CN105158224B - A kind of method and system for improving multi-photon imaging signal intensity - Google Patents
A kind of method and system for improving multi-photon imaging signal intensity Download PDFInfo
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Abstract
The present invention is applied to bio-photon field there is provided a kind of method for improving multi-photon imaging signal intensity, including：Obtain the signal intensity of multi-photon imaging；In object lens, strengthen the signal intensity of the multi-photon imaging by changing object lens fill factor, curve factor.Present invention also offers a kind of system for improving multi-photon imaging signal intensity.The present invention can improve maximized signal intensity in deep layer biological tissue high-order multi-photon micro-imaging, so as to obtain more big imaging depth.
Description
Technical field
The present invention relates to bio-photon field, more particularly to a kind of method for improving multi-photon imaging signal intensity and it is
System.
Background technology
Multi-photon micro-imaging (i.e. multi-photon is imaged) technology can carry out the deep tissues of non-intrusion type to living body biological
Imaging, with subcellular fraction resolution and three-dimensional imaging ability, is mainly used in the fields such as neurology, embryology, oncology
Researcher can in organism or vitro tissue form and physiological structure carry out visible.
Multi-photon imaging technique needs n (n is more than or equal to 2, and n is integer) individual photon to excite sample simultaneously, then launches
Go out a fluorescent photon signal, such as two-photon micro-imaging technique is a kind of the most commonly used multi-photon imaging technique, however,
Using the technology, its imaging depth most depth is 1mm for the intravital mouse Brian Imaging of uniform labelling, because should at this
The signal of depth focal point is flooded (i.e. its signal to background ratio is equal to 1) by reasons for its use fluorescence at surface, that is to say, that at this
Its signal run out of under depth.
At present, the signal tcam-exhaustion under big imaging depth limits maximum imaging depth.Therefore, how further to increase
Signal intensity is exactly that industry needs improved target badly all the time to realize the further imaging depth that improves.
The content of the invention
In view of this, the purpose of the embodiment of the present invention be to provide a kind of method for improving multi-photon imaging signal intensity and
System, it is intended to solve in the prior art due to exist under big imaging depth signal exhaust and lead to not further improve imaging
The problem of depth.
The embodiment of the present invention is achieved in that a kind of method for improving multi-photon imaging signal intensity, including：
Obtain the signal intensity of multi-photon imaging；
In object lens, strengthen the signal intensity of the multi-photon imaging by changing object lens fill factor, curve factor.
It is preferred that, the multi-photon imaging includes three-photon imaging and four photon imagings.
It is preferred that, it is described obtain multi-photon imaging signal intensity the step of include：
Using numerical simulation mode, the signal intensity that respectively obtains the three-photon imaging is calculated by below equation and described
The signal intensity of four photon imagings：
Wherein, S3, S4 represent the signal intensity of the three-photon imaging and the signal intensity of four photon imagings respectively,The light distribution in cylindrical coordinates is represented, V is represented to volume integral.
It is preferred that, described in object lens, the signal for strengthening the multi-photon imaging by changing object lens fill factor, curve factor is strong
The step of spending includes：
In object lens, under different type in-field keep object lens after power invariability, and by change object lens filling because
Son calculates intensity distribution of the offline polarized incident light in different type in-field near focal point, wherein, the different type enters
Penetrating field includes plane wave and Gaussian beam；
Using the offline polarized incident light in different type in-field plane wave is obtained in the intensity distribution of near focal point most
The optimal fill factor, curve factor of excellent fill factor, curve factor or Gaussian beam；And
Strengthen the signal intensity of the multi-photon imaging according to the optimal fill factor, curve factor under different type in-field.
It is preferred that, the linear polarization incident light of the plane wave is specifically calculated in the intensity distribution of near focal point by below equation
Obtain：
The linear polarization incident light of the Gaussian beam is obtained in specific calculated by below equation of intensity distribution of near focal point：
Wherein, Einc(α) represents linear polarization incident light in the intensity distribution of near focal point, ldepthRepresent imaging depth, leWith
Exciting light feature attenuation length is represented, β represents the fill factor, curve factor of object lens filling extent, αmaxThe angular integral upper limit is represented, α represents light
The angle of line and optical axis.
On the other hand, the present invention also provides a kind of system for improving multi-photon imaging signal intensity, including：
Signal acquisition module, the signal intensity for obtaining multi-photon imaging；
Signal enhancing module, in object lens, strengthening the multi-photon imaging by changing object lens fill factor, curve factor
Signal intensity.
It is preferred that, the multi-photon imaging includes three-photon imaging and four photon imagings.
It is preferred that, the signal acquisition module, specifically for utilizing numerical simulation mode, is calculated by below equation and obtained respectively
The signal intensity and the signal intensity of four photon imaging being imaged to the three-photon：
Wherein, S3, S4 represent the signal intensity of the three-photon imaging and the signal intensity of four photon imagings respectively,The light distribution in cylindrical coordinates is represented, V is represented to volume integral.
It is preferred that, the signal enhancing module is specifically included：
Calculating sub module, in object lens, the power invariability after object lens being kept under different type in-field, and pass through
Change object lens fill factor, curve factor to calculate intensity distribution of the offline polarized incident light in different type in-field near focal point, wherein,
The different type in-field includes plane wave and Gaussian beam；
Select submodule, for using the offline polarized incident light in different type in-field near focal point intensity distribution come
Obtain the optimal fill factor, curve factor of plane wave or the optimal fill factor, curve factor of Gaussian beam；And
Strengthen submodule, for strengthening the multi-photon imaging according to the optimal fill factor, curve factor under different type in-field
Signal intensity.
It is preferred that, the linear polarization incident light of the plane wave is specifically calculated in the intensity distribution of near focal point by below equation
Obtain：
The linear polarization incident light of the Gaussian beam is obtained in specific calculated by below equation of intensity distribution of near focal point：
Wherein, Einc(α) represents linear polarization incident light in the intensity distribution of near focal point, ldepthRepresent imaging depth, leWith
Exciting light feature attenuation length is represented, β represents the fill factor, curve factor of object lens filling extent, αmaxThe angular integral upper limit is represented, α represents light
The angle of line and optical axis.
The present invention is demonstrated in different l by numerical simulationdepth/leThere is optimal laser beam fill factor, curve factor under ratio,
In object lens, strengthen the signal intensity of multi-photon imaging by changing object lens fill factor, curve factor, and then it is deep further to improve imaging
Degree.
Brief description of the drawings
Fig. 1 is the method flow diagram of raising multi-photon imaging signal intensity in an embodiment of the present invention；
The detailed substeps flow chart that Fig. 2 is step S12 shown in Fig. 1 in an embodiment of the present invention；
Fig. 3 is the signal intensity and fill factor, curve factor that multi-photon is imaged in plane wave illumination in an embodiment of the present invention
Emulation testing comparison diagram；
Fig. 4 be an embodiment of the present invention in when Gaussian beam irradiate multi-photon imaging signal intensity and fill factor, curve factor
Emulation testing comparison diagram；
Fig. 5 is different in ratio with Gaussian beam irradiation in plane wave illumination respectively in an embodiment of the present invention
ldepth/leUnder the conditions of optimal fill factor, curve factor changing trend diagram；
Fig. 6 is the system structure diagram of raising multi-photon imaging signal intensity in an embodiment of the present invention；
Fig. 7 is the internal structure schematic diagram of signal enhancing module 12 shown in Fig. 6 in an embodiment of the present invention.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The specific embodiment of the invention provides a kind of method for improving multi-photon imaging signal intensity, mainly includes as follows
Step：
S11, the signal intensity for obtaining multi-photon imaging；
S12, in object lens, strengthen the signal intensity of multi-photon imaging by changing object lens fill factor, curve factor.
A kind of method for improving multi-photon imaging signal intensity provided by the present invention, is demonstrated not by numerical simulation
Same ldepth/leThere is optimal laser beam fill factor, curve factor under ratio, it is many to strengthen by changing object lens fill factor, curve factor in object lens
The signal intensity of photon imaging, and then further improve imaging depth.In the present embodiment, the raising multi-photon imaging signal
The method of intensity can apply to each place in bio-photon field, for example, can apply in scattering and organism-absorbing sample
In.
A kind of method for improving multi-photon imaging signal intensity provided by the present invention will be described in detail below.
Referring to Fig. 1, the method flow diagram to improve multi-photon imaging signal intensity in an embodiment of the present invention.
In step s 11, the signal intensity of multi-photon imaging is obtained.
In the present embodiment, the multi-photon imaging includes three-photon imaging and four photon imagings, still, described many
Photon imaging is not limited to three-photon imaging and four photon imagings, in the present embodiment, and the multi-photon imaging can also be wrapped
Include the n-photon imaging (n=5 or 6 or 7 or 8 or 9 ..., etc.) of higher order, such as five photon imagings, six light
Sub- imaging, seven photon imagings ..., etc. the like.In the present embodiment, 1700nm wave bands three-photon fluorescent micro-imaging
Technology (i.e. three-photon is imaged) can break through this imaging depth limitation of 1mm in the prior art, and compared with its all band, this three
Photon imaging technology reduces the decay of exciting light, and is high-order nonlinear technology because three-photon is excited, therefore its signal
Background ratio is lifted, and for example the present invention obtains the imaging depth more than 1.4mm in intravital mouse brain, in adult mice
Lamina albae is passed through in brain and its imaging depth reaches hippocampus, moreover, in identical wave band, the present invention have also been demonstrated than three
The four photon fluorescence micro-imaging techniques (i.e. four photon imagings) of photon higher order, four photon imagings have more than three-photon imaging
High signal to background ratio, and be imaged available for conventional green fluorescent protein.
In the present embodiment, the step S11 of the signal intensity for obtaining multi-photon imaging is specifically included：
Using numerical simulation mode, the signal intensity that respectively obtains the three-photon imaging is calculated by below equation and described
The signal intensity of four photon imagings：
Wherein, S3, S4 represent the signal intensity of the three-photon imaging and the signal intensity of four photon imagings respectively,The light distribution in cylindrical coordinates is represented, V is represented to volume integral.
In the present embodiment, above-mentioned formula (1) and (2) are to eliminate incoherent constant.
In step s 12, in object lens, the signal for strengthening the multi-photon imaging by changing object lens fill factor, curve factor is strong
Degree.In the present embodiment, the object lens are a kind of high-NA objectives, and in the present embodiment, numerical aperture is more than
Or high-NA can be referred to as equal to 0.9.
Step S12 specifically includes these three sub-steps of step S121-S123, as shown in Figure 2.
Referring to Fig. 2, shown in Fig. 1 in an embodiment of the present invention step S12 detailed substeps flow chart.
In step S121, in object lens, the power invariability after object lens is kept under different type in-field, and by changing
Become object lens fill factor, curve factor to calculate intensity distribution of the offline polarized incident light in different type in-field near focal point, wherein, institute
Stating different type in-field includes plane wave and Gaussian beam.
In the present embodiment, after incoherent constant is omitted, the vector of high numerical aperture (NA) objective focal length is electric
Field distribution is obtained by below equation (3)-(6)：
Wherein, Einc(α) represents intensity distribution of the linear polarization incident light near focal point, and ρ represents radial distance,Represent
Angle in master coordinate system, z represents axial distance, and k represents the wave vector in water, αmaxRepresent the angular integral upper limit, α represent light with
The angle of optical axis.
In the present embodiment, the linear polarization incident light of the plane wave is specific by following in the intensity distribution of near focal point
Formula (7) is calculated and obtained：
The linear polarization incident light of the Gaussian beam is specifically calculated in the intensity distribution of near focal point by below equation (8)
Arrive：
Wherein, Einc(α) represents linear polarization incident light in the intensity distribution of near focal point, ldepthRepresent imaging depth, leWith
Exciting light feature attenuation length is represented, β represents the fill factor, curve factor of object lens filling extent, αmaxThe angular integral upper limit is represented, α represents light
The angle of line and optical axis.
In step S122, obtained using the offline polarized incident light in different type in-field in the intensity distribution of near focal point
Take the optimal fill factor, curve factor of plane wave or the optimal fill factor, curve factor of Gaussian beam.
In step S123, strengthen the multi-photon imaging according to the optimal fill factor, curve factor under different type in-field
Signal intensity.
Referring to Fig. 3, in an embodiment of the present invention in plane wave illumination multi-photon be imaged signal intensity with filling out
Fill the emulation testing comparison diagram of the factor.
In the present embodiment, the present invention has the intensity of decay and undamped lower multi-photon imaging signal with filling out by contrast
Fill the change of the factor to illustrate the method that there is optimal filling, as shown in figure 3, two curves of arrow to the left are represented flat in figure
Face ripple irradiates arrow in the intensity and the variation tendency of fill factor, curve factor of photon imaging signal in lower and zero-decrement excitation beam, figure
Two curves to the right represent under plane wave illumination and there is the intensity of photon imaging signal in the excitation beam of decay with filling out
Fill the variation tendency of the factor.
As shown in figure 3, under plane wave illumination and in zero-decrement excitation beam, three-photon imaging signal S3With four photons
Imaging signal S4(i.e. β=1) has reached maximum in the case of maximum filling, and this just illustrates to be imaged in undamped lower multi-photon
There is optimal filling with the change of fill factor, curve factor in the intensity of signal.
As shown in figure 3, under plane wave illumination and existing in the excitation beam decayed, three-photon imaging signal S3With four light
Sub- imaging signal S4Peak turns to less fill factor (i.e. in figure shown in two curves of arrow right), for example,
In ldepth/leWhen=3.82, three-photon imaging signal S3With four photon imaging signal S4Optimal fill factor, curve factor (i.e. βopt) respectively
For 0.68 and 0.79, at this moment, for le=365 μm, corresponding imaging depth ldepthFor 1.4mm, this is equal to 1 with full packing β
Situation compares, S3And S4Be promoted to original 1.7 times and 1.5 times respectively, it was demonstrated that for three-photon imaging and four photons into
Picture, it is to avoid being filled up completely with for object lens dorsal pore footpath can strengthen signal intensity really.
Referring to Fig. 4, in an embodiment of the present invention when Gaussian beam is irradiated multi-photon be imaged signal intensity with
The emulation testing comparison diagram of fill factor, curve factor.
In the present embodiment, the present invention has the intensity of decay and undamped lower multi-photon imaging signal with filling out by contrast
Fill the change of the factor to illustrate the method that there is optimal filling, as shown in figure 4, two curves of arrow to the left are represented in height in figure
This light beam irradiates arrow in the intensity and the variation tendency of fill factor, curve factor of photon imaging signal in lower and zero-decrement excitation beam, figure
Two curves of head to the right represent in the case where Gaussian beam is irradiated and there is the intensity of photon imaging signal in the excitation beam of decay
With the variation tendency of fill factor, curve factor.
As shown in figure 4, under Gaussian beam irradiation and in zero-decrement excitation beam, three-photon imaging signal S3With four light
Sub- imaging signal S4Occur slight decline after reaching a maximum value, and with β increase, its respective signal intensity tends to one
The value of individual fixation, because Gaussian beam irradiation is intended to plane wave illumination under maximum conditions (i.e. β → ∞).Although such as
This, (the i.e. l in the case where there is decaydepth/le=3.82), however it remains make three-photon imaging signal S3With four photon imagings letter
Number S4The optimal fill factor, curve factor (i.e. in figure shown in two curves of arrow right) of maximum intensity value is reached, this feelings with plane wave
Condition is approximate, for three-photon imaging signal S3With four photon imaging signal S4For, corresponding optimal fill factor, curve factor βoptIt is not divided into
0.62 and 0.73.
Referring to Fig. 5, in an embodiment of the present invention respectively in plane wave illumination and Gaussian beam irradiation in ratio
Different ldepth/leUnder the conditions of optimal fill factor, curve factor changing trend diagram.
As shown in figure 5, whether under plane wave either Gaussian beam irradiation, for three-photon imaging signal S3With four light
Sub- imaging signal S4, with ldepth/leRatio increase, the light of more wide-angles is attenuated, it is therefore desirable to smaller filling
The factor come avoid excessive loss so that signal maximize, for example, for ldepth/le=7.63 (i.e. le=365 μm corresponding
Imaging depth ldepth=2.79mm), for S under plane wave illumination3, optimal fill factor, curve factor βoptOnly it is merely undamped for 0.52
Under the conditions of (i.e. β=1) when half or so.But, the S calculated in the case of optimal filling3And S4Respectively original 4.66
Again with 4.51 times, the numerical value in the case of maximum filling (β=1) is above.
Table 1 below shown in plane wave illumination, different ldepth/leUnder the conditions of ratio, under the conditions of optimal fill factor, curve factor
The ratio of signal and signal when being filled up completely with, this absolutely proves that optimal filling is favorably improved deep layer biological tissue high-order multi-photon
Imaging signal intensity.
Table 1
Above-mentioned table 1 is in different ldepth/leUnder ratio, S is obtained using optimal fill factor, curve factor method3And S4Signal with it is complete
Ratio relation during full packing (i.e. β=1).
A kind of method for improving multi-photon imaging signal intensity provided by the present invention, is demonstrated not by numerical simulation
Same ldepth/leThere is optimal laser beam fill factor, curve factor under ratio, it is many to strengthen by changing object lens fill factor, curve factor in object lens
The signal intensity of photon imaging, and then further improve imaging depth.
The specific embodiment of the invention also provides a kind of system 10 for improving multi-photon imaging signal intensity, mainly includes：
Signal acquisition module 11, the signal intensity for obtaining multi-photon imaging；
Signal enhancing module 12, in object lens, strengthening the multi-photon imaging by changing object lens fill factor, curve factor
Signal intensity.
A kind of system 10 for improving multi-photon imaging signal intensity provided by the present invention, is demonstrated by numerical simulation
Different ldepth/leThere is optimal laser beam fill factor, curve factor under ratio, in object lens, strengthened by changing object lens fill factor, curve factor
The signal intensity of multi-photon imaging, and then further improve imaging depth.
Referring to Fig. 6, showing the knot for the system 10 that multi-photon imaging signal intensity is improved in an embodiment of the present invention
Structure schematic diagram.In the present embodiment, improve multi-photon imaging signal intensity system 10 include signal acquisition module 11 and
Signal enhancing module 12.
Signal acquisition module 11, the signal intensity for obtaining multi-photon imaging.
In the present embodiment, the multi-photon imaging includes three-photon imaging and four photon imagings, still, described many
Photon imaging is not limited to three-photon imaging and four photon imagings, in the present embodiment, and the multi-photon imaging can also be wrapped
Include the n-photon imaging (n=5 or 6 or 7 or 8 or 9 ..., etc.) of higher order, such as five photon imagings, six light
Sub- imaging, seven photon imagings ..., etc. the like.
In the present embodiment, the signal acquisition module 11, specifically for utilizing numerical simulation mode, by below equation
Calculate the signal intensity for respectively obtaining the three-photon imaging and the signal intensity of four photon imaging：
Wherein, S3, S4 represent the signal intensity of the three-photon imaging and the signal intensity of four photon imagings respectively,The light distribution in cylindrical coordinates is represented, V is represented to volume integral.
In the present embodiment, above-mentioned formula (1) and (2) are to eliminate incoherent constant.
Signal enhancing module 12, in object lens, strengthening the multi-photon imaging by changing object lens fill factor, curve factor
Signal intensity.In the present embodiment, the object lens are a kind of high-NA objectives, and in the present embodiment, numerical value
Aperture, which is more than or equal to 0.9, can be referred to as high-NA.
In the present embodiment, signal enhancing module 12 specifically include calculating sub module 121, selection submodule 122 and
Strengthen submodule 123, as shown in Figure 7.
Referring to Fig. 7, showing in an embodiment of the present invention the internal structure signal of signal enhancing module shown in Fig. 6 12
Figure.
Calculating sub module 121, in object lens, keeping the power invariability after object lens under different type in-field, and
Intensity distribution of the offline polarized incident light in different type in-field near focal point is calculated by changing object lens fill factor, curve factor, its
In, the different type in-field includes plane wave and Gaussian beam；
In the present embodiment, after incoherent constant is omitted, the vector of high numerical aperture (NA) objective focal length is electric
Field distribution is obtained by below equation (3)-(6)：
Wherein, Einc(α) represents intensity distribution of the linear polarization incident light near focal point, and ρ represents radial distance,Represent
Angle in master coordinate system, z represents axial distance, and k represents the wave vector in water, αmaxRepresent the angular integral upper limit, α represent light with
The angle of optical axis.
In the present embodiment, the linear polarization incident light of the plane wave is specific by following in the intensity distribution of near focal point
Formula (7) is calculated and obtained：
The linear polarization incident light of the Gaussian beam is specifically calculated in the intensity distribution of near focal point by below equation (8)
Arrive：
Wherein, Einc(α) represents linear polarization incident light in the intensity distribution of near focal point, ldepthRepresent imaging depth, leWith
Exciting light feature attenuation length is represented, β represents the fill factor, curve factor of object lens filling extent, αmaxThe angular integral upper limit is represented, α represents light
The angle of line and optical axis.
Submodule 122 is selected, is divided for the intensity using the offline polarized incident light in different type in-field near focal point
Cloth obtains the optimal fill factor, curve factor of plane wave or the optimal fill factor, curve factor of Gaussian beam.
Strengthen submodule 123, for strengthening the multi-photon according to the optimal fill factor, curve factor under different type in-field
The signal intensity of imaging.
A kind of system 10 for improving multi-photon imaging signal intensity provided by the present invention, is demonstrated by numerical simulation
Different ldepth/leThere is optimal laser beam fill factor, curve factor under ratio, in object lens, strengthened by changing object lens fill factor, curve factor
The signal intensity of multi-photon imaging, and then further improve imaging depth.
In embodiments of the present invention, the technical scheme that the present invention is provided, is demonstrated in different l by numerical simulationdepth/le
There is optimal laser beam fill factor, curve factor under ratio, in object lens, strengthen multi-photon imaging by changing object lens fill factor, curve factor
Signal intensity, and then further improve imaging depth.
It is worth noting that, in above-described embodiment, included unit is simply divided according to function logic,
But above-mentioned division is not limited to, as long as corresponding function can be realized；In addition, the specific name of each functional unit
Only to facilitate mutually distinguishing, the protection domain being not intended to limit the invention.
In addition, one of ordinary skill in the art will appreciate that realizing all or part of step in the various embodiments described above method
It can be by program to instruct the hardware of correlation to complete, corresponding program can be stored in embodied on computer readable storage Jie
In matter, described storage medium, such as ROM/RAM, disk or CD.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Claims (8)
1. a kind of method for improving multi-photon imaging signal intensity, it is characterised in that methods described includes：
Obtain the signal intensity of multi-photon imaging；
In object lens, strengthen the signal intensity of the multi-photon imaging by changing object lens fill factor, curve factor；
Wherein, it is described in object lens, strengthen the step of the signal intensity of the multi-photon imaging by changing object lens fill factor, curve factor
Suddenly include：
In object lens, under different type in-field keep object lens after power invariability, and by change object lens fill factor, curve factor come
Intensity distribution of the offline polarized incident light in different type in-field near focal point is calculated, wherein, the different type in-field
For plane wave or Gaussian beam；
Filled out using the offline polarized incident light in different type in-field in the intensity distribution of near focal point to obtain the optimal of plane wave
Fill the optimal fill factor, curve factor of the factor or Gaussian beam；And
Strengthen the signal intensity of the multi-photon imaging according to the optimal fill factor, curve factor under different type in-field.
2. the method for multi-photon imaging signal intensity is improved as claimed in claim 1, it is characterised in that the multi-photon imaging
For three-photon imaging or four photon imagings.
3. the method for multi-photon imaging signal intensity is improved as claimed in claim 2, it is characterised in that the acquisition multi-photon
The step of signal intensity of imaging, includes：
Using numerical simulation mode, the signal intensity and four light for respectively obtaining the three-photon imaging are calculated by below equation
The signal intensity of son imaging：
Wherein, S3, S4 represent the signal intensity of the three-photon imaging and the signal intensity of four photon imagings respectively,The light distribution in cylindrical coordinates is represented, V is represented to volume integral.
4. the method for multi-photon imaging signal intensity is improved as claimed in claim 1, it is characterised in that the line of the plane wave
Polarized incident light is obtained in specific calculated by below equation of intensity distribution of near focal point：
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The linear polarization incident light of the Gaussian beam is obtained in specific calculated by below equation of intensity distribution of near focal point：
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</mrow>
Wherein, Einc(α) represents linear polarization incident light in the intensity distribution of near focal point, ldepthRepresent imaging depth, leWith expression
Exciting light feature attenuation length, β represents the fill factor, curve factor of object lens filling extent, αmaxRepresent the angular integral upper limit, α represent light with
The angle of optical axis.
5. a kind of system for improving multi-photon imaging signal intensity, it is characterised in that the raising multi-photon imaging signal intensity
System include：
Signal acquisition module, the signal intensity for obtaining multi-photon imaging；
Signal enhancing module, the signal in object lens, strengthening the multi-photon imaging by changing object lens fill factor, curve factor
Intensity；
Wherein, the signal enhancing module is specifically included：
Calculating sub module, in object lens, the power invariability after object lens to be kept under different type in-field, and by changing
Object lens fill factor, curve factor calculates intensity distribution of the offline polarized incident light in different type in-field near focal point, wherein, it is described
Different type in-field is plane wave or Gaussian beam；
Submodule is selected, for being obtained using the offline polarized incident light in different type in-field in the intensity distribution of near focal point
The optimal fill factor, curve factor of plane wave or the optimal fill factor, curve factor of Gaussian beam；And
Strengthen submodule, the letter for strengthening the multi-photon imaging according to the optimal fill factor, curve factor under different type in-field
Number intensity.
6. the system of multi-photon imaging signal intensity is improved as claimed in claim 5, it is characterised in that the multi-photon imaging
For three-photon imaging or four photon imagings.
7. the system of multi-photon imaging signal intensity is improved as claimed in claim 6, it is characterised in that the signal acquisition mould
Block, specifically for utilizing numerical simulation mode, calculated by below equation the signal intensity that respectively obtains three-photon imaging and
The signal intensity of four photon imaging：
Wherein, S3, S4 represent the signal intensity of the three-photon imaging and the signal intensity of four photon imagings respectively,The light distribution in cylindrical coordinates is represented, V is represented to volume integral.
8. the system of multi-photon imaging signal intensity is improved as claimed in claim 5, it is characterised in that the line of the plane wave
Polarized incident light is obtained in specific calculated by below equation of intensity distribution of near focal point：
<mrow>
<msubsup>
<mi>E</mi>
<mrow>
<mi>p</mi>
<mi>l</mi>
<mi>a</mi>
<mi>n</mi>
<mi>e</mi>
</mrow>
<mrow>
<mi>i</mi>
<mi>n</mi>
<mi>c</mi>
</mrow>
</msubsup>
<mrow>
<mo>(</mo>
<mi>&alpha;</mi>
<mo>)</mo>
</mrow>
<mo>=</mo>
<mfrac>
<mn>1</mn>
<mi>&beta;</mi>
</mfrac>
<mi>exp</mi>
<mrow>
<mo>(</mo>
<mo>-</mo>
<mfrac>
<msub>
<mi>l</mi>
<mrow>
<mi>d</mi>
<mi>e</mi>
<mi>p</mi>
<mi>t</mi>
<mi>h</mi>
</mrow>
</msub>
<mrow>
<mn>2</mn>
<msub>
<mi>l</mi>
<mi>e</mi>
</msub>
<mi>c</mi>
<mi>o</mi>
<mi>s</mi>
<mi>&alpha;</mi>
</mrow>
</mfrac>
<mo>)</mo>
</mrow>
<mo>;</mo>
</mrow>
The linear polarization incident light of the Gaussian beam is obtained in specific calculated by below equation of intensity distribution of near focal point：
<mrow>
<msubsup>
<mi>E</mi>
<mrow>
<mi>G</mi>
<mi>a</mi>
<mi>u</mi>
<mi>s</mi>
<mi>s</mi>
<mi>i</mi>
<mi>a</mi>
<mi>n</mi>
</mrow>
<mrow>
<mi>i</mi>
<mi>n</mi>
<mi>c</mi>
</mrow>
</msubsup>
<mrow>
<mo>(</mo>
<mi>&alpha;</mi>
<mo>)</mo>
</mrow>
<mo>=</mo>
<mfrac>
<mn>1</mn>
<mi>&beta;</mi>
</mfrac>
<msqrt>
<mfrac>
<mrow>
<mn>1</mn>
<mo>-</mo>
<mi>exp</mi>
<mrow>
<mo>(</mo>
<mo>-</mo>
<mn>2</mn>
<mo>)</mo>
</mrow>
</mrow>
<mrow>
<mn>1</mn>
<mo>-</mo>
<mi>exp</mi>
<mrow>
<mo>(</mo>
<mo>-</mo>
<mn>2</mn>
<mo>/</mo>
<msup>
<mi>&beta;</mi>
<mn>2</mn>
</msup>
<mo>)</mo>
</mrow>
</mrow>
</mfrac>
</msqrt>
<mi>exp</mi>
<mrow>
<mo>(</mo>
<mo>-</mo>
<mfrac>
<mrow>
<msup>
<mi>sin</mi>
<mn>2</mn>
</msup>
<mi>&alpha;</mi>
</mrow>
<mrow>
<msup>
<mi>&beta;</mi>
<mn>2</mn>
</msup>
<msup>
<mi>sin</mi>
<mn>2</mn>
</msup>
<msub>
<mi>&alpha;</mi>
<mrow>
<mi>m</mi>
<mi>a</mi>
<mi>x</mi>
</mrow>
</msub>
</mrow>
</mfrac>
<mo>)</mo>
</mrow>
<mi>exp</mi>
<mrow>
<mo>(</mo>
<mo>-</mo>
<mfrac>
<msub>
<mi>l</mi>
<mrow>
<mi>d</mi>
<mi>e</mi>
<mi>p</mi>
<mi>t</mi>
<mi>h</mi>
</mrow>
</msub>
<mrow>
<mn>2</mn>
<msub>
<mi>l</mi>
<mi>e</mi>
</msub>
<mi>cos</mi>
<mi>&alpha;</mi>
</mrow>
</mfrac>
<mo>)</mo>
</mrow>
</mrow>
Wherein, Einc(α) represents linear polarization incident light in the intensity distribution of near focal point, ldepthRepresent imaging depth, leWith expression
Exciting light feature attenuation length, β represents the fill factor, curve factor of object lens filling extent, αmaxRepresent the angular integral upper limit, α represent light with
The angle of optical axis.
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|---|---|---|---|---|
| US7943889B2 (en) * | 2008-03-21 | 2011-05-17 | Prairie Technologies, Inc. | Apparatus for improving detection efficiency of multiphoton microscopy systems by focus compensation, pupil image division, and parallel pupil rearrangement |
| CN103890633A (en) * | 2011-09-29 | 2014-06-25 | Fei公司 | Microscope device |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7943889B2 (en) * | 2008-03-21 | 2011-05-17 | Prairie Technologies, Inc. | Apparatus for improving detection efficiency of multiphoton microscopy systems by focus compensation, pupil image division, and parallel pupil rearrangement |
| CN103890633A (en) * | 2011-09-29 | 2014-06-25 | Fei公司 | Microscope device |
Non-Patent Citations (2)
| Title |
|---|
| "On the fundamental imaging-depth limit in two-photon microscopy";Patrick Theer,Winfried Denk;《Proceedings of SPIE》;20040908;第5463卷;第45-55页 * |
| "飞秒激光微纳加工数据模型研究";牛立刚;《中国博士学位论文全文数据库 信息科技辑》;20110915(第09期);论文全文 * |
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