, and setting when
Between the fluorescence signal of privileged site is collected in window.
Present device utilizes the delay time generator of PLC technology, control pulse excitation, delay fluorescence signal acquisition, letter
Number repeatedly accumulation, sample movement carried out automatically by preset program;The delay is 10ns to 10ms, and preferably 100ns is extremely
500ns.Analysis method and equipment of the invention can be applicable to measurement fluorescent nano probe or fluorescence nano carries medicine body analogue probe
Be distributed in animal body, transport or cancer target kinetic property in terms of.
Above-mentioned optical field distribution device and light field control diaphragm for the regulation of space conversion efficiency also belongs to the present invention
Protection scope.I mage analysis methods provided by the invention and equipment are in measurement fluorescent nano probe or fluorescence nano load medicine body mould
Quasi- probe is distributed in animal body, transport or cancer target kinetic property in terms of application also belong to protection model of the invention
It encloses.
Fluorescent nano probe analysis method and equipment in living animal body provided by the invention, overcome traditional in vivo animal
Penetration depth is small in fluorescence imaging analysis method, background fluorescence interference is big, is also easy to produce pseudomorphism, is difficult to internal fluorescent nano probe
The disadvantages of kinetic property carries out the analysis of accurate non-invasive quantitative is transported, there is big imaging analysis depth, high reliablity, detection spirit
The advantages that sensitivity is high quantitative analysis can inject the intracorporal fluorescent nano probe of animal in the way of hurtless measure by the technology
Transport kinetic property, the distribution of cancer target kinetic property and the fluorescent nano probe in vivo etc. it is accurate
Information (see embodiment 1) has significant application value in terms of the development of nanometer medicine, New Type of Diseases diagnostic techniques.Such as
Shown in embodiment 6, the process and apparatus of the present invention can carry out 100 fluorescent marker tumour cells below living animal epidermis clear
Chu's ground imaging analysis, and quantitative analysis can be carried out to the cell marked in animal body.
The present invention is additional not introducing by the provided optical field distribution device equipped with particular curve shape light field control diaphragm
It is in the round femtosecond pulse beam of gaussian-shape distribution by energy density in the case where dispersion (except dispersion caused by lens group)
It is shaped as the linear femtosecond pulse beam of flat field, this is that conventional interference type shaping device institute is irrealizable.The present invention makes flat field
Linear femtosecond laser beam is focused in the following detected part of living animal epidermis, is scanned two-photon excitation to detected part,
The epidermal tissue effectively reduced reasons for its use fluorescence under femtosecond laser excitation, while solving because of femtosecond laser gaussian-shape
Optical field distribution leads to excitation light power density unevenness, it is impossible to be used under big visual field accurately two-photon excitation delay detection fluorescence at
As the problem of quantitative analysis, the efficiency and reliability of the two-photon excitation imaging analysis of raising.The present invention is by being provided
Equipped with endless belt-shaped light field control diaphragm optical field distribution device, make femtosecond laser beam portion to be measured below living animal epidermis
Position focuses, and reduces interference of the fluorescent nano probe to analytical effect around animal cuticle and detected part, the analysis knot of raising
The reliability of fruit.
The present invention utilizes the long-term durability luminous probe of two-photon excitation efficient rare-earth, the distribution of delay fluorescence intensity, delay
The collaboration coupling effect of the multinomial technologies such as spectra collection parsing, realizes internal fluorescent nano probe or fluorescence nano load medicine body is defeated
The accurate quantitative analysis (referring to embodiment 1) of sport dynamics property and tumor tissues targeting binding kinetics property, this effect
It is that can not be expected and know from previously reported result.The present inventor's studies have shown that glimmering using existing commercially available living animal
Light imaging analysis equipment can not obtain accurate above- mentioned information.
Detailed description of the invention
Fig. 1 is a kind of schematic diagram of typical structure of optical field distribution device, in which: 1-1 is concavees lens, and 1-2 is convex lens, 1-
3 control diaphragm for light field, and 1-4 is cylindrical convex lens, and 1-5 is motorized precision translation stage.
Fig. 2 shows three kinds of forms of implementation of light field control diaphragm: a, curved shape diaphragm;B, strip-form diaphragm;C, annulus
Shape diaphragm.
Fig. 3 is two-photon excitation delay fluorescence detection living animal imaging analysis equipment schematic diagram, in which: 3-1 is femtosecond
Laser, 3-2 are optical field distribution device, and 3-3 is sample translation stage, and 3-4 is dichroscope, and 3-5 is objective lens, and 3-6 is to turn to reflection
Mirror, 3-7 are the first imaging lens group, and 3-8 is imaging spectrometer, and 3-9 is the first ICCD, and 3-10 is the second imaging lens group, 3-
11 be the 2nd ICCD.
Fig. 4 is the tactic pattern figure under two-photon excitation imaging pattern, in which: 3-1 is femtosecond laser, and 3-2 is light field point
Cloth device, 3-3 are sample translation stage, and 3-4 is dichroscope, and 3-5 is objective lens, and 3-6 is steering reflection mirror, and 3-10 is the second imaging
Lens group, 3-11 are the 2nd ICCD.
Fig. 5 is the tactic pattern figure under Two-photon excitation spectra imaging pattern, in which: 3-1 is femtosecond laser, and 3-2 is light
Field distribution device, 3-3 are sample translation stage, and 3-4 is dichroscope, and 3-5 is objective lens, and 3-6 is steering reflection mirror, 3-7 first
Imaging lens group, 3-8 are imaging spectrometer, and 3-9 is the first ICCD.
Fig. 6 is two-photon excitation delay fluorescence detection living animal imaging device entirety control structure schematic diagram, wherein 3-5
For objective lens, 3-3 is sample translation stage, and 3-7 is the first imaging lens group,.
Fig. 7 is in embodiment 1, and difference fluorescent nano probe fluorescence intensity changes over time curve in vivo, in which: a, Eu@
SMA-mPEG-RGD probe, illustration are enlarged drawing;B, Eu@SMA-Tf probe.
Fig. 8 is two-photon excitation delay fluorescence detection imaging and common double light in living animal imaging analysis in embodiment 2
Son excitation fluorescence imaging effect compares, in which: a, leg tumor-bearing mice light field figure;B, c is respectively before injecting fluorescent nano probe
Common Two-photon excitation fluorescence imaging result afterwards;D, e is respectively two-photon excitation delay fluorescence before and after injecting fluorescent nano probe
Detect imaging results;Scale is 2mm.
Fig. 9 shows i mage analysis methods investigation depth experimental result in embodiment 2, in which: a, adipose tissue simulated solution
(1%) fluorescence imaging;B is placed in the fluorescence imaging of the fluorescent nano probe under the adipose tissue simulated solution (1%) of 5mm thickness;c,
It is placed in the fluorescence imaging of the fluorescent nano probe under the adipose tissue simulated solution (1%) of 7mm thickness.
Figure 10 shows in embodiment 4, femtosecond laser optical field distribution regulating effect, wherein a, on perpendicular to beam direction
Incident femtosecond pulse conversion efficiency figure;B, the femtosecond pulse linear laser optical power after optical field distribution device shaping are close
Distribution map is spent, dotted line is the optical field distribution figure that line focus is directly carried out using cylindrical lens, and solid line is to control light through curved shape light field
The optical field distribution figure after cylindrical lens line focus is carried out after door screen again.
Figure 11 shows the fuel factor in embodiment 5 in two-photon excitation delay fluorescence detection imaging analysis.