CN109358004A - The method and apparatus of dual-wavelength difference non-marked micro-imaging - Google Patents
The method and apparatus of dual-wavelength difference non-marked micro-imaging Download PDFInfo
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- CN109358004A CN109358004A CN201811457587.3A CN201811457587A CN109358004A CN 109358004 A CN109358004 A CN 109358004A CN 201811457587 A CN201811457587 A CN 201811457587A CN 109358004 A CN109358004 A CN 109358004A
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/21—Polarisation-affecting properties
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/1717—Systems in which incident light is modified in accordance with the properties of the material investigated with a modulation of one or more physical properties of the sample during the optical investigation, e.g. electro-reflectance
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- G01N2021/1765—Method using an image detector and processing of image signal
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- G01N2021/178—Methods for obtaining spatial resolution of the property being measured
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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Abstract
The method and apparatus that the present invention discloses a kind of dual-wavelength difference non-marked micro-imaging, wherein method includes: to scan sample using the first laser and second laser with wavelength difference, wherein having beam of laser is hollow beam, another beam of laser is solid light beam；The signal light that sample issues is acquired, the signal light includes the first signal light by first laser reflection and the second signal light by the reflection of the second first laser；The data of first signal light and second signal light are carried out subtracting each other processing, obtain differential signal, and carry out reestablishment imaging using the differential signal.Using two close beam laser of wavelength, irradiating sample simultaneously detects the two beam laser reflected from sample surface to the present invention simultaneously simultaneously, and detect two signals are then done difference processing, obtain the higher image of resolution ratio.Lateral resolution of the invention is high, signal-to-noise ratio is high, image taking speed is fast, imaging time is long, while device is simple, facilitates operation.
Description
Technical field
The invention belongs to the micro- field of optical ultra-discrimination, in particular to the dual-wavelength difference micro-imaging of a kind of non-marked
Method and apparatus.
Background technique
Optical microscopy is with a long history, is a very ancient imaging technique, it observes microcosmos for people and provides
A kind of feasible method, enriches the visual field of people.But with the continuous improvement of scientific level, it is especially biomedical and
It is higher and higher to the resolution requirement of microtechnic in terms of material science, and due to the limitation of Abbe diffraction limit, traditional optical
The resolution ratio of microtechnic is extremely limited, so that traditional optical microscopy be not applicable in, so scientific
Family invented various super-resolution microtechnics.
For confocal microscopy other than having the advantages that lateral resolution is high, its longitudinal section ability is also very strong.It breaks through
The limitation of ordinary optical microscope diffraction limit, lateral resolution are the 1.4 of the ordinary optical microscope of identical numerical aperture
Times, longitudinal resolution can achieve sub-micrometer scale.
On the basis of confocal microscopic imaging, ZL201210331504.2 proposes that a kind of resolution ratio is higher, image taking speed
Faster fluorescence difference micro-imaging technique, the technology are received simultaneously first with a solid beam spot scans sample excitation fluorescence
Collect the fluorescence signal, recycles the hollow light spot of phase co-wavelength to scan sample excitation fluorescence, and collect the fluorescence signal simultaneously, then
Two fluorescence signals are done into difference processing, obtain the higher sample image of resolution ratio.Although the technology improves the resolution of imaging
Rate, but since it is desired that scanning limits image taking speed twice, so that image taking speed is not fast enough, capture is living less than certain physiology
It is dynamic.
Summary of the invention
The invention proposes a kind of method and apparatus of dual-wavelength difference non-marked micro-imaging, using wavelength it is close two
Irradiating sample simultaneously detects the two beam laser reflected from sample surface to Shu Jiguang simultaneously simultaneously, then makes the difference detect two signals
Divide processing, obtains the higher image of resolution ratio；Wherein beam of laser is solid light beam, and another beam of laser is hollow beam；The party
Method improves the defect of above-mentioned imaging method, it is only necessary to and it is primary to scan sample, while also not needing to use fluorochrome label sample, it is real
Show in the case where maintaining like high-resolution, has improved image taking speed.
The purpose of the invention is to not drop while improving the lateral resolution and signal-to-noise ratio of confocal microscope system
The damage of low image taking speed and reduction to sample.
The present invention is based on dual-wavelength difference non-marked micro-imaging principles to improve system lateral resolution, uses two beam wavelength
Close laser irradiating sample simultaneously, and at the same time the laser for the two beam different wave lengths that detection is reflected from sample surfaces, then right
Two signals detected do difference processing, carry out the reconstruction of image after processing again.
A kind of method of dual-wavelength difference non-marked micro-imaging, comprising:
Using first laser and second laser scanning sample with wavelength difference, wherein having beam of laser is hollow beam,
Another beam of laser is solid light beam；
The signal light that sample issues is acquired, the signal light includes by the first signal light of first laser reflection and by the
The second signal light of two first lasers reflection；
The data of first signal light and second signal light are carried out subtracting each other processing, obtain differential signal, and utilize institute
It states differential signal and carries out reestablishment imaging.
Preferably, the wavelength difference of the first laser and second laser is in 20-45nm.
Preferably, being greater than the second laser of the first laser as hollow beam using wavelength.In another equivalent side
It, can also be using the lesser first laser of wavelength as hollow beam in case.
Further preferably smaller hollow light spot is formed, is had as hollow beam using the lesser first laser of wavelength
Superior technique effect.
Preferably, the first laser is modulated to P light after collimation；The second laser is modulated to P after collimation
Light is simultaneously converted to the hollow beam；The P light of first laser and the hollow beam of second laser scan sample after closing beam.
It is scanned preferably, closing two beam laser after beam and focusing to sample surface after being converted to rotatory polarization.
It adopts with the aforedescribed process, specific step is as follows in the technical program:
(1) laser that the shorter first laser device of wavelength and longer wavelengths of second laser issue is all first respectively by the
One single mode optical fiber and the second Single-Mode Fiber Coupling, are collimated through the first collimation lens and the second collimation lens again respectively.
(2) laser beam shorter by the wavelength of collimation, first passes through the first half wave plate, by modulation of polarization direction
For P light, P light is reflected by the first dichroic mirror；It is anti-by the first reflecting mirror by the longer wavelengths of laser beam of collimation
It penetrates, reflected light is transmitted through by vortex phase plate, vortex phase plate cooperates quarter-wave plate will after the second half wave plate
The light modulation is at hollow beam；It is overlapped after the laser beam transmission of vortex phase plate crosses dichroic mirror with the shorter laser of wavelength；
The azimuth for two beam laser being overlapped is by the orderly change at any time of the first two-dimensional scanning mirrors, and then in sample Surface scan sample
Product；It is focused later by the first scanning lens, while calibrating the barrel distortion and pincushion distortion generated due to two-dimensional scanning mirrors；
Tube lens and scanning lens form 4f system, for relaying to laser；It is anti-by second by the laser beam of tube lens
It penetrates mirror and is reflected into quarter-wave on piece, laser beam is become rotatory polarization by quarter-wave plate；Rotatory polarization focuses sample by object lens
On face；The reflected light and scattering light of sample surface successively pass through object lens and quarter-wave plate along original optical path, become S light；S light again according to
It is secondary to pass through the second reflecting mirror, through tube lens and scanning lens, it is reflected at the first polarization beam apparatus；The shorter reflection of wavelength
Light is reflected in the second dichroic mirror, and longer wavelengths of reflected light is transmitted in the second dichroic mirror；The shorter reflected light of wavelength is narrow through first
Band filter is coupled into third single mode optical fiber at the third lens, the first fiber-optical probe detect the wavelength of third single mode optical fiber compared with
Short reflected light；Longer wavelengths of transmitted light is reflected by third reflecting mirror, by the second narrow band filter at the 4th lens coupling
It closes into the 4th single mode optical fiber, the second fiber-optical probe detects the longer wavelengths of reflected light of the 4th single mode optical fiber；
The data of (3) first fiber-optical probes and the second fiber-optical probe are acquired by data collecting card, send in computer into
Row data processing；
(4) computer after the data normalization of the first fiber-optical probe and the second fiber-optical probe, will carry out subtracting each other processing,
Obtain differential signal；
(5) sample to be tested is placed on example platform, for the two-dimensional measurement of complete paired samples, computer passes through control two
The deflection of the orderly change laser of dimension scanning galvanometer is in turn scanned sample；
(6) it scans through piece image and the signal after difference processing is subjected to 2-d reconstruction again later.
Wherein, the scanning range of the range setting two-dimensional scanning mirrors measured as needed.
It wherein, is to make collimation laser by there is no energy when the first polarization beam apparatus for P light by two beam Laser Modulations
Loss.
Wherein, notice that the direction of vortex phase plate will cooperate the direction of quarter-wave plate to guarantee longer wavelengths of laser quilt
It is modulated to hollow beam.
Wherein, the effect of quarter-wave plate is first is that be modulated to rotatory polarization for line polarisation, second is that cooperation vortex phase plate will
Longer wavelengths of laser beam modulation is hollow beam.
Wherein, line polarisation is modulated to rotatory polarization to be scanned to be to make scanning light spot uniform intensity.
Wherein, the shorter solid laser beam of wavelength and longer wavelengths of hollow laser beam focus on same on sample surface simultaneously
Point, the shorter solid laser beam of wavelength and longer wavelengths of hollow laser beam reflected by sample surface is respectively by the first fiber-optic probe
Device and the second fiber-optical probe detect simultaneously, and the voltage signal that the first fiber-optical probe and the second fiber-optical probe detect is same
When by data collecting card acquire and be converted into digital signal, do difference processing again after two digital signal normalizations.
Wherein, vortex phase plate is also known as 0/2 π phase board, his effect is to combine quarter-wave plate by Airy disk energy
It is compressed to edge, forms the hot spot of central concave, i.e. hollow light spot, sample is carried out simultaneously using hollow light spot and solid hot spot
Then scanning obtains the hollow light spot and solid hot spot of sample surface reflection, the result of two hot spots is finally carried out calculus of differences,
Resolution ratio can be improved.
The principle of the present invention is as follows:
The shorter laser beam of the wavelength being emitted from laser and longer wavelengths of laser beam pass through single mode optical fiber respectively
It is collimated with collimation lens；Passing through half wave plate after the shorter laser beam of wavelength for its modulation of polarization direction is the direction P,
It is reflected again by the first dichroic mirror；Pass through reflecting mirror after longer wavelengths of laser beam to be reflected, then passes through half wave plate
It is the direction P by its modulation of polarization direction, P light is modulated to hollow beam after vortex phase plate, it is transmitted in the first dichroic mirror,
The laser beam shorter with wavelength is overlapped at this time；Two beam laser being overlapped are transmitted by first polarization beam apparatus, after pass through
It crosses two-dimensional scanning mirrors scanning and forms two-dimensional scanning light spot in sample surface, reflected using after 4f system by the second reflecting mirror,
Reflected light is converted into rotatory polarization after the first quarter-wave plate.
Rotatory polarization focuses on sample surface through object lens.The reflected light and scattering light of sample surface first pass through object lens collection, then pass through
Quarter-wave plate transmission is crossed, S light is switched to by rotatory polarization, is reflected after S light through the second reflecting mirror, the S light of reflection successively passes through again
It crosses through tube lens, scanning lens and two-dimensional scanning mirrors system, is reflected at the first polarization beam apparatus；The shorter reflection of wavelength
Light is reflected in the second dichroic mirror, and longer wavelengths of reflected light is transmitted in the second dichroic mirror；The shorter transmitted light of wavelength is narrow through first
Band filter is simultaneously coupled into third single mode optical fiber in the third lens, the first fiber-optical probe detect the wavelength of third single mode optical fiber compared with
Short transmitted light；Longer wavelengths of transmitted light is reflected by third reflecting mirror, by the second narrow band filter in the 4th Lens Coupling
Into the 4th single mode optical fiber, the second fiber-optical probe detects the longer wavelengths of reflected light of the 4th single mode optical fiber；
Upper sample is placed in example platform, sample is scanned using rotatory polarization.Benefit is computerizedd control two-dimensional scanning
Galvanometer accurately to adjust laser beam projection in the position on sample surface, carries out two-dimensional scanning to sample, passes through two fiber-optic probes
Device obtains the shorter solid hot spot and longer wavelengths of hollow light spot of wavelength of reflection respectively, corresponding response signal be V (v1,
) and V (v2, u2) u1；
Voltage signal is acquired by data collecting card and is become digital signal and send in computer；Computer is to two digital signals
It is normalized, then does calculus of differences；Repeat process above, until scanning through piece image, then using computer into
Row image reconstruction.
Wherein, the reason for being converted into P light is in order to which energy will not lose when making collimation laser through the first polarization beam apparatus.
It wherein, is to make collimation laser by there is no energy when the first polarization beam apparatus for P light by two beam Laser Modulations
Loss.
Wherein, notice that the direction of vortex phase plate will cooperate the direction of quarter-wave plate to guarantee longer wavelengths of laser quilt
It is modulated to hollow beam.
Wherein, the effect of quarter-wave plate is first is that be modulated to rotatory polarization for line polarisation, second is that cooperation vortex phase plate will
Longer wavelengths of laser beam modulation is hollow beam.
Wherein, the reason of being converted into rotatory polarization is to make scanning light spot uniform intensity.
Wherein, the scanning range of the range setting two-dimensional scanning mirrors measured as needed.
Wherein, the shorter solid laser beam of wavelength and longer wavelengths of hollow laser beam focus on same on sample surface simultaneously
Point, the shorter solid laser beam of wavelength and longer wavelengths of hollow laser beam reflected by sample surface is respectively by the first fiber-optic probe
Device and the second fiber-optical probe detect simultaneously, are detected to obtain voltage signal by the first fiber-optical probe and the second fiber-optical probe same
When by data collecting card acquire and be converted into digital signal, two digital signals do difference processing after being normalized again,
Difference processing can effectively improve the resolution ratio of microscopic system；Irradiating sample and the detection simultaneously simultaneously of two beam laser is from sample surface
Two beam laser of reflection, can effectively improve image taking speed.
Wherein, vortex phase plate is also known as 0/2 π phase board, he cooperates quarter-wave plate by Airy disk energy compression to side
Edge forms the hot spot of central concave, i.e. hollow light spot, is scanned simultaneously to sample using hollow light spot and solid hot spot, so
The reflection hollow light spot and solid hot spot for obtaining sample surface afterwards will carry out calculus of differences after the result normalization of two hot spots, can
To improve resolution ratio.
Wherein, the principle of difference is that the PSF that microscopical practical PSF is equivalent to the shorter solid laser beam of wavelength is subtracted
The PSF of the hollow laser beam of 450 wavelength, the PSF become narrower, so that improving microscopic system obtains resolution ratio.
The present invention also provides a kind of devices of dual-wavelength difference non-marked micro-imaging, comprising:
First laser device and second laser, output has the first laser and second laser of wavelength difference respectively；
Modulation element in the optical path of the first laser or second laser, modulating corresponding laser is hollow light
Beam；
First laser and second laser are focused to sample stage and collect the signal light reflected from sample by object lens；
First fiber-optical probe and the second fiber-optical probe, collect respectively by first laser reflection the first signal light and by
The second signal light of second first laser reflection；
And computer, the data of first signal light and second signal light are carried out subtracting each other processing, obtain differential signal,
And reestablishment imaging is carried out using the differential signal.
Preferably, the wavelength difference of the first laser and second laser is greater than described first in 20-45nm, with wavelength
The second laser of laser is hollow beam.
It, can also be using the lesser first laser of wavelength as hollow beam in another equivalent scheme.Further preferably adopt
It uses the lesser first laser of wavelength as hollow beam, forms smaller hollow light spot, there is superior technique effect.
It, will be to stress preferably, be equipped with half wave plate in the optical path of the first laser and second laser
The modulation of polarization direction of light light beam is P light；Modulation element in second laser optical path is the vortex phase plate set gradually and four
The second laser is modulated to hollow beam by/mono- wave plate, mating reaction.
Preferably, being modulated to the first laser of P light and being modulated to the second laser of hollow beam through the first dichroic mirror
After close beam, sample is scanned by the two-dimensional scanning mirrors in optical path.
Preferably, respectively enteing the first fiber-optical probe after the second dichroic mirror beam splitting by the signal light that object lens are collected
With the second fiber-optical probe.
More specifically, the device of the dual-wavelength difference non-marked micro-imaging of the technical program includes:
First laser device and second laser, for issuing the shorter laser beam of wavelength and longer wavelengths of laser beam；
First single mode optical fiber and the first collimation lens, collimate for the laser beam shorter to wavelength；
Second single mode optical fiber and the second collimation lens, for being collimated to longer wavelengths of laser beam；
First half wave plate, for being P light by the shorter laser beam modulation of wavelength, when making it through polarization beam apparatus
There is no energy loss；
First reflecting mirror, for reflecting longer wavelengths of laser beam；
Second half wave plate, for being P light by longer wavelengths of laser beam modulation, when making it through polarization beam apparatus
There is no energy loss；
First vortex phase plate forms hollow light spot for carrying out Spatial transmission to longer wavelengths of laser beam；
First dichroic mirror, the laser beam shorter for reflection wavelength and the longer laser beam of transmission peak wavelength；
First polarization beam apparatus is carried out for transmiting to the line polarisation, and to the reflected light that object lens are collected with scattering light
Reflection；
First two-dimensional scanning mirrors, for carrying out azimuthal change to the laser, so that the scanning of sample surface is carried out,
And solution scanning is carried out to the laser reflected from sample；
First scanning lens eliminates barrel distortion and pillow for calibrating to the laser beam for being scanned galvanometer
Shape distortion；
First tube lens form 4f system for the laser beam to be expanded and collimated, while with scanning lens；
Second reflecting mirror, for reflecting the laser beam；
Quarter-wave plate makes scanning light spot optical power detection for the line polarisation to be modulated to rotatory polarization；
Object lens for being focused to the rotatory polarization, and then are scanned sample, and be collected simultaneously example platform
Reflected light and scattering light；
Second dichroic mirror, the shorter laser reflection of wavelength for will be reflected from sample surface, while will be reflected from sample surface
Longer wavelengths of laser transmission；
First narrow band filter slice, for filtering out extra longer wavelengths of laser；
The third lens, for by the shorter laser coupled of wavelength into third single mode optical fiber；
Third single mode optical fiber functions simultaneously as the burnt aperture of copolymerization for conducting the shorter laser of wavelength；
First fiber-optical probe is measured for the laser shorter to wavelength, obtains voltage signal；
Second narrow band filter slice, for filtering out the shorter laser of extra wavelength；
4th lens, for by longer wavelengths of laser coupled into third single mode optical fiber；
4th single mode optical fiber functions simultaneously as the burnt aperture of copolymerization for conducting longer wavelengths of laser；
Second fiber-optical probe obtains voltage signal for measuring to longer wavelengths of laser；
Example platform, for placing sample；
Data collecting card, for acquiring the voltage signal of the first fiber-optical probe and the second fiber-optical probe, and by its turn
Digital signal is changed to send in computer；
Main control computer the voltage signal of capture card and carries out difference processing and sample image and rebuilds for receiving data,
Simultaneously for controlling the movement of two-dimensional scanning mirrors, sample is scanned；
The present invention, which compares prior art, has following innovative point:
(1) dual-wavelength difference micro-imaging technique is used, the lateral resolution and signal-to-noise ratio of system are improved.
(2) two beam laser irradiating sample, and capture card while the data for acquiring two detectors simultaneously, improves imaging
Speed；
The present invention compares prior art and has the advantage that
(1) system signal noise ratio and high resolution: the detection method that difference is subtracted each other two-by-two can inhibit the state difference of environment
Noise reduces PSF to improve signal-to-noise ratio to improve lateral resolution.
(2) image taking speed is fast: run-down sample only being needed to can be obtained by the image of solid hot spot and hollow light spot；
(3) apparatus structure is succinct.
Detailed description of the invention
Fig. 1 is the schematic device of dual-wavelength difference non-marked micro-imaging of the present invention；
Fig. 2 is the PSF of the shorter solid laser beam of wavelength；
Fig. 3 is the PSF of longer wavelengths of hollow laser beam；
Fig. 4 be microscopic system practical PSF, be subtracted by the PSF of the shorter solid laser beam of wavelength it is longer wavelengths of
What the PSF of hollow laser beam was obtained；
Fig. 5 is the result figure of common confocal microscope system shooting；
Fig. 6 is the result figure of dual-wavelength difference non-marked micro imaging system shooting.
In figure: longer wavelengths of first laser device 1, the first single mode optical fiber 2, the first collimation lens 3, the first reflecting mirror 4, the
Two half wave plates 5, the first vortex phase plate 6, the first dichroic mirror 7, the shorter first laser device 8 of wavelength, the second single-mode optics
Fibre 9, the second collimation lens 10, the first half wave plate 11, polarization beam apparatus 12, two-dimensional scanning mirrors system 13, scanning are saturating
Mirror 14, tube lens 15, the second reflecting mirror 16, quarter-wave plate 17, object lens 18, sample stage 19, the second dichroic mirror 20, first
Narrow band filter slice 21, the third lens 22, third single mode optical fiber 23, the first fiber-optical probe 24, third reflecting mirror 25, the second narrowband
Filter plate 26, the 4th lens 27, the 4th single mode optical fiber 28, the second fiber-optical probe 29, data collecting card and computer 30, display
Screen 31.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be described in detail, but the present invention is not limited to this.
A kind of device of dual-wavelength difference non-marked micro-imaging, as shown in Figure 1, comprising: longer wavelengths of first laser
Device 1, the first single mode optical fiber 2, the first collimation lens 3, the first reflecting mirror 4, the second half wave plate 5, the first vortex phase plate
6, the first dichroic mirror 7, the shorter first laser device 8 of wavelength, the second single mode optical fiber 9, the second collimation lens 10, the first half
Wave plate 11, polarization beam apparatus 12, two-dimensional scanning mirrors system 13, scanning lens 14, tube lens 15, the second reflecting mirror 16, four
/ mono- wave plate 17, object lens 18, sample stage 19, the second dichroic mirror 20, the first narrow band filter slice 21, the third lens 22, third list
Mode fiber 23, the first fiber-optical probe 24, third reflecting mirror 25, the second narrow band filter slice 26, the 4th lens 27, the 4th single-mode optics
Fibre 28, the second fiber-optical probe 29, data collecting card and computer 30, display screen 31.Wherein, the outgoing of the first single mode optical fiber 2
End face is located near the object focus of the first collimation lens 3, and the outgoing end face of the second single mode optical fiber 9 is located at the second collimation lens 10
Object focus near；Example platform 19 is placed on the focal plane of object lens 18, and third single mode optical fiber 23 is placed on the third lens
22 6 focal planes, the 4th single mode optical fiber 28 are placed on the focal plane of the 4th lens 27；Computer and data collecting card composition 30,
Computer and data collecting card 30 are connected with the first fiber-optical probe 24 and the second fiber-optical probe 29, computer and data acquisition
Card 30 is connected with two-dimensional scanning mirrors, and computer and data collecting card 30 are connected with display screen 31.
In the present embodiment, the optical maser wavelength that longer wavelengths of first laser device 1 exports is 445, and wavelength is shorter first to swash
The optical maser wavelength that light device 8 exports is 405.
Using device shown in FIG. 1, the method for lateral resolution is improved such as using dual wavelength non-marked difference micro-imaging
Under:
The longer wavelengths of laser beam issued from first laser device 1, is first introduced into single mode optical fiber 2, from single mode optical fiber 2
The longer wavelengths of laser beam of outgoing, completes primary collimation by collimation lens 3；Longer wavelengths of by primary collimation swashs
Light light beam passes through the first reflecting mirror 4 again and is reflected, 90 degree of the angular deflection of longer wavelengths of laser beam；By the wave of angular deflection
Long longer laser beam is by the second half wave plate 5 by bits of modulation P light；P light carries out position using vortex phase plate 6 and mutually adjusts
System, becomes hollow beam；Hollow beam is transmitted by the first dichroic mirror 7, and the laser beam shorter with wavelength is overlapped.From first laser
The shorter laser beam of the wavelength that device 8 issues, is first introduced into single mode optical fiber 9, and the wavelength being emitted from single mode optical fiber 9 is shorter to swash
Light light beam completes primary collimation by collimation lens 10；Pass through first again by the shorter laser beam of the wavelength of primary collimation
Half wave plate is modulated to P light；It is reflected after P light in the first dichroic mirror 7, is overlapped with longer wavelengths of laser beam.
Transmitted through the first polarization beam apparatus 12 after two beam laser beams being overlapped, wherein the first half wave plate and second
The purpose of half wave plate is the polarization side for guaranteeing the polarization direction of two beam laser and the first polarization beam apparatus 12 and allowing to transmit
To identical, laser beam can be made not generated a large amount of energy damage by reflection when through the first polarization beam apparatus 12 in this way
It loses.
P light transmission is crossed after the first polarization beam apparatus 12, and two-dimensional scanning mirrors 13, the fortune of two-dimensional scanning mirrors are then passed through
Dynamic rail mark is controlled by computer 30, and scanning mode is raster scanning, it is therefore an objective to laser beam be made in sample surface to be also raster pattern
Scanning sample, to obtain the biggish sample image of visual field.
It is made up of by the laser beam that two-dimensional scanning mirrors change deflection a scanning lens 14 and tube lens 15
4f system, wherein the effect of scanning lens 14 is to calibrate the barrel distortion and pincushion distortion generated due to two-dimensional scanning mirrors, mirror
Cylinder lens 15 are to relay and collimated laser beam.
Laser beam by 4f system, which is then reflected by the second reflecting mirror 16, reaches quarter-wave plate 17, a quarter
The effect of wave plate 17 makes to scan the hot spot of sample more evenly, second is that cooperation vortex phase plate 6 first is that be rotatory polarization by P light modulation
By longer wavelengths of laser beam modulation position hollow beam.Rotatory polarization is focused on sample surface using object lens 18.
The reflected light of sample surface and scattering light become S light transmitted through quarter-wave plate 17 by the collection of object lens 18, and in
It reflects at second reflecting mirror 16, is scanned using after 4f system by two bit scan galvanometer solutions, wherein solution scanning is to make to reflect
Light and scattering light are glancing incidences when being incident on the third lens 22 and four lens 27, guarantee that laser beam passes through the third lens 22
It is behind fixed point at focal plane after being focused with the 4th lens 27.
It is reflected after the first polarization beam apparatus 12 by the reflected light of solution scanning and scattering light；Reflected light passes through the two or two
When Look mirror 20, the shorter laser beam of wavelength is reflected, and longer wavelengths of laser beam is transmitted, to separate two beam laser.
The shorter laser beam of the wavelength reflected passes through the first narrow band filter 21, filters out extra longer wavelengths of laser
Beam, wherein the central wavelength of the first narrow band filter 21 is the central wavelength of the shorter laser of wavelength；By the wavelength of optical filter
Shorter laser beam focuses on back focal plane using the third lens poly- 22, and then is coupled into third single mode optical fiber 23, wherein the
The end face of three single mode optical fibers 23 is located in the focus of the third lens poly- 22；Third single mode optical fiber 23 send laser beam in the first detection
Device 24 is detected, output voltage signal.
The longer wavelengths of laser beam being transmitted is reflected by third reflecting mirror 25, and reflected light is filtered using the second narrowband
Wave device 26 filters out the shorter laser beam of extra wavelength, wherein the central wavelength of the second narrow band filter 26 is longer wavelengths of
The central wavelength of laser beam；Longer wavelengths of laser beam after optical filter is burnt after focusing on using the 4th lens poly- 27
On face, so that the 4th single mode optical fiber 28 is coupled into, wherein the end face of the 4th single mode optical fiber 28 is located at the focus of the 4th lens poly- 27
On；Laser beam is sent and is detected in the second detector 29 by the 4th single mode optical fiber 28, output voltage signal.
Two voltage signals of output are acquired by data collecting card 30, are transmitted to computer 30 later and are handled, wherein first
It is moved by computer control two-dimensional scanning mirrors 13 and then laser is made to carry out raster scanning to sample, while scanning, acquisition
Two channels of card acquire the voltage signal of the first detector 24 and the first detector 29 simultaneously respectively, have acquired piece image
After data, the data in two channels are first normalized, and then carry out difference processing, and differentiated data carry out permutatation,
A two-dimensional array is formed, by two-dimensional array by being then displayed on display screen 31 after data processing.
Since the shorter solid laser beam of wavelength and longer wavelengths of hollow laser beam have been carried out difference processing, improving
It is also improved while resolution ratio signal-to-noise ratio (noise that can inhibit the state difference of environment), while image taking speed also very fast (phase
Than for scanning through piece image with solid hot spot and then being scanned with hollow light spot speed improve one times).
Fig. 2 to Fig. 4 shows the burnt principle of double-colored difference copolymerization, the PSF of the shorter solid light beam of wavelength subtract wavelength compared with
Long hollow beam obtains a smaller solid light beam, which is equivalent to the microscopical practical PSF, smaller
PSF means better resolution ratio.
After Fig. 5 to Fig. 6 shows the method and apparatus using dual-wavelength difference non-marked micro-imaging, imaging resolution is obtained
It is promoted.Fig. 5 is the result figure of the fluorescent microsphere of common confocal microscope system shooting, and Fig. 6 is that dual-wavelength difference non-marked is aobvious
The result figure of micro- imaging system shooting, wherein what is shot is the same area of sample.
The foregoing is merely preferable implementation examples of the invention, are not intended to restrict the invention, it is all in spirit of that invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method of dual-wavelength difference non-marked micro-imaging characterized by comprising
Using first laser and second laser scanning sample with wavelength difference, wherein having beam of laser is hollow beam, it is another
Shu Jiguang is solid light beam；
The signal light that sample issues is acquired, the signal light includes by the first signal light of first laser reflection and by the two the
The second signal light of one laser reflection；
The data of first signal light and second signal light are carried out subtracting each other processing, obtain differential signal, and utilize the difference
Sub-signal carries out reestablishment imaging.
2. the method for dual-wavelength difference non-marked micro-imaging as described in claim 1, which is characterized in that the first laser
Wavelength difference with second laser is in 20-45nm.
3. the method for dual-wavelength difference non-marked micro-imaging as claimed in claim 1 or 2, it is characterised in that: big with wavelength
In the first laser second laser be hollow beam.
4. the method for dual-wavelength difference non-marked micro-imaging as claimed in claim 3, it is characterised in that: the first laser
P light is modulated to after collimation；The second laser is modulated to P light after collimation and is converted to the hollow beam；First swashs
The P light of light and the hollow beam of second laser scan sample after closing beam.
5. the method for dual-wavelength difference non-marked micro-imaging as claimed in claim 4, it is characterised in that: two beams after closing beam
Laser focuses to sample surface after being converted to rotatory polarization and is scanned.
6. a kind of device of dual-wavelength difference non-marked micro-imaging, it is characterised in that: include:
First laser device and second laser, output has the first laser and second laser of wavelength difference respectively；
Modulation element in the optical path of the first laser or second laser, modulating corresponding laser is hollow beam；
First laser and second laser are focused to sample stage and collect the signal light reflected from sample stage by object lens；
First fiber-optical probe and the second fiber-optical probe are collected respectively by the first signal light of first laser reflection and by second
The second signal light of first laser reflection；
And computer, the data of first signal light and second signal light are carried out subtracting each other processing, obtain differential signal, and benefit
Reestablishment imaging is carried out with the differential signal.
7. the device of dual-wavelength difference non-marked micro-imaging as claimed in claim 6, it is characterised in that: the first laser
Wavelength difference with second laser is greater than the second laser of the first laser as hollow beam using wavelength in 20-45nm.
8. the device of dual-wavelength difference non-marked micro-imaging as claimed in claim 7, it is characterised in that: the first laser
With half wave plate is equipped in the optical path of second laser, be P light by the modulation of polarization direction of corresponding laser beam；
Modulation element in second laser optical path is the vortex phase plate and quarter-wave plate set gradually, and mating reaction is by institute
The second laser stated is modulated to hollow beam.
9. the device of dual-wavelength difference non-marked micro-imaging as claimed in claim 8, it is characterised in that: be modulated to P light
First laser and the second laser for being modulated to hollow beam close beam after the first dichroic mirror, pass through the two-dimensional scanning mirrors in optical path
Sample is scanned.
10. the device of dual-wavelength difference non-marked micro-imaging as claimed in claim 9, it is characterised in that: collected by object lens
Signal light respectively enter the first fiber-optical probe and the second fiber-optical probe after the second dichroic mirror beam splitting.
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