CN108051909A - A kind of extended focal depth micro imaging system of combination optical tweezer function - Google Patents
A kind of extended focal depth micro imaging system of combination optical tweezer function Download PDFInfo
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- 238000003384 imaging method Methods 0.000 title claims abstract description 35
- 238000012576 optical tweezer Methods 0.000 title claims abstract description 29
- 239000000523 sample Substances 0.000 claims description 78
- 230000005540 biological transmission Effects 0.000 claims description 19
- 239000002245 particle Substances 0.000 claims description 9
- 238000007493 shaping process Methods 0.000 claims description 6
- 239000012472 biological sample Substances 0.000 claims description 4
- 230000005284 excitation Effects 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 3
- 230000005611 electricity Effects 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 238000009738 saturating Methods 0.000 claims description 3
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000000571 coke Substances 0.000 claims description 2
- 238000002310 reflectometry Methods 0.000 claims description 2
- 230000010358 mechanical oscillation Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000006073 displacement reaction Methods 0.000 description 4
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 238000000324 molecular mechanic Methods 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001246 colloidal dispersion Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000000651 laser trapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 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
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
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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
-
- 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
-
- 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
-
- 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
Abstract
The invention discloses a kind of extended focal depth micro imaging system of combination optical tweezer function, including：First laser device, first speculum, first spectroscope, first lens, second lens, first acousto-optic deflection device, 3rd lens, 4th lens, 5th lens, second acousto-optic deflection device, 6th lens, second spectroscope, first dichroic mirror, second dichroic mirror, first object lens, sample stage, second object lens, 3rd dichroic mirror, 3rd spectroscope, 4th spectroscope, 5th spectroscope, first four-quadrant position sensor, second four-quadrant position sensor, position sensor, LED light source, first detector, second laser, first cylindrical lens, set of cylindrical lenses, 4th dichroic mirror, scanning galvanometer, first telescopic system, automatically controlled lens, second telescopic system, second speculum, 3rd telescopic system, slit, 3rd speculum, second cylindrical lens, second detector.System utilizes automatically controlled lens scan difference depth of focus plane, no mechanical oscillation and image quality height.
Description
Technical field
The present invention relates to micro-imaging, optical tweezer fields, and in particular to a kind of extended focal depth of combination optical tweezer function it is micro- into
As system.
Background technology
In the research of cell biology, it is often necessary at the same to the mechanics of biomolecule in cell, mechanical property and
Cell three-dimensional structure is carried out at the same time research.Therefore, during practical study, generally use optical tweezer module and fluorescent microscopic imaging mould
The unimolecule mechanics microscopic system that block is combined is tested.When being operated using unimolecule mechanics microscopic system to sample,
Optical tweezer module therein can realize the measurement to monomolecular dynamics in biological cell, while fluorescent microscopic imaging mould
Block can obtain biological structure, extracellular or the various dimensions such as intracellular information.
The real-time three-dimensional biological structure of sample is realized using the fluorescent microscopic imaging module in unimolecule mechanics microscopic system
During imaging, fluorescent microscopic imaging module is realized usually using total internal reflectance microscope or confocal laser scanning microscope, CLSM.2004
Year, Lang Matthew J et al. exist《Nature Methods》It is delivered on periodical entitled《Simultaneous,
coincident optical trapping and single-molecule fluorescence》Article in propose one
The unimolecule mechanics microscopic system that kind is combined using total internal reflectance microscope and optical tweezer, total internal reflectance microscope is using when being totally reflected
Light energy penetration depth in optically thinner medium is limited and light energy is only along the characteristic of interface propagation, and selective excitation is located at fluorescence
The fluorescent marker on surface obtains the information of sample surfaces, evanescent wave penetration depth by capturing the evanescent wave of sample surfaces
Below 200 nanometers, so total internal reflectance microscope is not suitable for the scanning of the depth direction of micron dimension, therefore it can not realize
The three-dimensional structure imaging of micron dimension biological sample；The same year, Vossen Dirk L. J et al. exists《Review of
Scientific Instruments》It has been delivered on periodical entitled《Optical tweezers and confocal
microscopy for simultaneous three-dimensional manipulation and imaging in
concentrated colloidal dispersions.》Article in propose it is a kind of using common focus point migration it is micro-
The unimolecule mechanics microscopic system that mirror is combined with optical tweezer, and the focussing plane of confocal laser scanning microscope, CLSM is fixed, is led to
The micro-displacement platform for crossing mobile example or the position for manipulating object lens can realize the scanning in sample depth direction.But use carries
The confocal laser scanning microscope, CLSM of micro-displacement platform, by mobile micro-displacement platform come when realizing to sample depth scanning direction, with
Vertically moving for sample stage, the connection of the particle and pressure sensor of optical tweezer detection can be interrupted, this just directly affects list
Measurement of the optical tweezer module to molecular mechanics and mechanical property in molecular mechanics microscopic system；And micro-displacement platform is kept to fix, pass through
Control object lens in the position in sample depth direction come when scanning the sample of different depth, for the object lens of large-numerical aperture, this is not
It is suitble to carry out fluorescence imaging to unimolecule, while particle is captured and to molecule using unimolecule mechanics microscopic system
Mechanics and measuring mechanical characteristics are when operations, as the movement of object lens causes the offset for capturing light, finally influence optical tweezer and catch
Obtain intended particle.These technical problems all significantly limit the scanning in unimolecule mechanics microscopic system sample depth direction
Imaging, causes it that can not complete the three-dimensional imaging of biological micro-structure..
The content of the invention
Existing sample when the present invention is directed to existing unimolecule mechanics microscopic system to sample biological structure real time three-dimensional imaging
A kind of limited problem of product depth direction scan depths, it is proposed that the extended focal depth micro-imaging system of combination optical tweezer function
System.The system combination optical tweezer can accurately manipulate particle, and shake while three-dimensional imaging is realized without machinery
The dynamic error introduced, image quality higher are more rapidly stablized, and cost is relatively low.
A kind of extended focal depth micro imaging system of combination optical tweezer function, including：First laser device, the first speculum,
One spectroscope, the first lens, the second lens, the first acousto-optic deflection device, the 3rd lens, the 4th lens, the 5th lens, the second acousto-optic
Deflector, the 6th lens, the second spectroscope, the first dichroic mirror, the second dichroic mirror, the first object lens, sample stage, the second object lens,
Three dichroic mirrors, the 3rd spectroscope, the 4th spectroscope, the 5th spectroscope, the first four-quadrant position sensor, the second four-quadrant extreme position
Detector, position sensor, LED light source, the first detector, second laser, the first cylindrical lens, set of cylindrical lenses, the 4th
It is dichroic mirror, scanning galvanometer, the first telescopic system, automatically controlled lens, the second telescopic system, the second speculum, the 3rd telescopic system, narrow
Seam, the 3rd speculum, the second cylindrical lens, the second detector.
The first laser device output beam incides into the first spectroscope after the reflection of the first speculum, and the first spectroscope will
Light beam is divided into the first light beam and the second light beam；After wherein the first light beam is transmitted through the first spectroscope, through the first lens, second thoroughly
After mirror, the first acousto-optic deflection device, the 3rd lens the first dichroic mirror is reached from the second dichroic mirror；Second light beam is through the first light splitting
Mirror reflection after, after the 4th lens, the 5th lens, the second acousto-optic deflection device, the 6th lens from the second spectroscope transmission equally to
Up to the first dichroic mirror.First light beam and the second light beam are reflected through the first dichroic mirror, the transmission of the second dichroic mirror, finally by the first object lens
Two photo potential traps of formation on sample stage are focused on, the particle in sample is captured and measured；First light beam and the second light beam
After sample, collected by the second object lens and be split through the 3rd dichroic mirror reflection the 3rd spectroscope of arrival；First light beam and second
The first four-quadrant position sensor, the first light beam are reached by part the 4th spectroscope transmission of the 3rd spectroscope transmission in light beam
With the 4th dichroic mirror in-position detector of part transmitted in the second light beam by the 3rd spectroscope；First light beam and
The second four-quadrant position sensor, the first light are reached by part the 5th spectroscope transmission of the 3rd dichroic mirror in two light beams
By the 5th dichroic mirror in-position detector of part of the 3rd dichroic mirror in beam and the second light beam；First four-quadrant
Position sensor, the second four-quadrant position sensor, position sensor concurrent working can measure photo potential trap accurate three-dimensional position.
The second laser output beam reaches set of cylindrical lenses after the first cylindrical lens expands, by set of cylindrical lenses
After one-dimensional shaping, from the 4th dichroic mirror reflection incide on scanning galvanometer, after scanned vibration mirror reflected by the first telescopic system,
The first object lens are reflected by the second speculum and the second dichroic mirror after automatically controlled lens, the second telescopic system, the first object lens are by second
Laser Output Beam is focused on sample, and the irradiation excitation of light beam that sample is exported through second laser generates fluorescence, fluorescence by
After first object lens are collected, then respectively by the reflection of the second dichroic mirror and the second speculum, looking in the distance then in turn through second is
System, automatically controlled lens, the first telescopic system incide into the 3rd and look in the distance and be by overscanning vibration mirror reflected, the 4th dichroiscopic transmission
System, is equipped with slit in the 3rd telescopic system, and slit is accurate by the 3rd telescopic system by after the fluorescence filter outside focussing plane
Directly, the 3rd speculum reflects, then fluorescent foci is most collected and is imaged through the second detector afterwards by the second cylindrical lens；Institute
LED light source is stated through in the 3rd dichroic mirror, the second object lens back lighting to sample, after collected by the first object lens, the second dichroic mirror and the
The transmission of one dichroic mirror is incided into be imaged on the first detector.
In the present invention, the first laser device, second laser are point light source.
In the present invention, first, second acousto-optic deflection device is respectively used to change the deflection angle of first, second light beam,
The change of the inner focusing of the focussing plane in the sample position of first, second light beam is realized respectively, i.e., realizes first, second light respectively
The regulation and control of photo potential trap position of the beam in focussing plane.
In the present invention, first lens and the 4th lens can be adjusted along the relative position on paths direction,
The different position that the first light beam focuses on sample depth direction can be manipulated by wherein adjusting the position of the first lens, that is, adjust first
The position of light beam focussing plane；The difference that the second light beam focuses on sample depth direction can be manipulated by adjusting the position of the second lens
Position adjusts the position of the second light beam focussing plane.
In the present invention, second lens, the 3rd lens are for collimating the first light beam, and the 5th lens, the 6th are thoroughly
Mirror is used to collimate the second light beam.
In the present invention, the light beam that first dichroic mirror exports first laser device shows as height instead.Two or two color
The light beam and fluorescence that mirror exports second laser show as it is high anti-, the light beam of first laser device output is shown as it is high thoroughly.
3rd dichroic mirror shows as fluorescence high anti-.4th dichroic mirror shows as second laser output beam height instead,
Fluorescence is shown as high saturating.The height thoroughly, refers to transmissivity more than 98%；Described height is anti-, refer to reflectivity 98% with
On, it is specially 98% ~ 99.9%.
In the present invention, the first four-quadrant position sensor is used to detect photo potential trap on sample focussing plane direction
Horizontal coordinate, the second four-quadrant position sensor are used to detect vertical coordinate of the photo potential trap on sample focussing plane direction；Position
Detector is put for detecting position of the photo potential trap on sample depth direction.First four-quadrant position sensor, the second four-quadrant
Position sensor and position sensor concurrent working can determine the accurate three-dimensional position of photo potential trap.
In the present invention, the set of cylindrical lenses includes two oppositely positioned cylindrical lenses, for will be from second laser
The light beam of output carries out one-dimensional shaping.
In the present invention, it is poly- that the one-dimensional shaping refers to that cylindrical lens only has second laser output beam in one direction
Burnt effect so that the focal beam spot in the focussing plane of second laser output beam in the sample is line.
In the present invention, the scanning galvanometer is used to implement the line scanning to the sample in sample focussing plane, scanning
Direction is vertical with the line of focal beam spot in sample focussing plane.
In the present invention, the automatically controlled lens are controlled by voltage, when automatically controlled lens voltage is less than threshold voltage, automatically controlled lens measure
It is now negative lens, the outgoing beam by automatically controlled lens is in divergent shape；When automatically controlled lens voltage is equal to threshold voltage, automatically controlled lens
Plane mirror is shown as, the outgoing beam by automatically controlled lens is collimated light beam；It is automatically controlled when automatically controlled lens voltage is more than threshold voltage
Lens show as positive lens, by the outgoing beam of automatically controlled lens in convergence shape；By the outgoing of the different conditions of automatically controlled lens
Light beam finally converges at the different depth of sample；The face on the basis of exiting parallel light beam focussing plane, in the light for assembling shape outgoing
Beam finally focuses on reference plane close to the one side of the first object lens, and the light beam being emitted in divergent shape finally focuses on reference plane close to the
The one side of two object lens.
In the present invention, first telescopic system, the second telescopic system and the 3rd telescopic system are reversely put comprising two
The cylindrical lens put；Wherein, the first telescopic system and the second telescopic system, for eliminating the apparent amplification that automatically controlled lens introduce
Rate deviation, the 3rd telescopic system are used to carry out beam-expanding collimation to fluorescence.
In the present invention, the slit is placed on the confocal face in the 3rd telescopic system, is put down for filter scan imaging and focusing
Fluorescence outside face, so as to fulfill imaging is only scanned to focussing plane.
In the present invention, first detector is dot matrix CCD.
In the present invention, second detector is line array CCD, and linear scan imaging is carried out to fluorescence.
In the present invention, the LED light source is for illuminating sample, to provide apparent visual field in experimentation.
Preferably, the wavelength of the first laser device output beam is 1064 nanometers, biological sample is to the wavelength absorption
It is smaller.
Preferably, the wavelength of the second laser output beam is 532 nanometers, fluorescent can be effectively excited.
Preferably, first object lens select the oil immersion objective of the model UPlanSApo of Olympus Corp's production,
Enlargement ratio is 100 times, numerical aperture 1.4.
Preferably, second object lens select the object lens of the model LUMPlanFLN of Olympus Corp's production, put
Big multiplying power is 60 times, numerical aperture 1.0.
Preferably, the scanning galvanometer selects the optics of the model 6231H of Cambridge Technology productions
Scanning galvanometer, line sweep length are 15 millimeters.
Preferably, the slit selects the slit of the model S100R of Thorlab companies production.
Preferably, second detector selects the highly sensitive EMCCD phases of the model iXon3 of Andor companies production
Machine.
Compared with the prior art, the present invention has technique effect beneficial below：.
1st, optical optical tweezers system is focused on laser scanning microscope system and combined by the present invention together, is being realized to particle manipulation
It is also completed simultaneously to sample real time imagery, system structure is compact.
2nd, present invention introduces the realizations of automatically controlled lens to be scanned the focussing plane of sample difference depth of focus, will not move object lens
Will not mobile example platform, no mechanical oscillation, image quality is high, image stabilization, and cost is relatively low.
3rd, the present invention is realized to sample using multiple cylindrical lenses and line array CCD into line scanning imagery, scanning imagery
Speed is fast, high sensitivity.
Therefore, technical scheme is compared with original technology, while molecular dynamics characteristic test is carried out, energy
Enough realize quiveringly quickly scans the biological sample of sample different depth without machinery and completes three-dimensional imaging.
Description of the drawings
Fig. 1 is a kind of one embodiment of the extended focal depth micro imaging system structure of combination optical tweezer function of the present invention
Index path；
Wherein：1st, first laser device；2nd, the first speculum；3rd, the first spectroscope；4th, the first lens；5th, the second lens；6th, first
Acousto-optic polarizer；7th, the 3rd lens；8th, the 4th lens；9th, the 5th lens；10th, the second acousto-optic deflection device；11st, the 6th lens；12、
Second spectroscope；13rd, the first dichroic mirror；14th, the second dichroic mirror；15th, the first object lens；16th, sample stage；17th, the second object lens；18、
3rd dichroic mirror；19th, the 3rd spectroscope；20th, the 4th spectroscope；21st, the 5th spectroscope；22nd, the first four-quadrant position sensor；
23rd, the second four-quadrant position sensor；24th, position sensor；25th, LED light source；26th, the first detector；27th, second laser；
28th, the first cylindrical lens；29th, set of cylindrical lenses；30th, the 4th dichroic mirror；31st, scanning galvanometer；32nd, the first telescopic system；33rd, it is electric
Control lens；34th, the second telescopic system；35th, the second speculum；36th, the 3rd telescopic system；37th, slit；38th, the 3rd speculum；
39th, the second cylindrical lens；40th, the second detector.
Fig. 2 is that different outgoing states is presented after the automatically controlled lens of different control voltages in collimated light beam；Wherein, Fig. 2 a
When representing that control voltage is less than threshold voltage, automatically controlled lens show as negative lens, and outgoing beam is in divergent shape, and Fig. 2 b represent control
When voltage is equal to threshold voltage, automatically controlled lens show as plane mirror, and outgoing beam is collimated light beam, and Fig. 2 c represent that control voltage is big
When threshold voltage, automatically controlled lens show as positive lens, and outgoing beam is in convergence shape.
Fig. 3 is the oscillogram that automatically controlled lens scan controls voltage.
Fig. 4 is the oscillogram of scanning galvanometer scanning voltage.
Specific embodiment
With reference to Figure of description, the present invention will be described in detail, but the present invention is not limited thereto.
It is an a kind of implementation of extended focal depth micro imaging system structure of combination optical tweezer function of the present invention as shown in Figure 1
The index path of example, the system of the embodiment include：
First laser device 1；First speculum 2；First spectroscope 3；First lens 4；Second lens 5；First acousto-optic polarizer 6；
3rd lens 7；4th lens 8；5th lens 9；Second acousto-optic deflection device 10；6th lens 11；Second spectroscope 12；One or two
Look mirror 13；Second dichroic mirror 14；First object lens 15；Sample stage 16；Second object lens 17；3rd dichroic mirror 18；3rd spectroscope 19；
4th spectroscope 20；5th spectroscope 21；First four-quadrant position sensor 22；Second four-quadrant position sensor 23；Position
Detector 24；LED light source 25；First detector 26；Second laser 27；First cylindrical lens 28；Set of cylindrical lenses 29；4th
Dichroic mirror 30；Scanning galvanometer 31；First telescopic system 32；Automatically controlled lens 33；Second telescopic system 34；Second speculum 35；The
Three telescopic systems 36；Slit 37；3rd speculum 38；Second cylindrical lens 39；Second detector 40.
Wherein, first laser device 1 is the LDH-TA-595 type lasers of PicoQuant companies, and second laser 27 is
The LDH-P-C-650B type lasers of PicoQuant companies.
For 1 output beam of first laser device after the reflection of the first speculum 2, the direction of propagation changes 90 °, incides into the first light splitting
Mirror 3, the light beam through the transmission of the first spectroscope 3 are the first light beam, and the light beam through the reflection of the first spectroscope 3 is the second light beam.First
Light beam reaches the first acousto-optic deflection device 6 after the first lens 4, the second lens 5, wherein, the first lens 4 are along paths side
Upward relative position can be adjusted, and can adjust the first light beam by the relative position for adjusting the first lens 4 focuses on sample
The different position of depth direction adjusts the position of the first light beam focussing plane；First acousto-optic deflection device 6 can manipulate the first light
The deflection angle of beam makes it collimate by the 3rd lens 7, the reflection and the two or two of the second spectroscope 12 and the first dichroic mirror 13
Any position on sample stage 16 on sample focussing plane direction can be focused on after the transmission of Look mirror 14 through the first object lens 15；The
One lens 4 and the work of 6 cooperation of the first acousto-optic deflection device, can adjust the first light beam and focus on different positions three-dimensional in sample
It puts.Second light beam reaches the second acousto-optic deflection device 10 after the 4th lens 8, the 5th lens 9, wherein, the 4th lens 8 are along light
Relative position on the direction of propagation of road can be adjusted, and can adjust the second light beam by the relative position for adjusting the 4th lens 8 gathers
Coke adjusts the position of the second light beam focussing plane in the different position in sample depth direction；Second acousto-optic deflection device 10 can be with
The angular deflection of the second light beam is manipulated, it is made to be collimated by the 6th lens 11, the transmission of the second spectroscope 12 and the first dichroic mirror
Appointing on sample focussing plane direction can be focused on after 13 reflection and the transmission of the second dichroic mirror 14 through the first object lens 15
One position；4th lens 8 and the work of 10 cooperation of the second acousto-optic deflection device, can adjust the second light beam and focus on three in sample
The different position of dimension.By manipulation the first light beam and the second light beam can be made to be reflected through the first dichroic mirror 13, the second dichroic mirror 14
Transmission, is finally focused on by the first object lens 15 on sample stage that intended particle is formed about two photo potential traps in sample, to particle into
The measurement of row mechanics and mechanical property.First light beam and the second light beam are collected by the second object lens 17 through the three or two after sample
The reflection of Look mirror 18 reaches the 3rd spectroscope 19；The light beam transmitted by the 3rd spectroscope 19 transmits arrival first through the 4th spectroscope 20
Four-quadrant position sensor 22, the light beam transmitted by the 3rd spectroscope 19 reflect arrival position sensor through the 4th spectroscope 20
24, it is transmitted by the light beam that the 3rd spectroscope 19 reflects through the 5th spectroscope 21 and reaches the second four-quadrant position sensor 23, by the
The light beam of three spectroscopes 19 reflection is reflected through the 5th spectroscope 21 reaches position sensor 24；First four-quadrant position sensor 22
For detecting horizontal coordinate of the photo potential trap on sample focussing plane direction, the second four-quadrant position sensor 23 is used to detect light
Vertical coordinate of the potential well on sample focussing plane direction；Position sensor 24 is used to detect photo potential trap in sample depth direction
Position.First four-quadrant position sensor 22, the second four-quadrant position sensor 23 and 24 concurrent working of position sensor can
To determine the accurate three-dimensional position of photo potential trap.
27 output beam of second laser is realized by set of cylindrical lenses 29 to second after the first cylindrical lens 28 expands
The one-dimensional shaping of 27 output beam of laser namely 27 output beam of second laser incide into the 4th dichroic mirror 30, scanning galvanometer
31st, the first telescopic system 32, automatically controlled lens 33, the second telescopic system 34, the second speculum 35, the focusing light of the second dichroic mirror 14
Spot and by the focal beam spot in the first object lens 15 focussing plane be in the sample finally line, while electromagnetic radiation fluorescence is through the
The second dichroic mirror 14, the second speculum 35, the second telescopic system 34, the prestige of automatically controlled lens 33, first are incided into the collection of one object lens 15
Remote system 32, scanning galvanometer 31, the 4th dichroic mirror 30, the 3rd telescopic system 36, the 3rd speculum 38, the second cylindrical mirror 39 it is poly-
Burnt hot spot becomes line.It is illustrated in figure 3 the scanning voltage waveform figure of scanning galvanometer 31, in the present embodiment, scanning galvanometer 31 is swept
Frequency is retouched as 50 hertz；The light beam for being scanned through the reflection of galvanometer 31 looks in the distance through the first telescopic system 32, automatically controlled lens 33, second and is
The 34, second speculum 35 of system, the second dichroic mirror 14 most afterwards through the first object lens 15 focus on sample stage 16 in sample to sample into
Row Express Order Wire scans, and scanning direction is in sample focussing plane and vertical with the line of focal beam spot.Second laser 27 therebetween
When output beam passes through automatically controlled lens 33, as shown in Fig. 2, controlling the voltage of automatically controlled lens 33 that can change to go out from automatically controlled lens 33
The state of irradiating light beam.In the present embodiment, as shown in Figure 2 a, when automatically controlled 33 voltage of lens is less than 3 volts, automatically controlled lens 33 are shown as
Negative lens is in divergent shape from automatically controlled 33 outgoing beam of lens；As shown in Figure 2 b, when automatically controlled 33 voltage of lens is equal to 3 volts,
Automatically controlled lens 33 show as plane mirror, are collimated light beams from automatically controlled 33 outgoing beam of lens；As shown in Figure 2 c, automatically controlled 33 electricity of lens
When pressure is more than 3 volts, automatically controlled lens 33 show as positive lens, from automatically controlled 33 outgoing beam of lens in convergence shape；Different conditions
Outgoing beam finally converges at the different depth of sample, the face on the basis of the focussing plane of exiting parallel light beam, goes out in shape is assembled
Irradiating light beam finally focuses on reference plane close to the one side of the first object lens 15, finally focuses on reference plane in divergent shape outgoing beam and leans on
The one side of nearly second object lens 17.As shown in figure 4, in the present embodiment, the scan-control voltage frequency of automatically controlled lens 33 is 10 hertz,
When the scan-control voltage to automatically controlled lens 33 is scanned with 10 hertz of frequency between -3 volts and 3 volts, can realize
In the Express Order Wire scanning of the focussing plane of the different depth of sample.Control the scan frequency of scanning galvanometer 31 for 50 hertz simultaneously and
Automatically controlled lens 33 control the scan frequency of voltage as 10 hertz, can be with the quick scanning of the three-dimensional of complete paired samples.By quickly sweeping
The fluorescence that the sample excitation retouched goes out is collected through the first object lens 15, then in turn through the second dichroic mirror 14, the second speculum 35,
Second telescopic system 34, automatically controlled lens 33, the first telescopic system 32, scanning galvanometer 31, the 4th dichroic mirror 30, the 3rd telescopic system
36th, the 3rd speculum 38 most focuses on the second detector 40 afterwards through the second cylindrical mirror 39 and carries out real time three-dimensional imaging.
LED light source 25 is converged to by the second object lens 17 on sample after the 3rd dichroic mirror 18, after collected by the first object lens 15,
Detection imaging on the first detector 26 is incided into second dichroic mirror 14 and the transmission of the first dichroic mirror 13, and wherein LED light source 25 is through the
Two object lens 17, which are converged on sample, to provide visual field for sample real-time three-dimensional scanning imagery.
It is last it should be noted that embodiment of above is only to illustrate the technical solution of patent and unrestricted, this field
Those of ordinary skill for do not depart from this patent principle on the premise of, several variations and modifications can also be made, this should also be regarded
For the protection domain of this patent.
Claims (10)
1. a kind of extended focal depth micro imaging system of combination optical tweezer function, including：First laser device, the first speculum, first
Spectroscope, the first lens, the second lens, the first acousto-optic deflection device, the 3rd lens, the 4th lens, the 5th lens, the second acousto-optic are inclined
Turn device, the 6th lens, the second spectroscope, the first dichroic mirror, the second dichroic mirror, the first object lens, sample stage, the second object lens, the 3rd
Dichroic mirror, the 3rd spectroscope, the 4th spectroscope, the 5th spectroscope, the first four-quadrant position sensor, the second four-quadrant extreme position are visited
Survey device, position sensor, LED light source, the first detector, second laser, the first cylindrical lens, set of cylindrical lenses, the four or two
It is Look mirror, scanning galvanometer, the first telescopic system, automatically controlled lens, the second telescopic system, the second speculum, the 3rd telescopic system, narrow
Seam, the 3rd speculum, the second cylindrical lens, the second detector；Detected sample is placed on the sample stage；
It is characterized in that：
The first laser device output beam incides into the first spectroscope after the reflection of the first speculum, and the first spectroscope is by light beam
It is divided into the first light beam and the second light beam；After wherein the first light beam is transmitted through the first spectroscope, through the first lens, the second lens,
After first acousto-optic deflection device, the 3rd lens the first dichroic mirror is reached from the second dichroic mirror；Second light beam is through the first spectroscope
After reflection, similary reach is transmitted from the second spectroscope after the 4th lens, the 5th lens, the second acousto-optic deflection device, the 6th lens
First dichroic mirror.First light beam and the second light beam are reflected through the first dichroic mirror, the transmission of the second dichroic mirror, are finally gathered by the first object lens
Coke is captured and measured to the particle in sample to two photo potential traps of formation on sample stage；First light beam and the second light beam warp
After crossing sample, collected by the second object lens and be split through the 3rd dichroic mirror reflection the 3rd spectroscope of arrival；First light beam and the second light
The first four-quadrant position sensor is reached by part the 4th spectroscope transmission of the 3rd spectroscope transmission in beam, the first light beam and
The 4th dichroic mirror in-position detector of part transmitted in second light beam by the 3rd spectroscope；First light beam and second
The second four-quadrant position sensor, the first light beam are reached by part the 5th spectroscope transmission of the 3rd dichroic mirror in light beam
With in the second light beam by the 5th dichroic mirror in-position detector of part of the 3rd dichroic mirror；First four-quadrant /V
Put detector, the second four-quadrant position sensor, position sensor concurrent working can measure photo potential trap accurate three-dimensional position；
The second laser output beam reaches set of cylindrical lenses after the first cylindrical lens expands, one-dimensional by set of cylindrical lenses
After shaping, incided into from the reflection of the 4th dichroic mirror on scanning galvanometer, by the first telescopic system, automatically controlled after scanned vibration mirror reflected
The first object lens are reflected by the second speculum and the second dichroic mirror after lens, the second telescopic system, the first object lens are by second laser
Device output beam is focused on sample, and the light beam irradiation excitation that sample is exported through second laser generates fluorescence, and fluorescence is by first
After object lens are collected, then respectively by the reflection of the second dichroic mirror and the second speculum, then in turn through the second telescopic system, electricity
Lens, the first telescopic system are controlled, the 3rd telescopic system is incided by overscanning vibration mirror reflected, the 4th dichroiscopic transmission, the
It will be after the fluorescence filter outside focussing plane, by the 3rd telescopic system collimation, the 3rd equipped with slit, slit in three telescopic systems
Speculum reflects, then fluorescent foci is most collected and is imaged through the second detector afterwards by the second cylindrical lens；The LED light
On the 3rd dichroic mirror of source, the second object lens back lighting to sample, after collected by the first object lens, the second dichroic mirror and the first dichroic mirror
Transmission is incided into be imaged on the first detector.
2. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 1；It is characterized in that：Institute
It states the light beam that the first dichroic mirror exports first laser device and shows as height instead.What second dichroic mirror exported second laser
Light beam and fluorescence show as height instead, the light beam of first laser device output are shown as high saturating.3rd dichroic mirror is to fluorescence
It shows as high anti-.4th dichroic mirror shows as second laser output beam height instead, fluorescence is shown as high saturating.It is described
Height thoroughly, refer to transmissivity more than 98%；The height is anti-, refers to reflectivity more than 98%, is specially 98% ~ 99.9%.
3. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 1；It is characterized in that：Institute
The first four-quadrant position sensor is stated for detecting horizontal coordinate of the photo potential trap on sample focussing plane direction, the second four-quadrant
Position sensor is used to detect vertical coordinate of the photo potential trap on sample focussing plane direction；Position sensor is used to detect photo potential
Position of the trap on sample depth direction.First four-quadrant position sensor, the second four-quadrant position sensor and position sensing
Device concurrent working can determine the accurate three-dimensional position of photo potential trap.
4. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 1；It is characterized in that：Institute
It states set of cylindrical lenses and includes two oppositely positioned cylindrical lenses, the light beam for will be exported from second laser carries out one-dimensional whole
Shape.
5. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 4；It is characterized in that：Institute
It states one-dimensional shaping and refers to that cylindrical lens only has focusing effect in one direction to second laser output beam so that second laser
Focal beam spot in the focussing plane of device output beam in the sample is line.
6. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 1；It is characterized in that：Institute
It states scanning galvanometer to be used to implement to the scanning of the line of the sample in sample focussing plane, scanning direction is in sample focussing plane
It is vertical with the line of focal beam spot.
7. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 1；It is characterized in that：Institute
It states automatically controlled lens to be controlled by voltage, when automatically controlled lens voltage is less than threshold voltage, automatically controlled lens show as negative lens, and process is automatically controlled
The outgoing beam of lens is in divergent shape；When automatically controlled lens voltage is equal to threshold voltage, automatically controlled lens show as plane mirror, by electricity
The outgoing beam for controlling lens is collimated light beam；When automatically controlled lens voltage is more than threshold voltage, automatically controlled lens show as positive lens, warp
The outgoing beam of automatically controlled lens is crossed in convergence shape；Sample is finally converged at by the outgoing beam of the different conditions of automatically controlled lens
Different depth；The face on the basis of exiting parallel light beam focussing plane finally focuses on reference plane in the light beam for assembling shape outgoing and leans on
The one side of nearly first object lens, the light beam being emitted in divergent shape finally focus on reference plane close to the one side of the second object lens.
8. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 1；It is characterized in that：Institute
It states the first telescopic system, the second telescopic system and the 3rd telescopic system and includes two oppositely positioned cylindrical lenses；Wherein,
One telescopic system and the second telescopic system, for eliminating the apparent magnifying power deviation that automatically controlled lens introduce, the 3rd telescopic system
For carrying out beam-expanding collimation to fluorescence.
9. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 1；It is characterized in that：Institute
The wavelength of first laser device output beam is stated as 1064 nanometers, biological sample is smaller to the wavelength absorption.
10. a kind of extended focal depth micro imaging system of combination optical tweezer function according to claim 1；It is characterized in that：
The wavelength of the second laser output beam is 532 nanometers, can effectively excite fluorescent.
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