CN102540447B - Trapping and detecting multiplexed scanning optical-tweezers system - Google Patents
Trapping and detecting multiplexed scanning optical-tweezers system Download PDFInfo
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- CN102540447B CN102540447B CN201210037312.0A CN201210037312A CN102540447B CN 102540447 B CN102540447 B CN 102540447B CN 201210037312 A CN201210037312 A CN 201210037312A CN 102540447 B CN102540447 B CN 102540447B
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Abstract
The invention provides a trapping and detecting multiplexed scanning optical-tweezers system. The trapping and detecting multiplexed scanning optical-tweezers system works by the following steps of: scanning a conjugate surface of a back focal plane of an objective lens to trap light beams to form a pair of scanning optical tweezers; collecting trapped small ball scattering light spots on the back focal plane of the objective lens, and projecting the light spots of the back focal plane of the objective lens onto a four-quadrant detector in a conjugate imaging manner; multiplexing an optical path by a trapping optical path and a detecting optical path through a half-reflecting and half-transparent plane mirror; changing symmetry of light spots when particles deviate the center of a light trap so as to realize high-precision detection on the displacement of the particles; and realizing detection of the light beam on the displacement of the scanning optical tweezers because the conjugate image central position of the light spots of the particles on the back focal plane of the objective lens is still placed on a target surface of a detector when the optical tweezers scan. The trapping and detecting multiplexed scanning optical-tweezers system provided by the invention simplifies a system design of the traditional scanning optical tweezers and also keeps the detection of the optical tweezers at different scanning positions so as to realize requirements on high-precision detection.
Description
Technical field
The invention belongs to the technical field of optical micro-manipulation system, particularly a kind ofly capture and survey multiplexing scan light tweezers system.
Background technology
There is light tweezer extensive application aspect research biomacromolecule of scan function, as Study on Protein foldable structure, DNA tensile properties and ligand-receptor interaction etc.In these application, utilize the ligh trap of light tweezer to capture the handle bead that is modified with biomacromolecule, move in the target such as biomacromolecule or cell that needs contact, then by ligh trap, handle bead is done to specific scanning motion, make biomacromolecule bear specific stress, just can study the mechanical characteristic of biological macromolecular structural change.There is the light tweezer of scan function, need to follow the trail of fast the position of ligh trap trap particles on optical optical tweezers system, therefore, in the design of scan light tweezer, not only need to realize the scan function of light tweezer, also need to realize to the ligh trap of scanning simultaneously and rapidly with high-precision detection.
In the detection of optical optical tweezers system, adopt 4 quadrant detector (Quadrant Photodiode, QPD) to realize by the location tracking of particle high-spatial and temporal resolution.QPD is made up of four standalone probe target surfaces, when the light beam irradiates that presents certain light distribution is when departing from target surface, the front and back difference of the difference result of the adjacent target surface signal of detector just can represent the displacement of BEAM SQUINT, realizes nano-grade displacement detected with high accuracy and the quick detection in the following time interval of sub-millisecond.
Optical optical tweezers system is generally set up and is formed based on commercial microscope, in traditional scan light tweezer design, adopts along the forward detection mode of capturing direction of beam propagation, by the scattering hot spot of particle on QPD probe microscope condenser back focal plane.The scattered light of particle can be that the light beam that ligh trap is captured produces, and can be also to irradiate ligh trap particle with detecting light beam in addition to produce.These two kinds of forward detections, based on the integrated scan light tweezer of microscope, all need to be built forward detection device at microscope condenser superjacent air space, and commercialization microscope top is built forward detection device and made whole scan light tweezers system seem complicated.
If utilize microscope self imaging function, along the scattered signal of capturing light beam reverse direction detection particle, be dorsad and survey, can simplify the design of light tweezer, light path is become simply, reduce the cost of system.Surveying dorsad in light path, currently used is all mode based on micro-imaging.While scanning for ligh trap, survey dorsad and can realize high-spatial and temporal resolution detection, therefore also have in the world the experimental provision of some light tweezers to adopt this detection mode.And in the time that light tweezer trap particles scans, imaging detection mode just can not be surveyed particle position by real-time follow-up dorsad.In the light tweezer of imaging detection dorsad, the axial micro-displacement that is captured particle will cause the picture of particle very large at Axial changes, and particle imaging size is also more big changes.Survey in order to meet limited detector target surface the picture that is captured particle, need to constantly regulate suitable light path parameter according to experiment.After particle size is excessive, its picture has exceeded detection target surface size, is difficult to realize effective detected with high accuracy.
In the design of the photodetection of scan light tweezer, forward detection device makes system complex, but the position of trap particles in can detection scanning ligh trap; Sniffer is simple dorsad, but the detection method of particle imaging is difficult to the application at scan light tweezer.
Summary of the invention
The object of the present invention is to provide a kind of multiplexing scan light tweezers system of capturing and survey, simplify the system of traditional scan light tweezer, the light tweezer that simultaneously remains on different scanning position is surveyed, and reaches the requirement of detected with high accuracy.
The present invention's solution is in order to achieve the above object:
Capture and survey a multiplexing scan light tweezers system, what it sent laser instrument capture, and laser beam utilizes lens combination to expand proper ratio becomes nearly parallel beam to propagate, through computer-controlled scanning mirror deflected beam; The light beam of deflection to trap position lens, focuses on microscope left channel center conjugate points C place by the light beam of horizontal infection through the lens reflecting of 45 degree half-reflection and half-transmissions; The part laser of transmission blocks with light barrier; The corresponding Amici prism of microscope inside left light mouth is replaced with to the dichroscope of spending total reflection for laser 45; Scanning mirror present position A and micro objective back focal plane B conjugation, light beam after these lens in light path after beam and focus size and object lens pupil in the same size, enter high-NA objective and focus on, so just formed scan light tweezer;
Detecting light beam is same laser beam with capturing light beam; In ligh trap, the back-scattering light of particle all passes through high-NA objective collection, along the light path former road backpropagation of capturing light beam; Concrete is: the scattering hot spot that is collected in B place through object lens, through lens and trap position lens, is imaged on the conjugate plane A ' of B place, directly surveys A ' hot spot with QPD and just realizes the detection to scan light tweezer;
Wherein, in light path, A and A ' are conjugate imaging relation with B, and A and A ' are respectively in the focal plane of trap position lens; C is the imaging conjugate points of microscope left side, infinite distance light hole; Position a and position b are respectively different scattering points on object lens imaging surface; Lens are imaged on A ' hot spot on 4 quadrant detector target surface.
In the detection design of optimizing at QPD, survey spot size most suitable in the half of target surface size, and the spot size that commercial various QPD target surface size is located with conjugate points A ' not necessarily mates, by suitable lens, A ' hot spot is imaged on QPD target surface with suitable enlargement ratio.
Advantage of the present invention and good effect are:
1, light tweezer of the present invention is in different angles scanning process, the situation that scanning light beam is truncated through the light beam of object lens back focal plane is identical, the laser power that enters object lens is consistent, hot spot symmetry does not have obvious variation in the time of small angle variation yet, the lateral attitude of light beam different angles incident major effect light tweezer trap particles, therefore in scanning process, the property retention of light tweezer is constant.
2, the present invention, in light tweezer scanning process, projects on detector target surface through a series of lens conjugate imagings by object lens back focal plane place's scattering hot spot, and particle has reflected in the change in location of the scattering hot spot at ligh trap center the distance that scanning ligh trap departs from.In light tweezer scanning process, the variation of scattering spot center can not make hot spot deviation detector target surface.
3, the present invention is at the light beam of capturing of special angle incident, and the position that center spot departs from has relatively reflected that particle departs from the displacement at ligh trap center.After demarcating by corresponding signal, so just realize the detection that departs from the high-precision detection of ligh trap the center displacement and scanning ligh trap the center displacement.
4, the present invention, in the time that light tweezer trap particles varies in size, surveys dorsad the mode of particle imaging and need to adjust detector position, and the mode of surveying dorsad back focal plane hot spot need not adjust detector position.In the conjugate plane of object lens back focal plane, scanning is captured light beam and is surveyed particle scattered light, get final product stable scan light tweezer and the high-precision detection of retention, simplify light forceps device, reduce cost, and solved the problem that need to readjust light path parameter for variable grain size aspect detection dorsad.
Accompanying drawing explanation
Fig. 1 captures and surveys the index path of multiplexing scan light tweezers system.
In figure, 1 is laser instrument; 2,3,6 and 11 is lens; 4 is scanning mirror; 5 is 4 quadrant detector; 7 is 45 degree half-reflection and half-transmission level crossings; 8 is light barrier; 9 is trap position lens; 10 for spending the dichroscope of total reflection for laser 45; 12 is high-NA objective; 13 is objective table; 14 is sample cell; 15 is condenser; 16 is microscope illumination light source; 17 is video monitoring camera.The conjugate plane at A and A ' representative and object lens back focal plane place, B is object lens back focal plane, the conjugate plane that C place is microscope imaging.
Fig. 2 object lens are collected the back-scattering light schematic diagram of focal plane diverse location.
In figure, F is object lens focus in object space; F ' is object lens rear focus; H
1and H
2for the principal plane of object lens equivalence; D is object lens back focal plane place diaphragm; A and b are respectively different scattering points on object lens imaging surface; The respectively corresponding object lens of a ' and b ' are collected the scattered beam by a and b.
Fig. 3 captures and surveys each conjugate plane graph of a relation of multiplexing light path.
In figure, 5 is 4 quadrant detector, and 6 and 11 is lens; 9 is trap position lens.L
1to L
4be respectively the light path of being separated by of each optical device in light path.A and A ' be and the conjugate plane at B place, the conjugate plane of position, C place microscope imaging.
Embodiment
Below in conjunction with the drawings and the specific embodiments, the present invention is described in detail, but protection scope of the present invention is not limited in embodiment below, should comprise the full content in claims.
Multiplexing light path design is captured and surveyed to scan light tweezer of the present invention is based on commercialization infinite distance microscopic system, and capturing light beam and detecting light beam is same laser beam, and schematic diagram is shown in Fig. 1.In optical optical tweezers system of the present invention, capturing light path design parameter need to meet the following conditions: (1) beam flying position and object lens back focal plane conjugation; (2) expanding than the designing requirement that relies on beam flying scope of scanning light beam spot size, scanning accuracy, the trap position focal length of lens and light beam; (3) enter object lens capture light beam spot size and object lens back focal plane is in the same size to the optical aperture of beam limit; (4) the trap position lens of control ligh trap and microscopical knot are as the confocal point of lens.Need to meet the following conditions for surveying light path design parameter: (1) is projected in the half that scattering hot spot on QPD target surface is about target surface size; (2) be projected in hot spot and the object lens back focal plane conjugation of QPD target surface.On whole optical optical tweezers system is integrated, except mechanical dimension can ensure that all optical elements can insert, will capture as far as possible light path and survey light path common optical elements, reduce the optical element of whole system.
The laser beam of capturing that laser instrument 1 is sent utilizes lens combination 2 and 3 to expand proper ratio to become nearly parallel beam to propagate, through computer-controlled scanning mirror 4 deflected beams.The light beam of deflection reflexes to trap position lens 9 through the level crossing 7 of 45 degree half-reflection and half-transmissions, focuses on conjugate points C place, microscope left channel center by the light beam of horizontal infection.The part laser of transmission blocks with light barrier 8.The corresponding Amici prism of microscope inside left light mouth is replaced with to the dichroscope 10 of spending total reflection for laser 45.Scanning mirror 4 present position A and micro objective back focal plane B conjugation, light beam after these lens in light path after beam and focus size and object lens pupil in the same size, enter high-NA objective 12 and focus on, so just formed scan light tweezer.
Detecting light beam is same laser beam with capturing light beam.In ligh trap, the back-scattering light of particle all passes through high-NA objective collection, along the light path former road backpropagation of capturing light beam.Because the extremely short high-NA objective of focal length is a complicated optical system, can represent imaging system with principal plane, see Fig. 2, H
1and H
2represent two principal planes of objective system, F and F ' represent the front and back focus of object lens.When particle is at position a and position b during in the scattering of ligh trap center, the scattered light of collecting by objective system hot spot symmetry on principal plane is consistent.Scanning distance is at micron order on microscope imaging face for general light tweezer, and therefore back focal plane F ' locates an also translation micron order distance of scattered beam spot center at (B point place).Spot center side-play amount in conjugate plane is also corresponding to micron order, and QPD target surface size is at grade, and therefore this micron order bias and detector allow to depart from grade hot spot and comparatively speaking can ignore.So just can realize the scanning of light tweezer time, survey hot spot still on detector target surface.
Fig. 3 is seen in each optical element relevant position that adopts QPD to survey scattering hot spot.Be collected in the scattering hot spot at B place through object lens through lens 11 and 9, be imaged on the conjugate plane A ' of B place, directly survey A ' hot spot with QPD and just realize the detection to scan light tweezer.In the detection design of optimizing at QPD, survey spot size most suitable in the half of target surface size, and the spot size that commercial various QPD target surface size is located with conjugate points A ' not necessarily mates, by suitable lens, A ' hot spot is imaged on QPD target surface with suitable enlargement ratio.
The essence that high precision displacement is surveyed is to reflect in ligh trap that Particle Phase is to the bias of ligh trap the center displacement.Capturing after light beam focuses on by high-NA objective of light tweezer, be submicron order at ligh trap position hot spot, this light beam irradiates is on micron/submicron grade particles, once particle departs from ligh trap center, nano level bias all can cause the scattering hot spot symmetry of being collected by object lens to change.In the time that QPD surveys this hot spot, on four target surfaces, survey light intensity different, just this slight change is carried out amplifying by Difference Calculation and surveyed.In scan light tweezer, as long as when scattering hot spot is still between the linear zone of detector while capturing beam flying, just can realize detected with high accuracy, therefore appropriate design is surveyed object lens principal plane or back focal plane hot spot, can effectively realize the detected with high accuracy of scan light tweezer.Because the principal plane of object lens is along with the parameter such as multiple and numerical aperture changes, and the design of microscopic system parfocalization causes object lens focal plane in same place, therefore surveys object lens back focal plane hot spot more convenient.Although spot center has trickle skew, this variation can not affect detection accuracy, and the only variation of beam flying position, reflection front and back.By suitable demarcation, the skew of spot center can also be served as the measuring amount of scan light tweezer scanning shift.
Can represent with the equivalent light path shown in Fig. 3 for the concrete lens position of capturing light path and survey in light path.In design when concrete optical parametric, suppose that the one dimension size in pupil footpath is respectively S after the hot spot of detector target surface, the bright dipping of laser instrument collimation and object lens
1, S
2and S
3; Capture laser beam expanding than being M through lens 2 and 3
1; The focal length of lens 6,9 and 11 is respectively f
1, f
2and f
3; Object lens enlargement ratio is M
obj; Scanning device angle scanning scope and resolution are respectively θ
0with Δ θ.If need to one-dimensional scanning scope be S in microscope is inspected when system
4, trap position focal length of lens f
2meet with scanning accuracy displacement S:
ΔS＝S
4Δθ/θ
0。⑵
By the known trap of infinite distance system position lens 9 and lens 11 at C place confocal point, L
3=f
2+ f
3.Microscope imaging conjugate plane C is fixed position in microscope left channel, and lens 11 are the lens that are fixed on microscope inside, and the focal length of the selected lens of different vendor is different.The beam expanding lens enlargement ratio that lens 9 and 11 form is M
2=f
3/ f
2, the bright dipping of laser instrument collimation expands and compares M
1=S
3/ (S
2m
2).Because beam flying angle is all very little, therefore A point is to the distance L of lens 9
ameet:
Make the design of light tweezer, parameter θ on as laser instrument, scanning mirror and microscopic system at apolegamy device
0, Δ θ, M
obj, S
2, S
3, f
3and L
4for fixed value, S
4for the requirement of design, L
alight path just can determine by formula (3).
In detection light path, A ' is consistent to the light path of lens 9 with A point.Object lens back focal plane hot spot S
3correspondence is S '=S at the spot size of conjugate plane
3/ M
2.By simple lens imaging, A ' is located to laser image spot and zoom in or out QPD target surface S
1half,
Therefore the selected hot spot that A order projects to the lens f3 of QPD target surface, lens to QPD target surface and trap position lens light path also just definite.
Capturing in different size particles experiments at scan light tweezer, is object lens back focal plane spot size due to what survey dorsad, therefore in experiment, need not adjust the variation that QPD target surface and enlargement ratio adapt to particle imaging size.Capture light beam and survey particle scattering hot spot by scanning on object lens back focal plane, just realized scan light tweezer and the high-precision detection of stable performance.
In the present invention, we are take Olympus IX7 microscopic system as example, apolegamy 100X oil immersion objective (NA1.4) laser focusing light beam forms ligh trap, the hot spot of laser instrument light-emitting window is the subparallel laser (Hong Kong Amonics AFL-1064-40-R-CL) of 4mm, the piezoelectric tilt platform S330.2SL of scanning mirror apolegamy PI company, 4 quadrant detector target surface is the S1557 of Bin Song company, and the ligh trap sweep limit of light tweezer is set as 1.8 μ m.According to institute's Chooser part and designing requirement, there is θ
0=2mrad, Δ θ=0.05 μ rad, M
obj=100, f
3=180mm, S
1=1mm, S
2=4mm, S
3=6mm, S
4=1.8 μ m, L
4=180mm.Can obtain f to (5) according to formula (1)
2=90mm, Δ S=0.045nm, L
3=270mm, M
2=2, M
1=0.75, L
a=90mm, S '=3mm.Select f
1=50mm, L
1=58.3mm, L
2=440mm.According to M
1focal length that can selector 2 is 100mm, and the focal length of device 3 is 75mm.
According to above parameter, scan light tweezer in theory Scan orientation precision can reach 0.045nm, but the precision of actual detection relies on the condition that concrete solution environmental and biomacromolecule meet with stresses.
The part that the present invention does not elaborate belongs to techniques well known.
Claims (2)
1. capture and survey multiplexing scan light tweezers system for one kind, it is characterized in that: laser beam scioptics group that what this system was sent laser instrument (1) capture expands proper ratio and becomes nearly parallel beam to propagate, this lens combination comprises first lens (2) and the second lens (3), through computer-controlled scanning mirror (4) deflected beam; The light beam of deflection reflexes to trap position lens (9) through the eyeglass (7) of 45 degree half-reflection and half-transmissions, focuses on conjugate points C place, microscope left channel center by the light beam of horizontal infection; The part of transmission for laser light barrier (8) block; The corresponding Amici prism of microscope inside left light mouth is replaced with to the dichroscope (10) of spending total reflection for laser 45; Scanning mirror present position A and micro objective back focal plane B conjugation, light beam after these lens in light path after beam and focus size and object lens pupil in the same size, enter high-NA objective (12) and focus on, so just formed scan light tweezer;
Detecting light beam is same laser beam with capturing light beam; In ligh trap, the back-scattering light of particle all passes through high-NA objective (12) collection, along the light path former road backpropagation of capturing light beam; Concrete is: the scattering hot spot that is collected in B place through object lens, through the 4th lens (11) and trap position lens (9), is imaged on the conjugate plane A ' of B place, directly surveys A ' hot spot with QPD and just realizes the detection to scan light tweezer;
Wherein, in light path, A and A ' are conjugate imaging relation with B, and A and A ' are respectively in the focal plane of trap position lens (9); C is the imaging conjugate points of microscope left side, infinite distance light hole; Position a and position b are respectively different scattering points on object lens imaging surface; The 3rd lens (6) are imaged on A ' hot spot on 4 quadrant detector target surface (5);
Can represent with the equivalent light path shown in Fig. 3 for the concrete lens position of capturing light path and survey in light path.In design when concrete optical parametric, suppose that the one dimension size in pupil footpath is respectively S after the hot spot of detector target surface, the bright dipping of laser instrument collimation and object lens
1, S
2and S
3; Capture laser beam expanding than being M1 through first lens (2) and the second lens (3); The focal length of the 3rd lens (6), trap position lens (9) and the 4th lens (11) is respectively f
1, f
2and f
3; Object lens enlargement ratio is M
obj; Scanning device angle scanning scope and resolution are respectively θ
0with Δ θ, if need to one-dimensional scanning scope be S in microscope is inspected when system
4, trap position focal length of lens f
2meet with scanning accuracy displacement S:
ΔS＝S
4Δθ/θ
0。⑵
By the known trap of infinite distance system position lens (9) and the 4th lens (11) at C place confocal point, L
3=f
2+ f
3, microscope imaging conjugate plane C is fixed position in microscope left channel, and the 4th lens (11) are the lens that are fixed on microscope inside, and the beam expanding lens enlargement ratio that trap position lens (9) and the 4th lens (11) form is M
2=f
3/ f
2, the bright dipping of laser instrument collimation expands and compares M
1=S
3/ (S
2m
2), because beam flying angle is all very little, therefore A point is to the distance L of trap position lens (9)
ameet:
Wherein, parameter θ
0, Δ θ, M
obj, S
2, S
3, f
3and L
4for fixed value, S
4for the requirement of design, L
alight path just can determine by formula (3).
2. a kind of multiplexing scan light tweezers system of capturing and survey according to claim 1, it is characterized in that: in the detection design of optimizing at QPD, survey spot size most suitable in the half of target surface size, and the spot size that commercial various QPD target surface size is located with conjugate points A ' not necessarily mates, by suitable lens, A ' hot spot is imaged on QPD target surface with suitable enlargement ratio.
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| CN102860845A (en) * | 2012-08-30 | 2013-01-09 | 中国科学技术大学 | Method and corresponding device for capturing and controlling in-vivo cells of living body animal |
| JP6172290B2 (en) | 2013-10-30 | 2017-08-02 | 株式会社ニコン | Total reflection microscope |
| CN103676126A (en) * | 2013-12-20 | 2014-03-26 | 同济大学 | Operation instrument for optical tweezers |
| CN104406528B (en) * | 2014-11-25 | 2017-11-07 | 中国科学技术大学 | A kind of method of the calibrated in situ piezotable displacement based on optical trap |
| CN105115476B (en) * | 2015-09-02 | 2017-08-25 | 合肥工业大学 | The flat board inclination measuring device detected based on multiple spot defocus |
| CN108051909B (en) * | 2017-11-20 | 2023-11-21 | 中国计量大学 | Extended focal depth microscopic imaging system combining optical tweezers function |
| CN109633858B (en) * | 2019-02-19 | 2020-07-07 | 浙江大学 | Device and method for aligning focal points of correlation light beams in optical tweezers |
| US12066367B2 (en) * | 2020-03-13 | 2024-08-20 | Brigham Young University | Electromagnetic trap multiplexing |
| CN113050265B (en) * | 2021-03-25 | 2022-03-01 | 中山大学 | Device and method for correcting drift of optical tweezers by using laser reflection light spots |
| CN113701998B (en) * | 2021-08-02 | 2022-10-21 | 浙江大学 | Focusing lens arrangement error correction device and method in optical tweezers system |
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| US6416190B1 (en) * | 2001-04-27 | 2002-07-09 | University Of Chicago | Apparatus for using optical tweezers to manipulate materials |
| JP5309867B2 (en) * | 2008-10-14 | 2013-10-09 | 株式会社ニコン | Nonlinear optical microscope and adjustment method thereof |
| CN101799482A (en) * | 2010-01-28 | 2010-08-11 | 哈尔滨工业大学 | Nano operating device with near-field optical tweezers and AFM probe |
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