CN101587238A - Dual-color two-photon fluorescence imaging method and device - Google Patents
Dual-color two-photon fluorescence imaging method and device Download PDFInfo
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Abstract
The invention provides a dual-color two-photon fluorescence imaging method and a device, which uses six dual-color beams for emitting along plus and minus X, plus and minus Y, plus and minus Z six directions into the sample 3, thereby forming a three-dimensional lattice for exciting fluorescence, and uses a two-dimension small hole array 6 for filtering background fluorescence in the detecting light path, uses femtosecond pulsed laser with two different kinds of frequencies for composing a dual-color beam, wherein the two are coaxial, and the wavelengths are lambada 1 and lambada 2 respectively, moreover, lambada 1 is equal to lambada 2. Two kinds of light with different wavelength form two different three-dimensional lattice structures in the sample 3, and delay line 10 is modulated for causing the six beams to have the same optical path, only in the zone where two kinds of three-dimensional lattices coincide mutually, the dual-color two-photon fluorescence can be excitated. The device provided by the invention can effectively improve resolution response and imaging speed of three-dimensional imaging, and can be conveniently operated.
Description
Technical field
The present invention is relevant with the biological tissue micro-imaging, relate to two-photon fluorescence imaging, the particularly a kind of dual-color two-photon fluorescence imaging method and device that can improve spatial resolution and image taking speed is to be applicable in the medical science detection to biological living cells dynamic process.
Background technology
In recent years, the two-photon fluorescence excitation imaging has caused people's extensive interest (Denk W, Strickler J, WebbW., Science, Vol.248,73～76,1997).Two-photon excitation (being designated hereinafter simply as TPE) is a kind of third-order non-linear process, the characteristics of this non-linear process, make two-photon excitation need higher exciting power, and square being directly proportional of fluorescence signal intensity and excitating light strength, thereby can be with the generation local of two-photon fluorescence at focus area.Therefore the two-photon imaging has the natural ability to the three-dimensional sample tomography.Compare with traditional single photon cofocus scanning fluorescent microscopic imaging (CSFM), two-photon excitation has advantages such as the filtering of being easy to, low bleaching and high image contrast.Its non-linear characteristics that excite can reduce the yardstick of point spread function, and it is original that halfwidth is kept to
But excitation wavelength double to have offset this effect (referring to M.Gu, C.J.R.Sheppard., J.Microsc., Vol.177,128～137,1995 and P.D.Higdon, P.Torok, T.Wilson., J.Microsc., Vol.193,127～141,1999), lateral resolution is with respect to CSFM even decline to some extent, horizontal and vertical resolution be about 200nm and 400nm (referring to G.J.R.Sheppard, M.Gu., Optik, Vol.86,104～106,1990).
In addition, image taking speed also is relatively to pay close attention among the TPE, and for the dynamic process of observing bulky objects and some living things systems (as cell differentiation etc.), we need higher image taking speed.Have three kinds of methods to improve image taking speed at present: first is to use high speed scanner, as polygon mirror scanner (K.H.Kim, C.Buehler, P.T.C.So., Appl.Opt., Vol.38,6004～6009,1999), in the method, thereby the raising of sweep velocity certainly will bring the reduction of time shutter to weaken fluorescence signal intensity, will reduce signal to noise ratio (snr).The secondth, only to interested limited bulk scanning.The 3rd is multifocal multiphoton microscopic method (be called for short MMM), and this method excites with many focuses and substitutes single focus and excite and improve image taking speed, is practical a kind of scheme (J.Bewersdorf, R.Pick, S.W.Hell, Opt.Lett., Vol.23,655～657,1998).1998, A.H.Buist etc. utilized two-dimensional array of micro-lenses to realize that bidimensional focus array comes fluorescence excitation to realize the TPE imaging; 2000, propositions such as D.N.Fittinghoff utilized etalon that femtosecond light is divided into multi beam, and further formed one dimension focus array to carry out the TPE imaging; 2003, L.Sacconi etc. utilized diffraction optical element that the beam splitting of femtosecond light is also further formed two-dimentional focus array to realize the TPE imaging, and horizontal and vertical resolution is about 250nm and 800nm.These schemes can both effectively improve image taking speed, are highly suitable for living imaging, but its spatial resolution remains further to be improved.
How to seek that a kind of suitable scheme improves resolution and image taking speed is the major issue that present optical microphotograph imaging field faces.
During two-photon excitation, the frequency of two photons can be identical, also can be different, and the biphotonic process of the photon excitation of two different frequencies is called dual-color two-photon and excites (being designated hereinafter simply as TCTPE).Two excitation wavelengths of TCTPE need satisfy 1/ λ
e=1/ λ
1+ 1/ λ
2, wherein: λ
eBe one-photon excitation wavelength, λ
1And λ
2Be two excitation wavelengths.TCTPE fluorescence signal intensity I
TC∝ I
1(λ
1) I
2(λ
2).TCTPE is that its optical characteristics in imaging is different with the maximum difference of TPE, and the two bundle exciting lights of TCTPE have different wavelength, belong to incoherent and excite, and TPE is the coherent excitation process.For TCTPE, have only two-beam crossover zone ability fluorescence excitation, the two-beam non-coherent addition, secondary lobe is much smaller, and is therefore higher with respect to the signal to noise ratio (S/N ratio) of TPE.In typical dual-color two-photon scheme, the θ incident that has a certain degree of the light of two kinds of different wave lengths focuses on same point fluorescence excitation in the sample with two object lens with two-beam respectively.Two-beam spatially is distinct before and after focus, and is only overlapping at focus area, therefore has only focus area can excite dual-color two-photon fluorescence, and ground unrest is very little.
At this, we combine the thought of TCTPE and MMM, propose a kind of dual-color two-photon fluorescence imaging scheme, can effectively improve image taking speed and resolution.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiency of above-mentioned existing biological tissue fast imaging aspect, a kind of dual-color two-photon fluorescence imaging method and device are provided, this device can improve the speed and the spatial resolution of three-dimensional imaging in the biological tissue, and easy to operate.
Technical solution of the present invention is as follows:
A kind of dual-color two-photon fluorescence imaging method, be characterized in adopting six bundle double color lasers, edge ± X respectively, ± Y, ± Z six direction incides in the sample, form three-dimensional lattice and come fluorescence excitation, and in surveying light path, use the method for a two-dimentional array of orifices wiping out background fluorescence, utilize the femtosecond pulse composition wavelength of two kinds of different frequencies to be respectively λ
1And λ
2Double color laser, both are coaxial,, and λ
1=2 λ
2, the light of two kinds of wavelength forms two kinds of different three-dimensional lattice structures in sample, regulate delay line and make six bundle light aplanatisms, only just can excite dual-color two-photon fluorescence in the zone that two kinds of three-dimensional lattices overlap.
A kind of dual-color two-photon fluorescence imaging device, this device comprise illumination section, survey and collect part and sweep test, are characterized in:
Described illumination section comprises six bundle double color lasers, and every bundle double color laser is λ by wavelength
1With wavelength be λ
2Light form both coaxial and λ
1=2 λ
2All be equipped with half-wave plate and delay line in every beam optical path, five bundle double color lasers respectively behind separately half-wave plate and delay line edge ± X, ± Y ,+five directions of Z incide sample, the 6th bundle double color laser through half-wave plate and delay line after inject sample, this dichroic mirror and the placement at 45 of this double color laser by-Z direction by the dichroic mirror reflection;
Described probe portion is made up of successively dichroic mirror, collecting lens, filter plate, two-dimentional array of orifices, CCD and described computing machine, described collecting lens places the transmitted light direction of described dichroic mirror, CCD places the focal plane of described collecting lens, described array of orifices is close to the input end face of described CCD and is placed, the fluorescence signal intensity that sample produces inputs to described computing machine after being obtained by described CCD, carry out data processing by computing machine, the three-dimensional microscopic image of the sample of recombinating out;
Described sweep test comprises the D translation platform of putting sample, and computer drives is also controlled moving of described D translation platform;
Described sample is through fluorescein-labeled sample.
The wavelength X of described double color laser
1And λ
2Be respectively 1200nm and 600nm.
Described dichroic mirror is to be the high reflection of light beam of 1200nm and 600nm to wavelength, and wavelength is the dichroic mirror of the high transmission of light beam of 500-550nm.
Technique effect of the present invention is:
1, uses six bundle double color lasers to interfere, form the space three-dimensional dot matrix, realize multi-point shooting, can improve image taking speed.
2, use the dual-color two-photon fluorescence excitation, the generation of fluorescence when overlapping on time and space, the light of two wavelength is just arranged, therefore the zone that overlaps of two three-dimensional lattices that only form at the light of two wavelength could produce fluorescence, can reduce excitation volume and improve resolution, and increased spacing between the phosphor dot, make the easier detection of signal.
3, can realize side's property resolution such as three-dimensional, promptly identical horizontal and vertical resolution can reach 150nm.
4, in surveying light path, use array of orifices, can effectively eliminate the outer background fluorescence of focus.
5, use infrared wavelength 1200nm, can reduce the influence of scattering, in addition, exciting light is provided by OPA, and its tunable wave length scope is 1160nm-2600nm, can free selective excitation wavelength.
Description of drawings
Fig. 1 is the index path of dual-color two-photon fluorescence imaging device specific embodiment of the present invention, and the direction of arrow is wherein represented the polarization direction of six bundle double color lasers.
Among the figure: 1 is half-wave plate, and 2 is dichroic mirror (anti-to 1200nm and 600nm, 500-550nm is saturating), and 3 is sample, 4 is collecting lens, and 5 is filter plate (saturating to 520-550nm), and 6 is the bidimensional array of orifices, and 7 is CCD, 8 is three-dimensional platform, and 9 is computing machine, and 10 is delay line.
Embodiment
The invention will be further described below in conjunction with embodiment and accompanying drawing, but should not limit protection scope of the present invention with this.
See also Fig. 1, Fig. 1 is the index path of dual-color two-photon fluorescence imaging device specific embodiment of the present invention, and as seen from the figure, dual-color two-photon fluorescence imaging device of the present invention, this device comprise illumination section, survey and collect part and sweep test:
Described illumination section comprises six bundle double color lasers, and each double color laser is λ by wavelength
1With wavelength be λ
2Light form both coaxial and λ
1=2 λ
2All be equipped with half-wave plate 1 and delay line 10 in every beam optical path, five bundle double color lasers respectively behind separately half-wave plate 1 and delay line 10 edge ± X, ± Y ,+five directions of Z incide sample 3, the 6th bundle double color laser through half-wave plate 1 and delay line 10 after inject sample 3, this dichroic mirror 2 and the placement at 45 of this double color laser by-Z direction by dichroic mirror 2 reflections; Described half-wave plate 1 and delay line 10, the polarization direction and the light path of control bundle respectively.
Described probe portion is made up of successively dichroic mirror 2, collecting lens 4, filter plate 5, two-dimentional array of orifices 6, CCD7 and described computing machine 9, described collecting lens 4 places the transmitted light direction of described dichroic mirror 2, CCD7 places the focal plane of described collecting lens 4, described array of orifices 6 is close to the input end face of described CCD7 and is placed, the fluorescence signal intensity that sample 3 produces inputs to described computing machine 9 after being obtained by described CCD7, carry out data processing by computing machine 9, the three-dimensional micro-image of the sample 3 of recombinating out.
Described sweep test comprises the D translation platform 8 of putting sample 3, and computing machine 9 drives and control moving of described D translation platform 8, to realize the scanning probe to the diverse location of sample 3;
Described sample 3 is through fluorescein-labeled sample.
Described double color laser, its wavelength X
1And λ
2Be respectively 1200nm and 600nm.Shown in arrow among Fig. 1, the double color laser of edge ± X-direction incident, its polarization direction is
The double color laser of edge ± Y direction incident, its polarization direction is
The double color laser of edge ± Z-direction incident, its polarization direction is
Described dichroic mirror 2 is to be that the light beam of 1200nm and 600nm is high anti-to wavelength, and wavelength is the high saturating dichroic mirror of the light beam of 500～550nm.
5 pairs of 520～550nm wave bands of described filter plate are saturating.
Be example with 1200nm and 600nm double color laser in the present embodiment, as shown in Figure 1, system mainly comprises illumination section, sweep test and survey and collect part.Six bundle double color laser edge ± X, ± Y, ± Z six direction incide in the sample 3, and its polarization direction is by polaroid 1 control, edge respectively
Three directions.The light of two kinds of wavelength forms two kinds of different three-dimensional lattice structures in sample, regulate delay line 10 and make six bundle light aplanatisms, and the zone that overlaps two kinds of three-dimensional lattices excites dual-color two-photon fluorescence, and excited fluorescent point also is three-dimensional lattice and distributes.The dual-color two-photon fluorescence that is excited sees through dichroic mirror 2, is collected lens 4 and collects, and through filter plate 5 and array of orifices 6, enter CCD 7.Array of orifices 6 can effectively be eliminated the outer background fluorescence of focus, has only the phosphor dot on the corresponding X-Y plane to be detected by CCD 7, by moving three dimension translation stage 8 can scanning samples diverse location.
The groundwork process is: six bundle double color laser edge ± X, and ± Y, ± Z six direction incide in the sample 3, and institute's excited fluorescent is the three-dimensional lattice structure, via dichroic mirror 2, collecting lens 4, filter plate 5 and array of orifices 6 enter CCD 7.Then, under the control of computing machine 9, by the diverse location of moving three dimension translation stage 8 scanning samples, simultaneously according to the data of CCD 7 records, can recombinate out the three-dimensional micro-image of sample of computing machine 9.In the enforcement, we with the direction of collecting fluorescence as the Z axle, with perpendicular to the direction of phosphor collection as X-Y plane.
For illustrating that this scheme can improve resolution, we get λ
1And λ
2Be respectively 1200nm and 600nm, collecting lens 4 is an oil immersion objective, and numerical aperture is NA=1.3, the refractive index of object lens and sample is 1.518, and as example, Theoretical Calculation draws, horizontal and vertical resolution is 150nm, obviously improves than traditional two-photon imaging.
The present invention uses six bundle double color laser air exercises, forms the space three-dimensional dot matrix, realizes multi-point shooting, has improved image taking speed and resolution greatly.The present invention is easy to operate, relatively flexibly, and can free selective excitation light wavelength.
Claims (4)
1, a kind of dual-color two-photon fluorescence imaging method, it is characterized in that adopting six bundle double color lasers, edge ± X respectively, ± Y, ± Z six direction incides in the sample, form three-dimensional lattice and come fluorescence excitation, and in surveying light path, use the method for a two-dimentional array of orifices wiping out background fluorescence, utilize the femtosecond pulse composition wavelength of two kinds of different frequencies to be respectively λ
1And λ
2Double color laser, both are coaxial, and λ
1=2 λ
2, the light of two kinds of wavelength forms two kinds of different three-dimensional lattice structures in sample, regulate delay line and make six bundle light aplanatisms, only just can excite dual-color two-photon fluorescence in the zone that two kinds of three-dimensional lattices overlap.
2, a kind of dual-color two-photon fluorescence imaging device, this device comprise illumination section, survey and collect part and sweep test, it is characterized in that:
Described illumination section comprises six bundle double color lasers, and every bundle double color laser is λ by wavelength
1With wavelength be λ
2Light form both coaxial and λ
1=2 λ
2All be equipped with half-wave plate (1) and delay line (10) in every beam optical path, five bundle double color lasers respectively behind separately half-wave plate (1) and delay line (10) edge ± X, ± Y ,+five directions of Z incide sample (3), the 6th bundle double color laser through half-wave plate (1) and delay line (10) after dichroic mirror (2) reflection is injected sample (3), this dichroic mirror (2) and the placement at 45 of this double color laser by-Z direction;
Described detection is collected part by successively dichroic mirror (2), collecting lens (4), filter plate (5), two dimension array of orifices (6), CCD (7) and computing machine (9) are formed, described collecting lens (4) places the transmitted light direction of described dichroic mirror (2), CCD (7) places the focal plane of described collecting lens (4), described two-dimentional array of orifices (6) is close to the input end face of described CCD (7) and is placed, the fluorescence signal intensity that sample (3) produces inputs to described computing machine (9) after being obtained by described CCD (7), carry out data processing by this computing machine (9), the three-dimensional micro-image of the sample of recombinating out (3);
Described sweep test comprises the D translation platform (8) of putting sample (3), and described computing machine (9) drives and control moving of described D translation platform (8);
Described sample (3) is through fluorescein-labeled sample.
3, dual-color two-photon fluorescence imaging device according to claim 2 is characterized in that the wavelength X of described double color laser
1And λ
2Be respectively 1200nm and 600nm.
4, dual-color two-photon fluorescence imaging device according to claim 3 is characterized in that described dichroic mirror (2) is is that the light beam of 1200nm and 600nm is high anti-to wavelength, and wavelength is the high saturating dichroic mirror of the light beam of 500～550nm.
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