CN103954598B - A kind of axial high-precision locating method based on evanescent wave illumination and device - Google Patents
A kind of axial high-precision locating method based on evanescent wave illumination and device Download PDFInfo
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- CN103954598B CN103954598B CN201410185352.9A CN201410185352A CN103954598B CN 103954598 B CN103954598 B CN 103954598B CN 201410185352 A CN201410185352 A CN 201410185352A CN 103954598 B CN103954598 B CN 103954598B
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Abstract
The invention discloses a kind of based on evanescent wave illumination axial high-precision locating method, comprise the following steps: 1) collimation after illuminating bundle through long-focus lens focus on, then by object lens be irradiated to sample surfaces occur total reflection generation evanescent wave, excite fluorescence；2) being divided into two-way light beam after the fluorescence line focus lens collected, a road light beam is received by gathering front detector, and another road light beam is received by gathering rear detector, obtains the Jiao Qiantu on sample top layer and defocused figure；3) control sample to move axially, obtain the Jiao Qiantu under sample different depth and defocused figure；4) to step 3) in Jiao Qiantu and defocused figure carry out difference processing, the degree of depth is fitted with difference result, obtains the calibration curve of the degree of depth and difference result；5) Jiao Qiantu on sample top layer and the difference result of defocused figure and calibration curve are utilized, it is achieved the axial location in each region, sample top layer.The invention also discloses a kind of axial high-precision positioner based on evanescent wave illumination.
Description
Technical field
The invention belongs to the field of the micro-micro-combination burnt with copolymerization of total internal reflection fluorescent, particularly relate to one
Plant axial high-precision locating method based on evanescent wave illumination and device.
Background technology
The axial hi-Fix of sample to be realized, it is necessary to obtained by the method for 3-D scanning
Each several part in sample just can axially be positioned by three-dimensionalreconstruction result, and the method is time-consuming and operating procedure
Complex.The present invention proposes a kind of axial localization method of the high accuracy without axial scan and device,
Use Both wide field illumination and the spot scan method of reseptance of Laser Scanning Confocal Microscope of total internal reflectance microscope, logical
Cross in the method for out of focus difference detecting realizes the sample surfaces thin layer that excites evanescent wave and carry out axial height
Precision positions.
Wherein utilizing total internal reflection fluorescence microscope is to be produced when sample surfaces experiences total internal reflection by light
Evanescent wave illumination sample, only excite the fluorophor in the range of sample surfaces thin layer, thus improve
Micro-imaging spatial resolution on the longitudinal axis, then cannot realize sample each several part at coating interior
Axially location.
Laser Scanning Confocal Microscope can by the 3-D scanning of sample being realized the three-dimensionalreconstruction to sample, thus
Obtain the axial information of sample, sample is carried out high-precision axial location.This method uses complete interior anti-
Penetrate the lighting system of fluorescence microscope and the fluorescence reception method of Laser Scanning Confocal Microscope, by Both wide field illumination side
Formula combines with spot scan method of reseptance, by the method for out of focus difference detecting, it is not necessary to axial scan,
Only can realize the axial hi-Fix in sample surfaces thin layer by transversal scanning.
Summary of the invention
The invention provides a kind of axial high-precision locating method based on evanescent wave illumination and device, phase
For other axial localization methods, this device is based on utilizing total internal reflection fluorescence microscope, lighting system letter
Single, only excite the skin layer of sample, greatly reduce background noise；At probe portion, based on point
A kind of method that the mode that scanning receives proposes out of focus difference detecting, eliminates the side of 3-D scanning
Formula, only i.e. may be implemented in the nanoscale of (submicron order) in sample thin layer by two-dimensional scan the highest
Precision track and localization.
A kind of axial high-precision locating method based on evanescent wave illumination, comprises the following steps:
1) illuminating bundle after collimation focuses on through long-focus lens, then is irradiated to sample table by object lens
Face occurs total reflection to produce evanescent wave, excites sample top layer to send fluorescence；
2) being divided into two-way light beam after the fluorescence line focus lens collected, a road light beam is by being positioned at focus point
Front detector receives, and another road light beam is received by the detector after being positioned at focus point, obtains sample table
The Jiao Qiantu of layer and defocused figure；
3) control sample move axially, repeat step 1) and step 2) in operation, obtain sample
Jiao Qiantu under different depth and defocused figure；
4) to step 3) in Jiao Qiantu and defocused figure carry out difference processing, by the degree of depth and difference result
It is fitted, obtains the calibration curve of the degree of depth and difference result；
5) Jiao Qiantu and defocused figure to sample top layer carry out difference processing, bent according to described demarcation
Line, it is achieved the axial location in each region, sample top layer.
In step 2) in, use scanning galvanometer to be scanned the light that sample different piece sends receiving.
In step 1) in, the illuminating bundle after collimation is after long-focus lens focuses on microcobjective
On focal plane so that the illuminating bundle through microcobjective is exiting parallel light, and incident sample surfaces
Angle more than critical angle.
Present invention also offers a kind of axial high-precision positioner based on evanescent wave illumination, including:
Laser instrument, collimating lens, long-focus lens, microcobjective and the nanometer being sequentially arranged along light path
Translation stage, this nanometer translation stage is placed with sample；
Along receive fluorescence light path be sequentially arranged the first scanning galvanometer, collecting lens, beam splitter, and
The first photodetector before laying respectively at the focus point of collecting lens and after focus point and the second photoelectricity
Detector；
With for controlling the computer that nanometer translation stage and data process.
Wherein, the first reflecting mirror, dichroscope it are sequentially provided with between described long-focus lens and microcobjective
With the second reflecting mirror, for changing the direction of long-focus lens emitting light path, make the structure of whole device
Compacter.
Meanwhile, the second scanning galvanometer it is provided with between described collimating lens and long-focus lens.
Meanwhile, first adapted respectively is also included with the first photodetector and the second photodetector
Detection aperture, the second detection aperture.
In the present invention: laser instrument is optical fiber laser, it is used for sending laser, it is achieved to fluorescent samples
Illumination excite；Collimating lens collimates for the diverging light sending optical fiber laser；Long-focus
Lens, constitute a 4f system together with collimating lens, for collimated light beam is focused on object lens
Back focal plane；Microcobjective, makes the excitation beam exiting parallel focusing on objective lens ' with necessarily
Angle is incident in sample surfaces；Nanometer translation stage, is used for controlling that sample is nano level to be moved axially；Sweep
Retouch galvanometer, by controlling scanning galvanometer, the difference on the lateral attitude on sample top layer is sent
Light is received by a detector, it is achieved the two-dimensional scan to sample top layer；Collecting lens, is used for assembling reception
The fluorescence arrived；First detection aperture, the second detection aperture, be respectively placed in the burnt front and burnt of collecting lens
After, filter the light that the point of other lateral attitudes sends；First photodetector, the second photodetector,
The intensity signal that reception the first detection aperture, the second detection aperture receive respectively；Computer, is used for
Control moving axially of nanometer translation stage, process the signal of two detectors.
For above-mentioned axial high-precision positioner, detailed process is as follows:
(1) luminous point of optical fiber laser is placed on the front focal plane of collimating lens and sends illuminating bundle,
After collimating lens collimates, collimated light beam focuses on through long-focus lens, in way through two reflecting mirrors and
One dichroscope reflection, focuses on the equivalent back focal plane of long-focus lens.Collimating lens back focal plane
Overlap with the front focal plane of long-focus lens, now the front focal plane of collimating lens and long-focus lens etc.
Effect back focal plane conjugation.By regulation optical fiber head in the position of the front focal plane of the first object lens i.e. scalable
Light beam focal position on the equivalent back focal plane of long-focus lens.Again the back focal plane of object lens is adjusted
To overlapping with the equivalent back focal plane of long-focus lens, focusing on light beam can be parallel after microcobjective
Outgoing.Change optical fiber head, in the position of collimating lens front focal plane, is equivalent to regulate exiting parallel optical illumination
The angle of incidence of sample.Sample is placed in the front focal plane of object lens, is adjusted to be more than by the angle of incidence of illumination light
Critical angle so that parallel illumination light occurs total reflection to produce evanescent wave, to fluorescent samples at sample surfaces
Skin layer excite.
(2) fluorescent samples is excited by evanscent field and sends fluorescence, arranges scanning in receiving light path part and shakes
The light that the point source of sample different piece sends is scanned receiving by mirror.It is placed in object lens front focal plane
The light that certain point on sample sends receives formation directional light through object lens and is reflected mirror reflection and two again
Scanning galvanometer is entered after color mirror transmission.Collect by being focused lens after scanning galvanometer, then through spectroscope
(PBS) be divided into two-way light beam, respectively two apertures are placed in before the focus point of two-way light beam with point after,
The light intensity that two apertures receive is separately detected with two detectors.The light that two detectors are detected
Signal is converted to the signal of telecommunication and passes to computer, by controlling scanning galvanometer, sample surfaces is carried out two dimension
Scanning, obtains the out-of-focus image on two sample top layers, is two-dimensional scan figure and defocused two dimension before Jiao respectively
Scanning figure.
(3) to be believed by the degree of depth of luminous point in the difference result of figure and defocused figure obtains sample before Jiao
Breath, it is necessary first to the degree of depth and difference result are demarcated.First a light-emitting zone the least is selected,
Realized the movement of the axial nanometer scale of luminous point by the nanometer translation stage controlling to be connected with sample, pass through
I.e. different depth light-emitting zone time two detectors obtain being in axially different position corresponding to sample
Jiao Qiantu and defocused figure, then the difference result of the degree of depth with Jiao Qiantu and defocused figure is fitted, thus
Obtain the calibration curve of the degree of depth and difference result.
(4) after having demarcated, nanometer translation stage is set back, by computer to sample top layer
The burnt front figure of two dimension and defocused figure that scanning draws carry out difference processing, further according to calibration curve, obtain sample
The nano level axial location of product top layer regional.
The principle of the invention is as follows:
The present invention, at illuminator section, uses the evanescent wave illumination side of general total internal reflectance microscope
Method, by long-focus lens by the laser beam focusing after collimation at the back focal plane of microcobjective
On, light beam is by the sample surfaces of directional light irradiation at a certain angle after object lens, by regulation light source position
Control the incident angle of light beam to ensure that incident illumination is totally reflected at sample surfaces.Regulation sample
The thin layer that is excited is near the focal plane of object lens.Receiving device part, employing focuses on identical together
The reception mode of spot scan.By each Mapping of sample surfaces by the way of vibration mirror scanning.
At probe portion, it is divided into two parts to respectively enter two detections by spectroscope the light beam received little
Hole, before one of them detection aperture is placed on the back focus of condenser lens, another detection aperture is placed
After the back focus of condenser lens, deduct the defocused light intensity detected by the light intensity detected before Jiao
The axial location of the luminous point that degree judgement sample is excited in thin layer.By this method can be general
Total internal reflection on the basis of, omit the step of axial scan, only can realize axle by two-dimensional scan
To hi-Fix.
Compared with prior art, the present invention has a following useful technique effect:
(1) high accuracy achieving axial no-raster axially positions.
(2) apparatus structure is simple, convenient data processing.
Accompanying drawing explanation
Fig. 1 is the axial high-precision positioner signal of evanescent wave based on common total internal reflection illumination
Figure；
Fig. 2 is axial high-precision positioner signal based on the illumination of Dynamic Annular total internal reflection evanescent wave
Figure；
Fig. 3 is that sample is excited top layer schematic diagram, and wherein region 1 represents and is placed in condenser lens focal plane
The light-emitting zone that aperture below can receive, region 2 represents the sample top layer excited by evanescent wave,
Region 2 represents and is placed in the light-emitting zone that the aperture before condenser lens focal plane can receive；
I in Fig. 41、I2Be respectively before Jiao, light intensity and the axle of luminous point in sample of defocused pinhole Detection
To the relation curve of the degree of depth, I3Relation curve for difference result Yu luminous point axial depth；
Fig. 5 (a) is the scan ring schematic diagram of focus point on microcobjective back focal plane in case study on implementation two；
Fig. 5 (b) is microcobjective partial devices enlarged diagram in case study on implementation two.
Detailed description of the invention
Describe the present invention in detail below in conjunction with embodiment and accompanying drawing, but the present invention is not limited to this.
Embodiment 1
As it is shown in figure 1, a kind of three-dimensional high-precision based on common total internal reflection microscope mode is followed the tracks of
Positioner, including optical fiber laser 1, collimating lens 2, long-focus lens 3, the first reflecting mirror 4,
Dichroscope 5, the second reflecting mirror 6, microcobjective 7, sample 8, nanometer translation stage 9, scanning galvanometer
10, condenser lens 11, beam splitter 12, the first detection aperture 13, the first photodetector 14, the
Two detection apertures 15, the second photodetector 16, computer 17.
Using the device shown in Fig. 1 to realize for fluorescent samples, the present embodiment illuminates based on evanescent wave
Axially high-precision locating method, its process is as follows:
(1) optical fiber laser 1 sends illumination light, after collimating lens 2 collimates, through long-focus
Lens 3 are assembled；
(2) front focal plane of long-focus lens overlaps with the back focal plane of collimating lens 2, and light beam is through focal length
After lens 3 are assembled, respectively after the first reflecting mirror 4, dichroscope 5 and the second reflecting mirror 6 reflect
Focus on plane C a bit, plane C is the equivalent back focal plane of long-focus lens 3.By image
Relation can obtain, and the front focal plane A of collimating lens 2 is (equivalent with the back focal plane B of long-focus lens 3
Back focal plane C) conjugation.By regulation optical fiber laser optical fiber head in collimating lens front focal plane (plane
A) position can change light beam focus at long-focus lens equivalence back focal plane (plane C)
Position.
(3) back focal plane of regulation microcobjective 7 and the equivalent back focal plane of long-focus lens 3 are (flat
Face C) overlap, focus on the light beam of plane C after microcobjective there is certain angle with optical axis
Sample 8 is irradiated in collimated light beam outgoing.Illumination light is controlled in the position of plane A by regulation optical fiber head
It is incident in the angle of sample surfaces so that it is exceed critical angle and occur total reflection generation suddenly to die at sample surfaces
Ripple excites fluorescent samples.
(4) sample being positioned at object lens front focal plane is inspired fluorescence, is collected by microcobjective 7,
Reflect through the second reflecting mirror 6, then after dichroscope is transmitted into scanning galvanometer, line focus lens 11
After convergence, be split device 12 light splitting.Two-beam is little with two defocused detections before being respectively disposed in Jiao
Hole receives, U in figureMFocal plane for condenser lens 11.
(5) as it is shown on figure 3, luminous point is positioned at the different degree of depth, burnt front and defocused detection aperture
The light intensity received is different.Be placed in the detection aperture reception before Jiao is such as region 1 size in Fig. 3
The light that sends of Airy disk region, be placed in that defocused detection aperture receives is as big in region 3 in Fig. 3
The light that little Airy disk region sends, is positioned at the light that the luminous point of the axially different degree of depth sends the most burnt
Light intensity I that front and defocused detection aperture receives1、I2Relation curve such as Fig. 4 institute with its axial depth
Show, by difference I2-I1Curve I can be obtained3.Can in the range of certain depth difference result I3With
Axial depth becomes relation one to one, and luminous point is positioned at the different degree of depth, its difference result obtained
It is different.Want the result by difference detecting and obtain the axial location of its luminous point, it is necessary first to
To difference result I3Demarcate with the luminous point degree of depth.
(6) first select a light-emitting zone the least, control it by nanometer translation stage relatively deep
Degree, and measure its corresponding I3, obtain I3A lot of to discrete point with degree of depth h, then click on discrete
Row matching, obtains an I3The matched curve of-h.
(7) after having demarcated, sample top layer is carried out two-dimensional scan, measure the I of each point3, can obtain
Obtain the degree of depth of each luminous point of sample top layer.
Embodiment 2.
As in figure 2 it is shown, a kind of three-dimensional super-resolution based on Dynamic Annular total internal reflection illumination mode is micro-
Device, including optical fiber laser 1, collimating lens 2, scanning galvanometer 18, long-focus lens 3,
One reflecting mirror 4, dichroscope 5, the second reflecting mirror 6, microcobjective 7, sample 8, nanometer translation stage
9, scanning galvanometer 10, condenser lens 11, beam splitter 12, the first detection aperture 13, the first photoelectricity
Detector 14, the second detection aperture 15, the second photodetector 16, computer 17.
The device shown in Fig. 2 is used to realize the one for fluorescent samples based on Dynamic Annular total internal reflection
The axial high-precision locating method of evanescent wave illumination, its process is as follows:
(1) optical fiber laser 1 sends illumination light, after collimating lens 2 collimates, enters scanning
Galvanometer 18, the zero diopter of scanned galvanometer 18 outgoing is focused on its back focal plane through long-focus lens 3；
(2) long-focus lens 3 is a long-focus lens, light beam through long-focus lens 3 assemble after,
The one of plane A is focused on respectively after the first reflecting mirror 4, dichroscope 5 and the second reflecting mirror 6 reflect
Point, plane A is the equivalent back focal plane of long-focus lens 4.
(3) back focal plane of regulation microcobjective 7 and the equivalent back focal plane of long-focus lens 3 are (flat
Face A) overlap, focus on the light beam of plane A after microcobjective there is certain angle with optical axis
Sample 8 is irradiated in collimated light beam outgoing.
(4) control light beam focal position in plane A by control scanning galvanometer 18, make light
Beam focus is formed shown in circular scanning such as Fig. 5 (a) of certain radius in plane A, and requirement
The radius of this circular scanning meets
r/f≥arctanθc=nSample/nOil
As shown in Fig. 5 (b), wherein r is the radius of plane A annular scanning, and f is microcobjective
Focal length, θcLight beam can occur the critical angle of total reflection, nSampleFor the refractive index of sample, nOilMicro-
The refractive index of object lens immersion oil.
Light beam irradiates sample surfaces at a certain angle by after microcobjective.Control by controlling galvanometer
The radius that focus point scans at plane A annular is so that light beam faces to exceed after microcobjective 7
The angle of incidence at angle, boundary is irradiated in sample surfaces experiences total internal reflection.Then sample surfaces is had same incidence
Angle, the light beam circulation illumination of different orientations.
(4) sample being positioned at object lens front focal plane is inspired fluorescence, is collected by microcobjective 7,
Reflect through the second reflecting mirror 6, then after dichroscope 5 is transmitted into scanning galvanometer 10, line focus is saturating
After mirror 11 is assembled, be split device light splitting.Two-beam is respectively disposed in before Jiao and two defocused detections
Aperture receives.
(5) Jiao Qiantu and defocused figure are obtained by the two-dimensional scan of scanning galvanometer 10, then by strictly according to the facts
Execute the identical method of example one to there being each luminous component in sample to carry out axial hi-Fix.
Claims (7)
1. an axial high-precision locating method based on evanescent wave illumination, it is characterised in that include
Following steps:
1) illuminating bundle after collimation focuses on through long-focus lens, then is irradiated to sample table by object lens
Face occurs total reflection to produce evanescent wave, excites sample top layer to send fluorescence；
In step 1) in, the illuminating bundle after collimation is after long-focus lens focuses on microcobjective
On focal plane so that the illuminating bundle through microcobjective is exiting parallel light；
2) being divided into two-way light beam after the fluorescence line focus lens collected, a road light beam is by being positioned at focus point
Front detector receives, and another road light beam is received by the detector after being positioned at focus point, obtains sample table
The Jiao Qiantu of layer and defocused figure；
3) control sample move axially, repeat step 1) and step 2) in operation, obtain sample
Jiao Qiantu under different depth and defocused figure；
4) to step 3) in Jiao Qiantu and defocused figure carry out difference processing, by the degree of depth and difference result
It is fitted, obtains the calibration curve of the degree of depth and difference result；
5) Jiao Qiantu and defocused figure to sample top layer carry out difference processing, bent according to described demarcation
Line, it is achieved the axial location in each region, sample top layer.
2. the axial high-precision locating method illuminated based on evanescent wave as claimed in claim 1, its
It is characterised by, in step 2) in, the light using scanning galvanometer to send sample different piece is swept
Retouch reception.
3. the axial high-precision locating method illuminated based on evanescent wave as claimed in claim 1, its
Being characterised by, the angle of illuminating bundle incidence sample surfaces is more than critical angle.
4. an axial high-precision positioner based on evanescent wave illumination, it is characterised in that including:
Laser instrument, collimating lens, long-focus lens, microcobjective and the nanometer being sequentially arranged along light path
Translation stage, this nanometer translation stage is placed with sample；
Along receive fluorescence light path be sequentially arranged the first scanning galvanometer, collecting lens, beam splitter, and
The first photodetector before laying respectively at the focus point of collecting lens and after focus point and the second photoelectricity
Detector；
With for controlling the computer that nanometer translation stage and data process.
5. the axial high-precision positioner illuminated based on evanescent wave as claimed in claim 4, its
It is characterised by, between described long-focus lens and microcobjective, is sequentially provided with the first reflecting mirror, dichroscope
With the second reflecting mirror.
6. the axial high-precision positioner illuminated based on evanescent wave as claimed in claim 4, its
It is characterised by, between described collimating lens and long-focus lens, is provided with the second scanning galvanometer.
7. the axial high-precision positioner illuminated based on evanescent wave as claimed in claim 4, its
It is characterised by, also includes first adapted with the first photodetector and the second photodetector respectively
Detection aperture, the second detection aperture.
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| CN106772923B (en) * | 2015-11-24 | 2021-01-01 | 睿励科学仪器（上海）有限公司 | Automatic focusing method and system based on inclined slit |
| CN107918184A (en) * | 2016-10-09 | 2018-04-17 | 睿励科学仪器（上海）有限公司 | Non-perpendicular autofocus system and corresponding optical instrument |
| CN107356566B (en) * | 2017-03-30 | 2019-07-30 | 浙江大学 | The positioning of wide field three-dimensional super-resolution and imaging method and device |
| CN108061965B (en) * | 2017-11-30 | 2019-09-20 | 浙江大学 | Three-dimensional super-resolution micro imaging method and device based on varied angle total internal reflection Structured Illumination |
| CN108956561A (en) * | 2018-06-07 | 2018-12-07 | 浙江大学 | Copolymerization coke and annular total internal reflection double mode microscopic system based on scanning galvanometer |
| CN108982455B (en) * | 2018-07-31 | 2020-08-18 | 浙江大学 | Multi-focus light section fluorescence microscopic imaging method and device |
| CN110202414B (en) * | 2019-05-21 | 2020-07-31 | 王勇 | Non-contact high-precision tool setting system based on optical evanescent wave |
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| CN101963582A (en) * | 2010-09-13 | 2011-02-02 | 深圳大学 | Three-dimensional fluorescence nano microscope imaging method and system, and image equipment |
| CN102589466A (en) * | 2012-01-17 | 2012-07-18 | 浙江大学 | Contour microscopic method and device |
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