CN107941770A - Zoom two-photon optical tweezer microscopic imaging device and method - Google Patents
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Abstract
Zoom two-photon optical tweezer microscopic imaging device and method, belong to optical microphotograph imaging and optical control technical field.The technical characterstic of patent of the present invention is:Device includes:Two-photon lighting module, two-photon scan module, two photon probing module, two-photon axial direction focusing module and optical tweezer focus module.The present invention in conventional two-photon microscopic system increase by polarization spectroscope, quarter-wave plate, low aperture objective, Guan Jing and plane speculum group into axial focus control, realize the axial movement of two-photon focal plane in light path optical tweezer Two Photon Fluorescence altogether, axial movement is realized so as to capture suspended sample, completes the imaging of double photon three dimension computed tomography scanning.The invention is simple with adjustment, and zoom and axial Tomography Velocity are fast, low to observe the advantages of cost is low.
Description
Technical field
The present invention relates to a kind of microscopic imaging device and method, and in particular to a kind of zoom two-photon optical tweezer micro-imaging dress
Put with method, it can be achieved that light path two-photon microscopic system separates focusing altogether, the three-dimensional two-photon scanning microscopy of completion, belongs to optics and show
Micro- imaging and optical control technical field.
Background technology
In conventional objective lens formula optical tweezer microscope, light forceps device is usually located at sample both sides with imaging device, convenient to carry out
Individually focusing, but (such as add and radiate in sample side in some microscopic observation applications for needing to be introduced into other environmental variances
Device is used to study response mechanism of the living cells to radiation), it is desirable to optical tweezer is located at sample homonymy with imaging device, at this time, due to
Optical tweezer shares same object lens with three-dimensional microscopy device, its focal plane is difficult to separate, and can not realize that 3-D scanning is imaged.Double
Zoom lens or DMD are added in photon microscopic system can change the focal position of two-photon microscopic system, solve the above problems,
But modulating speed is slower or costly.
The content of the invention
The brief overview on the present invention is given below, in order to provide on the basic of certain aspects of the invention
Understand.It should be appreciated that this general introduction is not the exhaustive general introduction on the present invention.It is not intended to determine the pass of the present invention
Key or pith, nor is it intended to limit the scope of the present invention.Its purpose only provides some concepts in simplified form,
In this, as the preamble in greater detail discussed later.
In consideration of it, in order to overcome above-mentioned technical problem, the present invention provides a kind of zoom two-photon optical tweezer micro-imaging dress
Put and method, can not only improve zoom and axial Tomography Velocity, but also observation cost can be reduced.
Scheme one:The present invention provides a kind of zoom two-photon optical tweezer microscopic imaging device, including two-photon lighting module,
Two-photon axial direction focusing module, two-photon scan module, two photon probing module and optical tweezer focus module:
The two-photon lighting module is followed successively by according to the light direction of propagation:Laser one, beam expanding lens, polarization spectroscope;
The two-photon axial direction focusing module is followed successively by according to the light direction of propagation:Polarization spectroscope, quarter-wave plate,
Object lens one and plane mirror;
The two-photon scan module is followed successively by according to the light direction of propagation:Scanning galvanometer, scanning lens, Guan Jingyi, two
To Look mirror one, dichroscope two and object lens two;
The two photon probing module is followed successively by according to the light direction of propagation:Object lens two, dichroscope two, optical filter and
PMT;
The optical tweezer focus module is followed successively by according to the light direction of propagation:Laser two, conduction optical fiber, collimating mirror, Guan Jing
3rd, pipe mirror two, dichroscope one, dichroscope two and object lens two;
The two-photon lighting module, two-photon axial direction focusing module share polarization spectroscope;
The two-photon scan module, optical tweezer focus module share dichroscope one and object lens two;
The two-photon scan module, optical tweezer focus module, two photon probing module also share dichroscope two;
The lower section of object lens two sets sample.
Further:The sample for be suspended in maximum gauge in culture dish be micron order or nanoscale it is unicellular,
The sample to be tested of the almost spherical such as cell mass or particulate.
Further:The optical tweezer focus module, it launches one-wavelength laser wavelength between 750nm-900nm, double light
Sub- lighting module, it launches monochromatic femtosecond laser wavelength between 950nm-1100nm, and pulse width is less than 150fs, passes through
Dichroscope one synthesizes light path, shares object lens two and clamps and observe sample.
Further:The polarization spectroscope reflection light polarization direction is identical with the outgoing of laser one light polarization direction.
Further:The axial maximum moving range of the plane mirror is equal to the depth of focus of object lens one.
Scheme two:A kind of zoom two-photon optical tweezer micro imaging method proposed by the present invention, this method are to be based on scheme one
What the zoom two-photon optical tweezer microscopic imaging device was realized, specific steps:
Step a, laser one sends exciting light, and directional light, directional light are formed after beam expanding lens and polarization spectroscope
By quarter-wave plate and object lens one be transmitted to plane mirror produce reflection laser, then be scanned through galvanometer, scanning lens,
Guan Jingyi, dichroscope one, dichroscope two and object lens two form focal beam spot, the focusing light after transmiting on sample
Spot excitation sample sends fluorescence;
Step b, laser two sends laser and produces directional light by conduction optical fiber and collimating mirror, then by Guan Jingsan, pipe
Mirror two, dichroscope one, dichroscope two and object lens two form focal beam spot, clamp sample;
Step c, plane mirror initial position is set to be located at the quasi- focal planes of object lens one 2., then two-photon focal position is located at
The quasi- focal plane of object lens two is 2. ', plane mirror axial scan scope D is set1+D2, then two-photon focal beam spot axial scan is corresponded to
Scope is D1’+D2', plane mirror position and two-photon focal position correspondence are D1/D1'=D2/D2'=(M1M2)2;Institute
State D1For the far burnt displacement of plane mirror, D1' for the closely burnt displacement of two-photon focal position, D2For the closely burnt displacement of plane mirror,
D2' for the far burnt displacement of two-photon focal position, M1For object lens one and the focal length ratio of scanning lens, M2For Guan Jingyi and object lens two
Focal length ratio;
Step d, it is N to set the scanning number of plies, then plane reflection scarnning mirror stepping is (D1+D2)/ N, two-photon focal beam spot are swept
It is (D to retouch stepping1’+D2')/N, so as to fulfill quick three-dimensional computed tomography scanning.
Beneficial effect:
In the common light path optical tweezer micro imaging system of tradition optical tweezer is carried out usually using the approach before varifocal mirror or DMD modulating waves
System is separated with the focal plane of micro imaging system, for three-dimensional imaging, but with adjustment is complicated, modulating speed is limited and cost
The drawbacks of expensive.The present invention is using by polarization spectroscope, quarter-wave plate, low aperture objective, caliber and plane speculum group
Into two-photon focusing module, it can be achieved that in the case where objective table and object lens are motionless, only by moving axially plane mirror
Two-photon focal plane is moved axially, completes the quick three-dimensional scanning imagery to optical tweezer crawl sample;Adjustment of the present invention is simple,
Zoom and axial Tomography Velocity can be improved, but also observation cost can be reduced.
Brief description of the drawings
Fig. 1 is the structure diagram of the zoom two-photon optical tweezer microscopic imaging device of the present invention.
Fig. 2 is the zoom two-photon optical tweezer micro imaging method flow chart of the present invention.
In figure:1 laser one, 2 beam expanding lens, 3 polarization spectroscopes, 4 quarter-wave plates, 5 object lens one, 6 plane mirrors,
7 scanning galvanometers, 8 scanning lenses, 9 pipe mirrors one, 10 dichroscopes one, 11 dichroscopes two, 12 object lens two, 13 samples, 14
Optical filter, 15 collecting lenses, 16PMT, 17 pipe mirrors two, 18 pipe mirrors three, 19 collimating mirrors, 20 conduction optical fiber, 21 lasers two.
Embodiment
The one exemplary embodiment of the present invention is described hereinafter in connection with attached drawing.For clarity and conciseness,
All features of actual implementation mode are not described in the description.It should be understood, however, that developing any this actual implementation
It must be made during example much specific to the decision of embodiment, to realize the objectives of developer, for example, symbol
Conjunction and system and those relevant restrictive conditions of business, and these restrictive conditions may have with the difference of embodiment
Changed.In addition, it will also be appreciated that although development is likely to be extremely complex and time-consuming, to having benefited from the present invention
For those skilled in the art of disclosure, this development is only routine task.
Herein, it is also necessary to which explanation is a bit, in order to avoid having obscured the present invention because of unnecessary details, in the accompanying drawings
The apparatus structure closely related with scheme according to the present invention and/or processing step are illustrate only, and is eliminated and the present invention
The little other details of relation.
Embodiment 1:A kind of zoom two-photon optical tweezer microscopic imaging device is present embodiments provided as shown in Figure 1, is used for
It is switched fast axial location and realizes three-dimensional computed tomography scanning.
A kind of zoom two-photon optical tweezer microscopic imaging device, including two-photon lighting module, two-photon axial direction focusing module,
Two-photon scan module, two photon probing module and optical tweezer focus module:
The two-photon lighting module is followed successively by according to the light direction of propagation:Laser 1, beam expanding lens 2, polarization spectroscope
3;
The two-photon axial direction focusing module is followed successively by according to the light direction of propagation:Polarization spectroscope 3, four/wave plate 4,
Object lens 1 and plane mirror 6;
The two-photon scan module is followed successively by according to the light direction of propagation:Scanning galvanometer 7, scanning lens 8, Guan Jingyi 9,
Dichroscope 1, dichroscope 2 11 and object lens 2 12;
The two photon probing module is followed successively by according to the light direction of propagation:Object lens 2 12, dichroscope 2 11, optical filter
14th, collecting lens 15 and PMT16;
The optical tweezer focus module is followed successively by according to the light direction of propagation:Laser 2 21, conduction optical fiber 20, collimating mirror
19th, Guan Jingsan 18, pipe mirror 2 17, dichroscope 1, dichroscope 2 11 and object lens 2 12;
The two-photon lighting module, two-photon axial direction focusing module share polarization spectroscope 3;
The two-photon scan module, optical tweezer focus module share dichroscope 1 and object lens 2 12;
The two-photon scan module, optical tweezer focus module, two photon probing module also share dichroscope 2 11;
The lower section of object lens 2 12 sets sample 13.
More specifically:The object lens 1 are low aperture objective, and aperture is less than 0.4.
More specifically:The sample 13 is the micron order or nanoscale suspension cell or particle being placed in culture dish
Shape sample to be tested.
More specifically:The optical tweezer focus module, it launches one-wavelength laser wavelength between 750nm-900nm, double
Photon lighting module, it launches monochromatic femtosecond laser wavelength between 950nm-1100nm, and pulse width is less than 150fs, warp
Cross dichroscope 1 and synthesize light path, share the clamping of object lens 2 12 and observation sample 13.
More specifically:The polarization spectroscope 3 reflects light polarization direction and is emitted light polarization direction phase with laser 1
Together.
More specifically:The axial maximum moving range of plane mirror 6 is equal to the depth of focus of object lens 1.
Embodiment 2:A kind of zoom two-photon optical tweezer micro imaging method is present embodiments provided as shown in Figures 1 and 2,
Three-dimensional computed tomography scanning is realized for being switched fast axial location.
A kind of zoom two-photon optical tweezer micro imaging method, this method are based on the zoom double-photon optical described in embodiment 1
What tweezer microscopic imaging device was realized, specific steps:
Step a, laser 1 sends exciting light, and directional light is formed after beam expanding lens 2 and polarization spectroscope 3, parallel
Light is transmitted to plane mirror 6 by quarter-wave plate 4 and object lens 1 and produces reflection laser, then is scanned through galvanometer 7, sweeps
Retouch to be formed on sample 13 after lens 8, Guan Jingyi 9, dichroscope 1, dichroscope 2 11 and object lens 2 12 transmit and gather
Burnt hot spot, the focal beam spot excitation sample 13 send fluorescence;
Step b, laser 2 21 sends laser and produces directional light by conduction optical fiber 20 and collimating mirror 19, then by Guan Jing
3 18, pipe mirror 2 17, dichroscope 1, dichroscope 2 11 and object lens 2 12 form focal beam spot, clamp sample 13;
Step c, 6 initial position of plane mirror is set to be located at the quasi- focal planes of object lens 1 2., then two-photon focal position position
In object lens 2 13 quasi- focal plane 2. ', plane mirror 6 axial scan scope D is set1+D2, then two-photon focal beam spot axis is corresponded to
It is D to scanning range1’+D2', 6 position of plane mirror and two-photon focal position correspondence are D1/D1'=D2/D2'=
(M1M2)2;The D1For the far burnt displacement of plane mirror 6, D1' for the closely burnt displacement of two-photon focal position, D2For plane mirror 6
Nearly Jiao's displacement, D2' for the far burnt displacement of two-photon focal position, M1For object lens 1 and the focal length ratio of scanning lens 8, M2For Guan Jing
1 with the focal length ratio of object lens 2 12;
Step d, it is N to set the scanning number of plies, then it is (D that plane mirror 6, which scans stepping,1+D2)/N, two-photon focal beam spot
Scanning stepping is (D1’+D2')/N, so as to fulfill quick three-dimensional computed tomography scanning.
More specifically:Two-photon lighting module sends laser and is synthesized by two-photon scan module with optical tweezer focus module
Light path, reflected light enter two photon probing module, in two-photon axial direction focusing module, plane mirror position 1. when, it is corresponding
1. optical tweezer focal position is ', plane mirror position 2. when, 2. corresponding optical tweezer focal position is ', plane mirror is in position
3. when, 3. corresponding optical tweezer focal position is '.
Plane mirror initial position is 2., 2. optical tweezer two-photon focal plane initial position is ', move axially plane mirror
1. 2. 3. locate position into figure, corresponding to be changed to through 2 12 focal beam spot position of object lens generation correspondence position in figure 1. ' 2. ' 3. '
Locate position, plane mirror and focal plane displacement distance correspondence are D1/D1'=D2/D2'=(M1M2)2。
More specifically:The throw light of object lens 2 12, it is laggard through dichroscope 2 11, optical filter 14 and collecting lens 15
Enter PMT16, the PMT is photomultiplier.
Although disclosed embodiment is as above, its content is only to facilitate understand the technical side of the present invention
Case and the embodiment used, are not intended to limit the present invention.Any those skilled in the art to which this invention pertains, not
On the premise of departing from disclosed core technology scheme, any modification can be made in the form and details of implementation and is become
Change, but the protection domain that the present invention is limited, the scope that the appended claims that must still be subject to limits.
Claims (6)
1. zoom two-photon optical tweezer microscopic imaging device, it is characterised in that:Axially focus including two-photon lighting module, two-photon
Module, two-photon scan module, two photon probing module and optical tweezer focus module:
The two-photon lighting module is followed successively by according to the light direction of propagation:Laser one (1), beam expanding lens (2), polarization spectroscope
(3);
The two-photon axial direction focusing module is followed successively by according to the light direction of propagation:Polarization spectroscope (3), quarter-wave plate
(4), object lens one (5) and plane mirror (6);
The two-photon scan module is followed successively by according to the light direction of propagation:Scanning galvanometer (7), scanning lens (8), Guan Jingyi
(9), dichroscope one (10), dichroscope two (11) and object lens two (12);
The two photon probing module is followed successively by according to the light direction of propagation:Object lens two (12), dichroscope two (11), optical filter
(14), collecting lens (15) and PMT (16);
The optical tweezer focus module is followed successively by according to the light direction of propagation:Laser two (21), conduction optical fiber (20), collimating mirror
(19), Guan Jingsan (18), pipe mirror two (17), dichroscope one (10), dichroscope two (11) and object lens two (12);
The two-photon lighting module, two-photon axial direction focusing module share polarization spectroscope (3);
The two-photon scan module, optical tweezer focus module share dichroscope one (10) and object lens two (12);
The two-photon scan module, optical tweezer focus module, two photon probing module also share dichroscope two (11);
The lower section of object lens two (12) sets sample (13).
2. zoom two-photon optical tweezer microscopic imaging device according to claim 1, it is characterised in that:The sample
(13) it is treating for the almost spherical such as micron order or nanoscale is unicellular, cell mass or particulate to be suspended in maximum gauge in culture dish
Sample.
3. zoom two-photon optical tweezer microscopic imaging device according to claim 1, it is characterised in that:The optical tweezer focuses on mould
Block, it launches one-wavelength laser wavelength between 750nm-900nm, two-photon lighting module, it launches monochromatic femtosecond laser ripple
It is long to synthesize light path by dichroscope one (10) between 950nm-1100nm, share object lens two (12) clamping and observation quilt
Sample (13).
4. zoom two-photon optical tweezer microscopic imaging device according to claim 3, it is characterised in that:The polarization spectroscope
(3) it is identical with laser one (1) outgoing light polarization direction to reflect light polarization direction.
5. zoom two-photon optical tweezer microscopic imaging device according to claim 4, it is characterised in that:The plane mirror
(6) axial maximum moving range is equal to the depth of focus of object lens one (5).
6. zoom two-photon optical tweezer micro imaging method, this method is to be based on any zoom two-photon in Claims 1 to 5
What optical tweezer microscopic imaging device was realized, it is characterised in that:Specific steps:
Step a, laser one (1) sends exciting light, and directional light is formed after beam expanding lens (2) and polarization spectroscope (3), puts down
Row light is transmitted to plane mirror (6) by quarter-wave plate (4) and object lens one (5) and produces reflection laser, then is scanned through
After galvanometer (7), scanning lens (8), Guan Jingyi (9), dichroscope one (10), dichroscope two (11) and object lens two (12) transmission
Focal beam spot is formed on sample (13), the focal beam spot excitation sample (13) sends fluorescence;
Step b, laser two (21) sends laser and produces directional light by conduction optical fiber (20) and collimating mirror (19), then by pipe
Mirror three (18), pipe mirror two (17), dichroscope one (10), dichroscope two (11) and object lens two (12) form focal beam spot, folder
Firmly sample (13);
Step c, plane mirror (6) initial position is set to be located at the quasi- focal planes of object lens one (5) 2., then two-photon focal position position
In object lens two (13) quasi- focal plane 2. ', set plane mirror (6) axial scan scope D1+D2, then correspond to two-photon and focus on light
Spot axial scan scope is D1’+D2', plane mirror (6) position and two-photon focal position correspondence are D1/D1'=D2/
D2'=(M1M2)2;The D1For the remote burnt displacement of plane mirror (6), D1' for the closely burnt displacement of two-photon focal position, D2For plane
The nearly burnt displacement of speculum (6), D2' for the far burnt displacement of two-photon focal position, M1For object lens one (5) and the focal length of scanning lens (6)
Ratio, M2For Guan Jingyi (9) and the focal length ratio of object lens two (12);
Step d, it is N to set the scanning number of plies, then plane mirror (6) scanning stepping is) (D1+D2)/N, two-photon focal beam spot are swept
It is (D to retouch stepping1’+D2')/N, so as to fulfill quick three-dimensional computed tomography scanning.
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