CN108051909B - Extended focal depth microscopic imaging system combining optical tweezers function - Google Patents
Extended focal depth microscopic imaging system combining optical tweezers function Download PDFInfo
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- 238000003384 imaging method Methods 0.000 title claims abstract description 38
- 238000012576 optical tweezer Methods 0.000 title claims abstract description 25
- 230000003287 optical effect Effects 0.000 claims description 27
- 238000000386 microscopy Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 9
- 238000007493 shaping process Methods 0.000 claims description 7
- 238000002310 reflectometry Methods 0.000 claims description 4
- 238000002834 transmittance Methods 0.000 claims description 4
- 230000005540 biological transmission Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 description 64
- 238000010586 diagram Methods 0.000 description 5
- 238000006073 displacement reaction Methods 0.000 description 4
- 239000012472 biological sample Substances 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 2
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001246 colloidal dispersion Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000000651 laser trapping Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000329 molecular dynamics simulation Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0036—Scanning details, e.g. scanning stages
- G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/008—Details of detection or image processing, including general computer control
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Abstract
The invention discloses an extended focal depth microscopic imaging system combining optical tweezers functions, which comprises: the laser comprises a first laser, a first reflecting mirror, a first spectroscope, a first lens, a second lens, a first acousto-optic deflector, a third lens, a fourth lens, a fifth lens, a second acousto-optic deflector, a sixth lens, a second spectroscope, a first dichroic mirror, a second dichroic mirror, a first objective lens, a sample stage, a second objective lens, a third dichroic mirror, a third spectroscope, a fourth spectroscope, a fifth spectroscope, a first four-quadrant position detector, a second four-quadrant position detector, a position detector, an LED light source, a first detector, a second laser, a first cylindrical lens, a cylindrical lens group, a fourth dichroic mirror, a scanning galvanometer, a first telescopic system, an electric control lens, a second telescopic system, a second reflecting mirror, a third telescopic system, a slit, a third reflecting mirror, a second cylindrical lens and a second detector. The system scans different focal depth planes by using the electric control lens, and has no mechanical vibration and high imaging quality.
Description
Technical Field
The invention relates to the field of microscopic imaging and optical tweezers, in particular to an extended focal depth microscopic imaging system combining the functions of the optical tweezers.
Background
In the research of cell biology, it is often necessary to simultaneously study the mechanical and mechanical properties of biomolecules in cells and the three-dimensional structure of cells. Therefore, in the practical research process, an experiment is usually performed by adopting a single-molecule mechanical microscopic system combining an optical tweezers module and a fluorescence microscopic imaging module. When the single-molecule mechanical microscopic system is used for operating the sample, the optical tweezers module can measure the dynamic characteristics of single molecules in biological cells, and meanwhile, the fluorescence microscopic imaging module can acquire multidimensional information such as biological structures, extracellular or intracellular information and the like.
When the fluorescent microscopic imaging module in the single-molecule mechanical microscopic system is used for realizing real-time three-dimensional biological structure imaging of a sample, the fluorescent microscopic imaging module is usually realized by using a total internal reflection microscope or a confocal laser scanning microscope. In 2004, lang Matthew J et al published in Nature Methods journal entitled "Simultaneous, coincident optical trapping and single-molecule fluorescence" put forward a single-molecule mechanical microscope system using a combination of a total internal reflection microscope and optical tweezers, where the total internal reflection microscope uses the characteristic that the penetration depth of optical energy in an optical-hydrophobic medium is limited and the optical energy propagates only along an interface during total reflection, to selectively excite a fluorescent mark on a fluorescent surface, that is, acquire information on the sample surface by capturing evanescent waves on the sample surface, and the penetration depth of the evanescent waves is below 200 nm, so that the total internal reflection microscope is not suitable for scanning in the depth direction of micrometer scale, and thus cannot realize three-dimensional structural imaging of micrometer scale biological samples; in the same year, vossen Dirk L.J et al, journal Review ofScientific Instruments, entitled "Optical tweezers and confocalmicroscopy for simultaneous three-dimensional manipulation and imaging inconcentrated colloidal dispersions," propose a single-molecule mechanical microscopy system using confocal laser scanning microscopy in combination with optical tweezers, where the focal plane of the confocal laser scanning microscopy is fixed, and scanning in the depth direction of the sample can be achieved by moving the micro-displacement stage of the sample or manipulating the position of the objective lens. However, when the confocal laser scanning microscope with the micro displacement table is used and the micro displacement table is moved to realize scanning of the sample in the depth direction, the connection between the particles detected by the optical tweezers and the pressure sensor is interrupted along with the vertical movement of the sample table, so that the measurement of the mechanical and mechanical properties of the molecules by the optical tweezers module in the single-molecule mechanical microscope system is directly influenced; the micro displacement table is kept fixed, when the position of the objective lens in the depth direction of the sample is controlled to scan samples with different depths, the objective lens with large numerical aperture is not suitable for fluorescence imaging of single molecules, and when the single-molecule mechanical microscope system is used for capturing particles, measuring mechanical properties of the molecules and the like, the movement of the objective lens can cause deflection of capturing light, and finally the optical tweezers are influenced to capture target particles. These technical problems limit scanning imaging of the single-molecule mechanical microscope system in the depth direction of the sample to a great extent, so that three-dimensional imaging of biological microstructures cannot be completed.
Disclosure of Invention
The invention provides an extended focal depth microscopic imaging system combining optical tweezers functions, aiming at the problem that the existing single-molecule mechanical microscopic system has limitation on the scanning depth of a sample in the depth direction when a sample biological structure is subjected to real-time three-dimensional imaging. The system can accurately control particles by combining optical tweezers, and has the advantages of higher imaging quality, rapidness and stability and lower cost, and no error caused by mechanical vibration while realizing three-dimensional imaging.
An extended depth of focus microscopy imaging system incorporating optical tweezers functionality, comprising: the laser comprises a first laser, a first reflecting mirror, a first spectroscope, a first lens, a second lens, a first acousto-optic deflector, a third lens, a fourth lens, a fifth lens, a second acousto-optic deflector, a sixth lens, a second spectroscope, a first dichroic mirror, a second dichroic mirror, a first objective lens, a sample stage, a second objective lens, a third dichroic mirror, a third spectroscope, a fourth spectroscope, a fifth spectroscope, a first four-quadrant position detector, a second four-quadrant position detector, a position detector, an LED light source, a first detector, a second laser, a first cylindrical lens, a cylindrical lens group, a fourth dichroic mirror, a scanning galvanometer, a first telescopic system, an electric control lens, a second telescopic system, a second reflecting mirror, a third telescopic system, a slit, a third reflecting mirror, a second cylindrical lens and a second detector.
The first laser output beam is reflected by the first reflecting mirror and then enters the first spectroscope, and the first spectroscope divides the beam into a first beam and a second beam; the first light beam is transmitted through the first spectroscope, and then reflected from the second spectroscope to the first dichroic mirror after passing through the first lens, the second lens, the first acousto-optic deflector and the third lens; the second light beam is reflected by the first spectroscope, transmitted by the second spectroscope through the fourth lens, the fifth lens, the second sound deflector and the sixth lens and also reaches the first dichroic mirror. The first light beam and the second light beam are reflected by the first dichroic mirror and transmitted by the second dichroic mirror, and finally focused on a sample table by the first objective lens to form two optical potential wells, so as to capture and measure particles in the sample; the first light beam and the second light beam are collected by the second objective lens after passing through the sample, reflected by the third dichroic mirror and reaching the third spectroscope to be split; the part of the first light beam and the second light beam transmitted by the third spectroscope is transmitted by the fourth spectroscope to reach the first four-quadrant position detector, and the part of the first light beam and the second light beam transmitted by the third spectroscope is reflected by the fourth spectroscope to reach the position detector; the part of the first light beam and the second light beam reflected by the third spectroscope is transmitted to the second four-quadrant position detector through the fifth spectroscope, and the part of the first light beam and the second light beam reflected by the third spectroscope is reflected to the position detector through the fifth spectroscope; the first four-quadrant position detector, the second four-quadrant position detector and the position detector work in parallel to measure the accurate three-dimensional position of the optical potential well.
The second laser output beam reaches the cylindrical lens group after being expanded by the first cylindrical lens, is reflected by the cylindrical lens group to be incident on the scanning vibrating mirror after being subjected to one-dimensional shaping, is reflected by the scanning vibrating mirror to be reflected by the first telescopic system, the electric control lens and the second telescopic system and is reflected by the second reflecting mirror and the second dichroic mirror to the first objective lens, the first objective lens focuses the second laser output beam on a sample, the sample is excited by the irradiation of the beam output by the second laser to generate fluorescence, the fluorescence is collected by the first objective lens, is reflected by the second dichroic mirror and the second reflecting mirror, is reflected by the second telescopic system, the electric control lens and the first telescopic system in sequence, is incident on the third telescopic system after being reflected by the scanning vibrating mirror and is transmitted by the fourth dichroic mirror, is provided with a slit on a confocal surface in the third telescopic system, the slit is used for filtering the fluorescence outside a focusing plane, is collimated by the third telescopic system and reflected by the third reflecting mirror, and is focused by the second cylindrical lens, and is finally collected by the second detector and imaged; the LED light source irradiates the sample after passing through the third dichroic mirror and the second objective, and is collected by the first objective, and the second dichroic mirror and the first dichroic mirror are transmitted and incident on the first detector for imaging.
In the invention, the first laser and the second laser are both point light sources.
In the invention, the first and second optical deflectors are respectively used for changing the deflection angles of the first and second light beams, so as to respectively realize the change of the focusing positions of the first and second light beams in a focusing plane in a sample, namely the regulation and control of the optical potential well positions of the first and second light beams in the focusing plane.
In the invention, the relative positions of the first lens and the fourth lens in the propagation direction along the light path can be adjusted, wherein the position of the first lens can be adjusted to control the first light beam to focus at different positions in the depth direction of the sample, namely, the position of the focusing plane of the first light beam is adjusted; adjusting the position of the second lens can control the second light beam to focus at different positions in the depth direction of the sample, i.e. adjust the position of the second light beam focusing plane.
In the invention, the second lens and the third lens are used for collimating the first light beam, and the fifth lens and the sixth lens are used for collimating the second light beam.
In the present invention, the first dichroic mirror exhibits high reflection to the light beam output from the first laser. The second dichroic mirror exhibits high reflection of the light beam and fluorescence output from the second laser and high transmission of the light beam output from the first laser. The third dichroic mirror exhibits high reflection of fluorescence. The fourth dichroic mirror exhibits high reflectivity for the second laser output beam and high transmissivity for fluorescence. The high transmittance means that the transmittance is more than 98%; the high reflectivity means that the reflectivity is more than 98 percent, and particularly 98 to 99.9 percent.
In the invention, the first four-quadrant position detector is used for detecting the horizontal coordinate of the optical potential well in the direction of the sample focusing plane, and the second four-quadrant position detector is used for detecting the vertical coordinate of the optical potential well in the direction of the sample focusing plane; the position detector is used for detecting the position of the optical potential well in the depth direction of the sample. The first four-quadrant position detector, the second four-quadrant position detector and the position detector work in parallel to determine the accurate three-dimensional position of the optical potential well.
In the present invention, the cylindrical lens group includes two oppositely disposed cylindrical lenses for one-dimensionally shaping the light beam output from the second laser.
In the invention, the one-dimensional shaping means that the cylindrical lens has a focusing effect on the output beam of the second laser in only one direction, so that a focusing light spot of the output beam of the second laser in a focusing plane in the sample is a line.
In the invention, the scanning galvanometer is used for realizing line scanning of the sample in the sample focusing plane, and the scanning direction of the scanning galvanometer is vertical to the line of the focusing light spot in the sample focusing plane.
In the invention, the electric control lens is controlled by voltage, and when the voltage of the electric control lens is smaller than the threshold voltage, the electric control lens is a negative lens, and the emergent light beam passing through the electric control lens is divergent; when the voltage of the electric control lens is equal to the threshold voltage, the electric control lens is a plane mirror, and the emergent light beam passing through the electric control lens is a parallel light beam; when the voltage of the electric control lens is larger than the threshold voltage, the electric control lens is a positive lens, and the emergent light beam passing through the electric control lens is convergent; the emergent light beams passing through the electric control lens in different states are finally converged at different depths of the sample; the parallel emergent beam focusing plane is used as a reference plane, the emergent beam in a convergent shape is finally focused on one side of the reference plane, which is close to the first objective lens, and the emergent beam in a divergent shape is finally focused on one side of the reference plane, which is close to the second objective lens.
In the invention, the first telescopic system, the second telescopic system and the third telescopic system all comprise two oppositely placed cylindrical lenses; the first telescopic system and the second telescopic system are used for eliminating obvious amplification deviation introduced by the electric control lens, and the third telescopic system is used for performing beam expansion collimation on fluorescence.
In the invention, the first detector is a lattice CCD.
In the invention, the second detector is a linear array CCD, and performs linear scanning imaging on fluorescence.
In the invention, the LED light source is used for illuminating a sample and providing a bright field of view for the experiment process.
Preferably, the wavelength of the output beam of the first laser is 1064 nm, and the biological sample absorbs less at this wavelength.
Preferably, the wavelength of the output beam of the second laser is 532 nm, so that the fluorescence of the sample can be effectively excited.
Preferably, the first objective lens is an oil immersed objective lens manufactured by Olympic corporation and having a model number UPlanSApo, the magnification is 100 times, and the numerical aperture is 1.4.
Preferably, the second objective lens is an objective lens with a model number of lumland fln manufactured by olympin corporation, the magnification is 60 times, and the numerical aperture is 1.0.
Preferably, the scanning galvanometer is an optical scanning galvanometer manufactured by Cambridge Technology and having a model number 6231H, and the line scanning width is 15 mm.
Preferably, the slit is a slit with a model number S100R manufactured by Thorlab.
Preferably, the second detector is a high-sensitivity EMCCD camera with model number iXon3 manufactured by Andor company.
Compared with the prior art, the invention has the following beneficial technical effects: .
1. The invention combines the optical tweezers system with the confocal laser scanning microscopic system, realizes particle manipulation and completes real-time imaging of a sample, and has compact system structure.
2. The invention introduces the electric control lens to realize scanning of focusing planes with different focal depths of the sample, does not move the objective lens or the sample table, has no mechanical vibration, high imaging quality, stable imaging and lower cost.
3. The invention realizes line scanning imaging on the sample by utilizing a plurality of cylindrical lenses and the linear array CCD, and has high scanning imaging speed and high sensitivity.
Therefore, compared with the prior art, the technical scheme of the invention can realize rapid scanning of biological samples with different depths of the sample without mechanical vibration and complete three-dimensional imaging while testing molecular dynamics characteristics.
Drawings
FIG. 1 is a schematic diagram of an exemplary embodiment of an extended depth of focus microscopy imaging system architecture incorporating optical tweezers functionality in accordance with the present invention;
wherein: 1. a first laser; 2. a first mirror; 3. a first spectroscope; 4. a first lens; 5. a second lens; 6. a first acousto-optic polarizer; 7. a third lens; 8. a fourth lens; 9. a fifth lens; 10. a second acoustic deflector; 11. a sixth lens; 12. a second beam splitter; 13. a first dichroic mirror; 14. a second dichroic mirror; 15. a first objective lens; 16. a sample stage; 17. a second objective lens; 18. a third dichroic mirror; 19. a third spectroscope; 20. a fourth spectroscope; 21. a fifth spectroscope; 22. a first four-quadrant position detector; 23. a second four-quadrant position detector; 24. a position detector; 25. an LED light source; 26. a first detector; 27. a second laser; 28. a first cylindrical lens; 29. a cylindrical lens group; 30. a fourth dichroic mirror; 31. scanning a vibrating mirror; 32. a first telescopic system; 33. an electric control lens; 34. a second telescopic system; 35. a second mirror; 36. a third telescopic system; 37. a slit; 38. a third mirror; 39. a second cylindrical lens; 40. and a second detector.
FIG. 2 shows different emergent states of parallel light beams after passing through electrically controlled lenses with different control voltages; in fig. 2a, when the control voltage is smaller than the threshold voltage, the electric control lens is a negative lens, the emergent beam is divergent, in fig. 2b, when the control voltage is equal to the threshold voltage, the electric control lens is a plane mirror, the emergent beam is a parallel beam, in fig. 2c, when the control voltage is larger than the threshold voltage, the electric control lens is a positive lens, and the emergent beam is convergent.
Fig. 3 is a waveform diagram of the electronically controlled lens scan control voltage.
Fig. 4 is a waveform diagram of a scanning voltage of a scanning galvanometer.
Detailed Description
The present invention will be described in detail below with reference to the drawings of the specification, but the present invention is not limited thereto.
An optical path diagram of an embodiment of an extended depth of focus microscopy imaging system architecture incorporating optical tweezers functionality of the present invention is shown in fig. 1, the system of this embodiment comprising:
a first laser 1; a first mirror 2; a first spectroscope 3; a first lens 4; a second lens 5; a first acousto-optic polarizer 6; a third lens 7; a fourth lens 8; a fifth lens 9; a second acoustic deflector 10; a sixth lens 11; a second beam splitter 12; a first dichroic mirror 13; a second dichroic mirror 14; a first objective lens 15; a sample stage 16; a second objective lens 17; a third dichroic mirror 18; a third spectroscope 19; a fourth spectroscope 20; a fifth spectroscope 21; a first four-quadrant position detector 22; a second four-quadrant position detector 23; a position detector 24; an LED light source 25; a first detector 26; a second laser 27; a first cylindrical lens 28; a cylindrical lens group 29; a fourth dichroic mirror 30; scanning galvanometer 31; a first telescopic system 32; an electronically controlled lens 33; a second telescopic system 34; a second mirror 35; a third telescopic system 36; a slit 37; a third mirror 38; a second cylindrical lens 39; a second detector 40. The first laser 1 is an LDH-TA-595 laser of PicoQuant company, and the second laser 27 is an LDH-P-C-650B laser of PicoQuant company.
After the output light beam of the first laser 1 is reflected by the first reflecting mirror 2, the propagation direction is changed by 90 degrees, the light beam enters the first spectroscope 3, the light beam transmitted by the first spectroscope 3 is a first light beam, and the light beam reflected by the first spectroscope 3 is a second light beam. The first light beam passes through the first lens 4 and the second lens 5 and then reaches the first acousto-optic deflector 6, wherein the relative position of the first lens 4 in the propagation direction along the light path can be adjusted, and the relative position of the first lens 4 can be adjusted to focus at different positions in the depth direction of the sample, namely the position of a first light beam focusing plane; the first acousto-optic deflector 6 can control the deflection angle of the first light beam, so that the first light beam can be focused on any position on the sample focusing plane direction on the sample stage 16 through the first objective lens 15 after being collimated by the third lens 7, reflected by the second beam splitter 12 and the first dichroic mirror 13 and transmitted by the second dichroic mirror 14; the first lens 4 and the first acousto-optic deflector 6 cooperate to adjust the focusing of the first light beam at three-dimensional different positions in the sample. The second light beam passes through the fourth lens 8 and the fifth lens 9 and then reaches the second optical deflector 10, wherein the relative position of the fourth lens 8 in the propagation direction along the optical path can be adjusted, and the second light beam can be adjusted to focus at different positions in the depth direction of the sample by adjusting the relative position of the fourth lens 8, namely, the position of a second light beam focusing plane is adjusted; the second optical deflector 10 can control the angle deflection of the second light beam, so that the second light beam can be focused at any position in the direction of the focusing plane of the sample through the first objective lens 15 after being collimated by the sixth lens 11, transmitted by the second beam splitter 12, reflected by the first dichroic mirror 13 and transmitted by the second dichroic mirror 14; the fourth lens 8 and the second optical deflector 10 cooperate to adjust the focusing of the second light beam at different positions in three dimensions in the sample. The first light beam and the second light beam can be reflected by the first dichroic mirror 13 and transmitted by the second dichroic mirror 14 through control, and finally, the first light beam and the second light beam are focused by the first objective lens 15 to the vicinity of target particles in a sample on a sample stage to form two optical potential wells, so that the mechanical and mechanical properties of the particles are measured. After the first light beam and the second light beam pass through the sample, the first light beam and the second light beam are collected by the second objective lens 17 and reflected by the third dichroic mirror 18 to reach the third spectroscope 19; the light beam transmitted by the third spectroscope 19 is transmitted to the first four-quadrant position detector 22 through the fourth spectroscope 20, the light beam transmitted by the third spectroscope 19 is reflected to the position detector 24 through the fourth spectroscope 20, the light beam reflected by the third spectroscope 19 is transmitted to the second four-quadrant position detector 23 through the fifth spectroscope 21, and the light beam reflected by the third spectroscope 19 is reflected to the position detector 24 through the fifth spectroscope 21; the first four-quadrant position detector 22 is used for detecting the horizontal coordinate of the optical potential well in the direction of the sample focusing plane, and the second four-quadrant position detector 23 is used for detecting the vertical coordinate of the optical potential well in the direction of the sample focusing plane; the position detector 24 is used to detect the position of the optical potential well in the depth direction of the sample. The first 22, second 23 and position 24 four-quadrant position detectors operate in parallel to determine the exact three-dimensional position of the optical potential well.
After the beam output by the second laser 27 is expanded by the first cylindrical lens 28, one-dimensional shaping of the beam output by the second laser 27 is realized by the cylindrical lens group 29, namely, the beam output by the second laser 27 is incident to the fourth dichroic mirror 30, the scanning galvanometer 31, the first telescopic system 32, the electric control lens 33, the second telescopic system 34, the second reflecting mirror 35, the focusing light spot of the second dichroic mirror 14 and finally the focusing light spot of the first objective lens 15 in the focusing plane of the sample are taken as lines, and meanwhile, the sample emitted fluorescence is collected by the first objective lens 15 and is incident to the second dichroic mirror 14, the second reflecting mirror 35, the second telescopic system 34, the electric control lens 33, the first telescopic system 32, the scanning galvanometer 31, the fourth dichroic mirror 30, the third telescopic system 36, the third reflecting mirror 38 and the focusing light spot of the second cylindrical lens 39 are taken as lines. As shown in fig. 3, which shows a waveform diagram of a scanning voltage of the scanning galvanometer 31, in this embodiment, the scanning frequency of the scanning galvanometer 31 is 50 hz; the light beam reflected by the scanning galvanometer 31 is focused on the sample stage 16 through the first objective lens 15 by the first telescopic system 32, the electric control lens 33, the second telescopic system 34, the second reflecting mirror 35 and the second dichroic mirror 14 to perform fast line scanning on the sample, wherein the scanning direction is in the focusing plane of the sample and is perpendicular to the line of the focusing light spot. While the output beam of the second laser 27 passes through the electrically controlled lens 33, as shown in fig. 2, controlling the voltage of the electrically controlled lens 33 can change the state of the beam exiting the electrically controlled lens 33. In this embodiment, as shown in fig. 2a, when the voltage of the electro-control lens 33 is less than 3 volts, the electro-control lens 33 acts as a negative lens, and the light beam emitted from the electro-control lens 33 is divergent; as shown in fig. 2b, when the voltage of the electro-controlled lens 33 is equal to 3 volts, the electro-controlled lens 33 acts as a plane mirror, and the light beam emitted from the electro-controlled lens 33 is a parallel light beam; as shown in fig. 2c, when the voltage of the electric control lens 33 is greater than 3 volts, the electric control lens 33 acts as a positive lens, and the light beam emitted from the electric control lens 33 is convergent; the outgoing beams in different states are finally converged at different depths of the sample, the focusing plane of the parallel outgoing beams is taken as a reference plane, the outgoing beams in a converging shape are finally focused on one side of the reference plane, which is close to the first objective lens 15, and the outgoing beams in a diverging shape are finally focused on one side of the reference plane, which is close to the second objective lens 17. As shown in fig. 4, in the present embodiment, the frequency of the scan control voltage of the electro-optic lens 33 is 10 hz, and when the scan control voltage of the electro-optic lens 33 is scanned between-3 volts and 3 volts at a frequency of 10 hz, rapid line scanning of the focal plane at different depths of the sample can be achieved. Meanwhile, the scanning frequency of the scanning galvanometer 31 is controlled to be 50 Hz, and the scanning frequency of the voltage controlled by the electric control lens 33 is controlled to be 10 Hz, so that the three-dimensional rapid scanning of the sample can be completed. Fluorescence excited by the rapidly scanned sample is collected by the first objective lens 15, then sequentially passes through the second dichroic mirror 14, the second reflecting mirror 35, the second telescopic system 34, the electric control lens 33, the first telescopic system 32, the scanning galvanometer 31, the fourth dichroic mirror 30, the third telescopic system 36 and the third reflecting mirror 38, and finally is focused on the second detector 40 by the second cylindrical mirror 39 for real-time three-dimensional imaging.
The LED light source 25 is converged on the sample by the second objective lens 17 after passing through the third dichroic mirror 18, and then collected by the first objective lens 15, the second dichroic mirror 14 and the first dichroic mirror 13 are transmitted and incident on the first detector 26 for detection imaging, wherein the LED light source 25 is converged on the sample by the second objective lens 17 to provide a field of view for real-time three-dimensional scanning imaging of the sample.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solution of the patent and not limiting, and that a person skilled in the art may make several variations and modifications without departing from the principles of the patent, which should also be regarded as the protection scope of the patent.
Claims (6)
1. An extended depth of focus microscopy imaging system incorporating optical tweezers functionality, comprising: a first laser, a first mirror, a first spectroscope, a first lens, a second lens, a first acousto-optic deflector, a third lens, a fourth lens, a fifth lens, a second acousto-optic deflector, a sixth lens, a second spectroscope, a first dichroic mirror, a second dichroic mirror, a first objective lens, a sample stage, a second objective lens, a third dichroic mirror, a third spectroscope, a fourth spectroscope, a fifth spectroscope, a first four-quadrant position detector, a second four-quadrant position detector, a position detector, an LED light source, a first detector, a second laser, a first cylindrical lens, a cylindrical lens group, a fourth dichroic mirror, a scanning galvanometer, a first telescopic system, an electronically controlled lens, a second telescopic system, a second mirror, a third telescopic system, a slit, a third mirror, a second cylindrical lens, a second detector; placing a sample to be detected on the sample table;
the method is characterized in that:
the first laser outputs a light beam with the wavelength of 1064 nanometers, the light beam is reflected by the first reflector and then enters the first spectroscope, and the first spectroscope divides the light beam into a first light beam and a second light beam; the first light beam is transmitted through the first spectroscope, and then reflected from the second spectroscope to the first dichroic mirror after passing through the first lens, the second lens, the first acousto-optic deflector and the third lens; the second light beam is reflected by the first spectroscope, and then transmitted by the second spectroscope through the fourth lens, the fifth lens, the second sound deflector and the sixth lens to the first dichroic mirror; the first light beam and the second light beam are reflected by the first dichroic mirror and transmitted by the second dichroic mirror, and finally focused on a sample table by the first objective lens to form two optical potential wells, so as to capture and measure particles in the sample; the first light beam and the second light beam are collected by the second objective lens after passing through the sample, reflected by the third dichroic mirror and reaching the third spectroscope to be split; the part of the first light beam and the second light beam transmitted by the third spectroscope is transmitted by the fourth spectroscope to reach the first four-quadrant position detector, and the part of the first light beam and the second light beam transmitted by the third spectroscope is reflected by the fourth spectroscope to reach the position detector; the part of the first light beam and the second light beam reflected by the third spectroscope is transmitted to the second four-quadrant position detector through the fifth spectroscope, and the part of the first light beam and the second light beam reflected by the third spectroscope is reflected to the position detector through the fifth spectroscope; the first four-quadrant position detector, the second four-quadrant position detector and the position detector work in parallel to measure the accurate three-dimensional position of the optical potential well;
the light beam with the output wavelength of 532 nanometers of the second laser reaches the cylindrical lens group after being expanded by the first cylindrical lens, is reflected by the cylindrical lens group to be incident on the scanning galvanometer after being subjected to one-dimensional shaping, is reflected by the scanning galvanometer and then is reflected by the second mirror and the second galvanometer to be reflected by the first objective lens after passing through the first telescopic system, the electric control lens and the second telescopic system, the first objective lens focuses the light beam output by the second laser onto a sample, the sample is excited by the light beam irradiation output by the second laser to generate fluorescence, the fluorescence is collected by the first objective lens, is respectively reflected by the second dichroic mirror and the second mirror, is sequentially reflected by the second telescopic system, the electric control lens and the first telescopic system, is incident on the third telescopic system after being reflected by the scanning galvanometer and is transmitted by the fourth telescopic system, a slit is arranged in the third telescopic system, the fluorescence outside a focusing plane is filtered by the slit and then is collimated and reflected by the third telescopic system, and is focused by the second cylindrical lens, and finally is collected and imaged by the second detector; the LED light source irradiates the sample after passing through the third dichroic mirror and the second objective, is collected by the first objective, and is transmitted by the second dichroic mirror and the first dichroic mirror to be incident on the first detector for imaging;
the scanning galvanometer is used for realizing line scanning of the sample in the sample focusing plane, and the scanning direction of the scanning galvanometer is vertical to the line of the focusing light spot in the sample focusing plane;
the cylindrical lens group comprises two oppositely arranged cylindrical lenses for one-dimensionally shaping the beam output from the second laser.
2. An extended depth of focus microscopy imaging system incorporating optical tweezers functionality according to claim 1; the method is characterized in that: the first dichroic mirror shows high reflection to the light beam output by the first laser; the second dichroic mirror shows high reflection to the light beam and fluorescence output by the second laser and high transmission to the light beam output by the first laser; the third dichroic mirror exhibits a high reflection of fluorescence; the fourth dichroic mirror shows high reflection to the output beam of the second laser and high transmission to fluorescence; the high transmittance means that the transmittance is more than 98%; the high reflection means that the reflectivity is more than 98%.
3. An extended depth of focus microscopy imaging system incorporating optical tweezers functionality according to claim 1; the method is characterized in that: the first four-quadrant position detector is used for detecting the horizontal coordinate of the optical potential well in the direction of the sample focusing plane, and the second four-quadrant position detector is used for detecting the vertical coordinate of the optical potential well in the direction of the sample focusing plane; the position detector is used for detecting the position of the optical potential well in the depth direction of the sample; the first four-quadrant position detector, the second four-quadrant position detector and the position detector work in parallel to determine the accurate three-dimensional position of the optical potential well.
4. An extended depth of focus microscopy imaging system incorporating optical tweezers functionality according to claim 1; the method is characterized in that: the one-dimensional shaping means that the cylindrical lens has a focusing effect on the output beam of the second laser in only one direction, so that a focusing light spot of the output beam of the second laser in a focusing plane in the sample is a line.
5. An extended depth of focus microscopy imaging system incorporating optical tweezers functionality according to claim 1; the method is characterized in that: the electric control lens is controlled by voltage, and when the voltage of the electric control lens is smaller than the threshold voltage, the electric control lens is a negative lens, and the emergent light beam passing through the electric control lens is divergent; when the voltage of the electric control lens is equal to the threshold voltage, the electric control lens is a plane mirror, and the emergent light beam passing through the electric control lens is a parallel light beam; when the voltage of the electric control lens is larger than the threshold voltage, the electric control lens is a positive lens, and the emergent light beam passing through the electric control lens is convergent; the emergent light beams passing through the electric control lens in different states are finally converged at different depths of the sample; the parallel emergent beam focusing plane is used as a reference plane, the emergent beam in a convergent shape is finally focused on one side of the reference plane, which is close to the first objective lens, and the emergent beam in a divergent shape is finally focused on one side of the reference plane, which is close to the second objective lens.
6. An extended depth of focus microscopy imaging system incorporating optical tweezers functionality according to claim 1; the method is characterized in that: the first telescopic system, the second telescopic system and the third telescopic system all comprise two oppositely placed cylindrical lenses; the first telescopic system and the second telescopic system are used for eliminating obvious amplification deviation introduced by the electric control lens, and the third telescopic system is used for performing beam expansion collimation on fluorescence.
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| CN111273433A (en) * | 2020-02-25 | 2020-06-12 | 中国科学院苏州生物医学工程技术研究所 | High-speed large-field-of-view digital scanning light-sheet microscopic imaging system |
| CN112880912B (en) * | 2021-01-08 | 2022-01-18 | 浙江大学 | Space resolution pressure measurement system and method based on vacuum holographic optical tweezers |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060033830A (en) * | 2004-10-16 | 2006-04-20 | 학교법인연세대학교 | Confocal laser--line scanning microscope with acousto-optic deflector and line scan camera |
| CN102540447A (en) * | 2012-02-17 | 2012-07-04 | 中国科学技术大学 | Trapping and detecting multiplexed scanning optical-tweezers system |
| KR20130026702A (en) * | 2011-09-06 | 2013-03-14 | 서울대학교산학협력단 | High-sensitivity and video-rate confocal fluorescence microscope |
| EP1946173B1 (en) * | 2005-11-08 | 2013-12-18 | Roland Kilper | Sample manipulation device |
| CN103940796A (en) * | 2014-04-22 | 2014-07-23 | 浙江大学 | Novel multi-angle and multi-mode quick switching circular optical illumination microscopic imaging system |
| CN105629454A (en) * | 2016-03-30 | 2016-06-01 | 中国计量学院 | Spatial light modulator-based dual-beam optical tweezers system |
| CN105954862A (en) * | 2016-07-08 | 2016-09-21 | 中国计量大学 | Microscopic lens and sample locking system based on 4Pi microscope framework |
| CN207440383U (en) * | 2017-11-20 | 2018-06-01 | 中国计量大学 | A kind of extended focal depth micro imaging system of combination optical tweezer function |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH699575A1 (en) * | 2008-10-06 | 2010-04-15 | Nectar Imaging S R L | An optical system for a confocal microscope. |
-
2017
- 2017-11-20 CN CN201711159419.1A patent/CN108051909B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060033830A (en) * | 2004-10-16 | 2006-04-20 | 학교법인연세대학교 | Confocal laser--line scanning microscope with acousto-optic deflector and line scan camera |
| EP1946173B1 (en) * | 2005-11-08 | 2013-12-18 | Roland Kilper | Sample manipulation device |
| KR20130026702A (en) * | 2011-09-06 | 2013-03-14 | 서울대학교산학협력단 | High-sensitivity and video-rate confocal fluorescence microscope |
| CN102540447A (en) * | 2012-02-17 | 2012-07-04 | 中国科学技术大学 | Trapping and detecting multiplexed scanning optical-tweezers system |
| CN103940796A (en) * | 2014-04-22 | 2014-07-23 | 浙江大学 | Novel multi-angle and multi-mode quick switching circular optical illumination microscopic imaging system |
| CN105629454A (en) * | 2016-03-30 | 2016-06-01 | 中国计量学院 | Spatial light modulator-based dual-beam optical tweezers system |
| CN105954862A (en) * | 2016-07-08 | 2016-09-21 | 中国计量大学 | Microscopic lens and sample locking system based on 4Pi microscope framework |
| CN207440383U (en) * | 2017-11-20 | 2018-06-01 | 中国计量大学 | A kind of extended focal depth micro imaging system of combination optical tweezer function |
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|---|---|
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