CN103292690A - Synthetic aperture microscopy method and device on basis of light field selection - Google Patents
Synthetic aperture microscopy method and device on basis of light field selection Download PDFInfo
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- CN103292690A CN103292690A CN2013102070729A CN201310207072A CN103292690A CN 103292690 A CN103292690 A CN 103292690A CN 2013102070729 A CN2013102070729 A CN 2013102070729A CN 201310207072 A CN201310207072 A CN 201310207072A CN 103292690 A CN103292690 A CN 103292690A
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Abstract
The invention discloses a synthetic aperture microscopy method on the basis of light field selection. The synthetic aperture microscopy method includes steps of (1) after processing probe light by means of expanding and deflecting, irradiating the probe light to a sample and collecting transmission light penetrates the sample by the aid of a microscopy imaging system, (2) combining and polarizing uniformly reference light with the transmission light collected by the microscopy imaging system after processing the reference light by means of expanding and deflecting, and recording an interference pattern of the two light beams through an image sensor, (3) changing irradiating angle of the probe light to the sample, repeating the steps (1) and (2) to acquire multiple interference patterns corresponding to multiple angles, and (4) obtaining phase distribution of each interference pattern by means of Fourier transform, and finally reconstructing to obtain microscopy images by a randon algorithm. The invention further discloses a synthetic aperture microscopy device on the basis of light field selection. By the synthetic aperture microscopy method with the light field selection, synthetic aperture of a microscope is increased effectively, lateral resolution and longitudinal resolution are improved, and super resolution effect is achieved.
Description
Technical field
The invention belongs to the micro-field of optical ultra-discrimination, particularly a kind of synthetic aperture microscopic method and device of selecting based on light field.
Background technology
21 century is the period of biology information technology develop rapidly, and is in this continuous process of development, also more and more urgent for the demand of the observation of the subcellular structure of living cells.At first, in order to realize to avoid the observation of living cells nature state the use of coloring agent as far as possible.Simultaneously owing to most of biological sample is that water white measurement by phase place is a kind of method that generally adopts at present, because microcobjective itself has limited the low-frequency information that can only receive sample light, be the important channel of improving resolution so how to enlarge the numerical aperture of microcobjective.
The Three-dimensional differe ntial interference contrast microscopy using synthetic aperture imaging that publishes thesis of Wonshik Choi team in 2012.Carry out the effect that light field simulation has realized enlarging the microcobjective virtual aperture by the mode of 2-D vibration mirror scanning.Improve the resolution of transmission-type phase microscope, and applied for relevant patent.But problem wherein is the two-dimensional scan galvanometer owing to do not stablize fixed point in the scanning process, so will inevitably introduce certain systematic error in the process of synthetic aperture.
Yann Cotte in 2013 Marker-free phase nanoscopy that publishes thesis.To import the light rotation by the method for using prism wedge, realize enlarging the effect of microcobjective virtual aperture.Improve resolution, and applied for relevant patent.Their scheme can be introduced certain angle of inclination from principle in prism wedge rotation, so and lack adjustability because the angle of wedge of prism wedge is fixed in the process of synthetic aperture.
Summary of the invention
In order to solve the present deficiency of optical ultra-discrimination field about existing in the synthetic aperture method, enrich the associative operation mode in synthetic aperture field simultaneously, the invention provides a kind of synthetic aperture microscopic method and device of selecting based on light field, effectively increased microscopical synthetic aperture by the method that adds the light field selection.The present invention is simple in structure, and lateral resolution and longitudinal frame all are significantly improved, and can be used for fields such as optical microphotograph field and biological subcellular structure information are obtained, high precision detection.
A kind of synthetic aperture microscopic method of selecting based on light field may further comprise the steps:
1) surveys light and shine on the sample after deviation is handled through expanding, utilize micro imaging system to collect the transmitted light that sees through sample;
2) reference light is handled transmission combiner and the unified polarization that back and micro imaging system are collected through expanding deviation, passes through the interference pattern of image recording sensor two-beam again;
3) change the angle that detection illumination is mapped to sample, repeating step 1) and step 2), many interference patterns corresponding with a plurality of angles obtained;
4) utilize Fourier transform method to obtain the PHASE DISTRIBUTION of every interference pattern, obtain micro-image by the reconstruct of randon algorithm again.
Described detection light and reference light are obtained by same laser beam light splitting, make reference light and survey that light frequency is identical, phasic difference is constant and direction of vibration is consistent, just can make the two-beam that closes behind the bundle produce interference.
It is different with the multiple that reference light enlarges to survey light, and the multiple that described detection light expands is 7～8 times, and the multiple that described reference light expands is 4～6 times.
Through expand that deviation handles-survey light after light field is selected, to shine on the sample, described light field is selected to be used for to focus on surveying the light center uniform parts.Light beam focuses on back focus and drops on the back focal plane of convergent lens, and the laser by convergent lens becomes directional light and shines on the sample like this.
The present invention also provides a kind of synthetic aperture microscope equipment of selecting based on light field, comprising:
First laser instrument and the beam expanding lens arranged successively along the reference light light path;
Along surveying second laser instrument, light field selector switch, sample platform and the micro imaging system that the light light path is arranged successively;
Be used for first polarization splitting prism with the transmission combiner of described reference light and micro imaging system collection;
Be used for making the reference light and the transmitted light that close behind the bundle to produce the analyzer of interfering;
Be used for gathering the image acquisition device of interference pattern;
And the computing machine that is used for the described image acquisition device of control and light field selector switch.
Described first laser instrument and second laser instrument are same laser instrument, and the laser beam that described laser instrument sends is divided into described detection light and reference light through second polarization splitting prism.The structure that single laser instrument makes whole microscope equipment is simple relatively, energy consumption is low, realize interfering for making reference light and transmitted light, need the frequency of two-beam identical, phasic difference is constant and direction of vibration is consistent, and the light beam that two laser instruments send is difficult to meet this requirement, but the reference light and the transmitted light that are obtained by single laser instrument light splitting more easily produce interference.
Described beam expanding lens is the 4f system, and this 4f system comprises the first preceding beam expanding lens, first catoptron and the first back beam expanding lens of arranging successively along light path.The 4f system is used for reference light is expanded, make reference light with a tight waist expand to the clear aperature of CCD close.
The light field selector switch has two kinds of selectable schemes, first kind is: described light field selector switch is made up of the beam-expanding system of arranging successively along light path and light field option board, described beam-expanding system comprises the second preceding beam expanding lens, second catoptron and the second back beam expanding lens of arranging successively along light path, described light field option board comprises rotating disk and the eccentric lens that are inlaid on the rotating disk, and described rotating disk is controlled by described computing machine; Second kind is: described light field selector switch comprises fiber coupler, polarization maintaining optical fibre, the optical fiber exit port of arranging successively along light path, and described optical fiber exit port off-centre is installed on the rotating disc, and described rotating disc is controlled by described computing machine.
In the light field selector switch of first kind of scheme, beam-expanding system is used for detecting light beam is extended to scope of activities can cover lens rotation on the light field selector switch time, and lens only to beam center relatively uniformly part carry out selective focus; Light field selector switch in second kind of scheme, fiber coupler couples light to detection in the polarization maintaining optical fibre, the other end of polarization maintaining optical fibre is connected with the optical fiber exit port, optical fiber exit port port also has micro lens to focus on again beam projecting light, this optical fiber exit port rotates with a rotating disc, be used for to change surveys illumination and is mapped to angle on the sample.
As preferably, described image acquisition device is CCD.
Principle of work of the present invention is as follows:
Laser shines to be divided on first polarization spectroscope surveys light and reference light, focus on the back focal plane of convergent lens after surveying light process light field selector switch, lens in the light field selector switch are with the 4f system of convergent lens formation from axle, when the lens eccentric position adjustment of light field selector switch can make light can focus on the different point of assembling back focal plane with following when rotating disk rotates together, the light that sees through convergent lens like this can be with different angular illumination to sample.Because the restriction in the aperture of microcobjective own, microcobjective can only receive the information of sample low frequency part during single angular illumination sample, and when multi-angle was shone, the sample message that angle of inclination incident obtains belonged to high-frequency information for straight incident.Transmitted light and reference light overlap at polarization splitting prism, realize the perfection interference of two-beam by the effect of analyzer.Survey the angle that illumination is mapped to sample by adjusting to change, and note the interferogram that respective angles is thrown light on high-speed CCD, use
Expression, wherein
Wave vector for irradiates light.
The first step is to the data that obtain
Carry out Fourier transform; In second step, the one-level frequency spectrum that obtains after adopting the shift frequency principle with Fourier transform moves on to the position of zero level frequency spectrum, adopts Hamming window to carry out filtering again, keeps the spectrum information of reflection object phase place; In the 3rd step, the information behind the shift frequency of second step acquisition is carried out inverse Fourier transform obtain PHASE DISTRIBUTION information Z
θ 
In the 4th step, pass through formula
Calculate the PHASE DISTRIBUTION under this wave vector angle; The 5th step, because each
All corresponding some angles is following
PHASE DISTRIBUTION, adopt the method for randon conversion to realize that three-dimensionalreconstruction obtains the three-dimensional micro-image of super-resolution.
The present invention is based on the three-dimensional microtechnic of synthetic aperture, by the effect that selection and simulation to light field have realized enlarging numerical aperture, realized the effect of super-resolution, and because system is more succinct, control conveniently, good economic advantages are arranged.
Compared with prior art, the present invention has following beneficial technical effects:
The error ratio of (1) bringing in light field simulation is less than existing systems;
(2) lateral resolution significantly improves than simple microscope, and lateral resolution can reach below the 200nm;
(3) apparatus structure is succinct, makes things convenient for the quick high accuracy adjustment, and economy is strong.
Description of drawings
Fig. 1 the present invention is based on the structural representation that light field is selected the microscope equipment of synthetic aperture.
Fig. 2 is a kind of embodiment of light field selector switch.
Fig. 3 is the structural representation of light field option board.
Fig. 4 is the another embodiment of light field selector switch.
Embodiment
Describe the present invention in detail below in conjunction with embodiment and accompanying drawing, but the present invention is not limited to this.
As shown in Figure 1, a kind of synthetic aperture microscope equipment of selecting based on light field comprises: laser instrument 1, focus lamp 2, aperture 3, collimation lens 4, reflective mirror 5, polarization splitting prism 6, light field selector switch 7, convergent lens 8, sample 9, microcobjective 10, field lens 11, preceding beam expanding lens 12, reflective mirror 13, back beam expanding lens 14, polarization splitting prism 15, polarization analyzer 16, high-speed CCD 17 and main control computer 18.
Light field selector switch 7 has two kinds of different structures.First kind of structure seen Fig. 2, comprises preceding beam expanding lens 19, catoptron 20, back beam expanding lens 21 and the light field option board 22 arranged successively along light path; As shown in Figure 3, the light field option board is made up of rotating disk 24 and the eccentric micro objective 23 that is inlaid on the rotating disk, and rotating disk is by the stepper motor driving and be controlled by main control computer 18.
As shown in Figure 4, the light field selector switch 7 of second kind of structure comprises light coupling mechanism 25, polarization maintaining optical fibre 26, optical fiber exit port 27 and the rotating disc 28 that connects successively, optical fiber exit port 27 is positioned on the rotating disc 28 and with its rotation, and rotating disc 28 is controlled by main control computer 18.
The course of work of said apparatus is as follows:
Laser instrument 1 sends laser beam, focus lamp 2, aperture 3 and collimation lens 4 are formed small filter, be used for laser beam is carried out filtering and collimation, polarization spectroscope 6 is divided into laser surveys light and reference light, reference light is through preceding beam expanding lens 12, catoptron 13 and back beam expanding lens 14 expand deviation, surveying light is incident in the light field selector switch 7, and focus on difference on convergent lens 8 front focal planes after expanding deviation by light field selector switch 7, the detection light that convergent lens 8 will focus on front focal plane converts directional light to and shines on the sample 9, and microcobjective 10 receives the transmitted light that sees through sample and cooperates the formation micro imaging system with field lens 11; Polarization splitting prism 15 is with transmitted light with reference to combiner, and the polarization of analyzer 16 unified two-beams is also realized perfect the interference, noted the interference pattern information of reference light and transmitted light with high-speed CCD 17.Main control computer 18 is connected with high-speed CCD 17 with light field selector switch 7, be used for coordinating the rotation of light field selector switch and the shooting of high-speed CCD 17, and deal with data obtains high-resolution sample image.
Wherein, in two kinds of structures of light field selector switch, if use the light field selector switch among Fig. 2, survey light by preceding beam expanding lens 19, catoptron 20, back beam expanding lens 21 is realized expanding and turning to, be mapped on the light field option board 22 through the detection illumination after expanding, the rotating disk 24 of light field option board rotates under the drive of stepper motor, and the part light that micro objective 23 will shine thereon focuses on again, focuses on the back focal plane of convergent lens 8; If use the light field selector switch among Fig. 4, surveying illumination is mapped on the fiber coupler 25, the at utmost coupling that focus lamp by adjusting fiber coupler and angle realize light beam, because polarization maintaining optical fibre 26 pliabilities can be adjusted to light the angle of wanting easily, the other end of polarization maintaining optical fibre links to each other with optical fiber exit port 27, and optical fiber exit port 27 embedded micro lens can focus on the light that polarization maintaining optical fibre 26 passes on the back focal plane of convergent lens 8.Optical fiber exit port 27 can be followed the dish 23 of walking around and be rotated together, realizes that laser focuses on the difference on convergent lens 8 back focal planes.
Above-mentioned device is realized the micro-method of synthetic aperture, and its process is as follows:
(1) laser instrument 1 is launched light beam, and line focus lens 2, aperture 3 and collimation lens obtain collimated light beam, and this collimated light beam is divided into through polarizing beam splitter 5 surveys light and reference light;
(2) employing light field selector switch as shown in Figure 2, surveying illumination is mapped on the light field selector switch 7, through preceding beam expanding lens 19, catoptron 20, back beam expanding lens 21 shines light beam expansion on the light field option board 22 then, the position of beam expanding lens 19 and back beam expanding lens 21 guarantees that the beam center homogeneous area that enlarges can cover the scope of micro objective 23 activities before adjusting, the part light that micro objective 23 will be radiated at above it focuses on again, the distance of adjusting between light field option board 22 and the convergent lens 8 guarantees that its focus is on the back focal plane of convergent lens 8, detection light becomes directional light through convergent lens 8 and is radiated on the sample 9, the transmitted light that sees through sample is received by microcobjective 10, cooperates by supporting with it microcobjective field lens 11 to shine on the polarization splitting prism 15;
(3) reference light expands through preceding beam expanding lens 12, catoptron 13 and back beam expanding lens 14 and turns to, the position of beam expanding lens 12 and catoptron 13 is positioned on the back focal plane of preceding beam expanding lens 12 reflecting surface before adjusting, the position of adjusting back beam expanding lens 14 guarantees that its front focus is at the reflecting surface place, can reduce the face shape of catoptron 13 like this to the influence on reference arm light wave corrugated as far as possible, shine on the polarization splitting prism 15 through expanding the reference light that turns to;
(4) adjust the angle of polarization splitting prism 15 and the angle of catoptron 13, guarantee that transmitted light and reference light overlap as far as possible, the illumination after the coincidence is mapped on the analyzer 16, adjusts the angle of analyzer 16, and transmitted light and reference light can perfectly be interfered;
(5) take the interference pattern of two-beam down with quick CCD17, be transferred to total control computer 18, total control computer 18 control light field selector switchs 7 carry out can realizing sample is carried out when light field is selected the transillumination of different angles, and record is surveyed illumination and is mapped to angle on the sample and the interference pattern corresponding with this angle.The light field selector switch rotates a circle, and finishes a required data acquisition of three-dimensionalreconstruction, obtains many interference patterns that mate with angle;
(6) 18 pairs of every interference patterns of main control computer utilize Fourier transform method to obtain PHASE DISTRIBUTION, obtain high-resolution micro-image by the randon conversion again.
Claims (10)
1. a synthetic aperture microscopic method of selecting based on light field is characterized in that, may further comprise the steps:
1) surveys light and shine on the sample after deviation is handled through expanding, utilize micro imaging system to collect the transmitted light that sees through sample;
2) reference light is handled transmission combiner and the unified polarization that back and micro imaging system are collected through expanding deviation, passes through the interference pattern of image recording sensor two-beam again;
3) change the angle that detection illumination is mapped to sample, repeating step 1) and step 2), many interference patterns corresponding with a plurality of angles obtained;
4) utilize Fourier transform method to obtain the PHASE DISTRIBUTION of every interference pattern, obtain micro-image by the reconstruct of randon algorithm again.
2. the synthetic aperture microscopic method of selecting based on light field as claimed in claim 1 is characterized in that the multiple that described detection light expands is 7～8 times, and the multiple that described reference light expands is 4～6 times.
3. the synthetic aperture microscopic method of selecting based on light field as claimed in claim 2 is characterized in that, the detection light through expanding the deviation processing shines on the sample after light field is selected, and described light field is selected to be used for to focus on surveying the light center uniform parts.
4. the synthetic aperture microscopic method of selecting based on light field as claimed in claim 1 is characterized in that described detection light and reference light are formed by same laser beam light splitting.
5. a synthetic aperture microscope equipment of selecting based on light field is characterized in that, comprising:
First laser instrument and the beam expanding lens arranged successively along the reference light light path;
Along surveying second laser instrument, light field selector switch, sample platform and the micro imaging system that the light light path is arranged successively;
Be used for first polarization splitting prism with the transmission combiner of described reference light and micro imaging system collection;
Be used for making the reference light and the transmitted light that close behind the bundle to produce the analyzer of interfering;
Be used for gathering the image acquisition device of interference pattern;
And the computing machine that is used for the described image acquisition device of control and light field selector switch.
6. the synthetic aperture microscope equipment of selecting based on light field as claimed in claim 5, it is characterized in that, described first laser instrument and second laser instrument are same laser instrument, and the laser beam that described laser instrument sends is divided into described detection light and reference light through second polarization splitting prism.
7. the synthetic aperture microscope equipment of selecting based on light field as claimed in claim 5 is characterized in that described beam expanding lens is the 4f system, and this 4f system comprises the first preceding beam expanding lens, first catoptron and the first back beam expanding lens of arranging successively along light path.
8. the synthetic aperture microscope equipment of selecting based on light field as claimed in claim 5, it is characterized in that, described light field selector switch is made up of the beam-expanding system of arranging successively along light path and light field option board, described beam-expanding system comprises the second preceding beam expanding lens, second catoptron and the second back beam expanding lens of arranging successively along light path, described light field option board comprises rotating disk and the eccentric lens that are inlaid on the rotating disk, and described rotating disk is controlled by described computing machine.
9. the synthetic aperture microscope equipment of selecting based on light field as claimed in claim 5, it is characterized in that, described light field selector switch comprises fiber coupler, polarization maintaining optical fibre, the optical fiber exit port of arranging successively along light path, the beam projecting device can be realized the beam projecting light beam is focused on again, described optical fiber exit port off-centre is installed on the rotating disc, and described rotating disc is controlled by described computing machine.
10. the synthetic aperture microscope equipment of selecting based on light field as claimed in claim 5 is characterized in that described image acquisition device is CCD.
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