CN108845408A - Based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method and apparatus - Google Patents

Based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method and apparatus Download PDF

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CN108845408A
CN108845408A CN201810712161.1A CN201810712161A CN108845408A CN 108845408 A CN108845408 A CN 108845408A CN 201810712161 A CN201810712161 A CN 201810712161A CN 108845408 A CN108845408 A CN 108845408A
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frequency
optical axis
carrier frequency
light beam
prism
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CN108845408B (en
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郭汉明
王秀花
王俊炜
郑思旭
柳阳
谢剑锋
黄斐
张肖肖
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University of Shanghai for Science and Technology
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University of Shanghai for Science and Technology
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    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052Optical details of the image generation
    • G02B21/0076Optical details of the image generation arrangements using fluorescence or luminescence
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036Scanning details, e.g. scanning stages
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036Scanning details, e.g. scanning stages
    • G02B21/0048Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B26/00Optical devices or arrangements for the control of light using movable or deformable optical elements
    • G02B26/08Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
    • G02B26/10Scanning systems
    • G02B26/12Scanning systems using multifaceted mirrors
    • G02B26/123Multibeam scanners, e.g. using multiple light sources or beam splitters

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

It is according to the present invention to be based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method and apparatus, device includes beam splitter, first frequency modulator, the first reflecting mirror, the second reflecting mirror, half wave plate, polarization splitting prism, second frequency modulator, dichroscope, polyhedral prism, 2-D vibration mirror, the first lens unit, focusing objective len, the second lens unit, pin hole component, detector.Beam splitter, the first reflecting mirror are arranged along primary optic axis, beam splitter, first frequency modulator, the second reflecting mirror are arranged along the second optical axis, first reflecting mirror, first frequency modulator, half wave plate, polarization splitting prism are arranged along third optical axis, second reflecting mirror, polarization splitting prism, second frequency modulator, dichroscope, polyhedral prism, 2-D vibration mirror are arranged along the 4th optical axis, 2-D vibration mirror, the first lens unit, focusing objective len are arranged along the 5th optical axis, and dichroscope, the second lens unit, pin hole component, detector are arranged along the 6th optical axis.

Description

Based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method and apparatus
Technical field
The invention belongs to optical technical fields, and in particular to one kind is high based on polyhedral prism and light beam frequency modulation high-resolution Fast imaging method and device.
Background technique
Optical imagery is widely used in life science, material science etc. and grinds due to having the characteristics that non-contact, non-destructive Study carefully field.And optical resolution is then an important indicator of optical imaging system, the higher the better for usual resolution ratio.Optical resolution Rate includes lateral resolution and axial resolution, can be limited each other between the two.I.e. lateral resolution is higher, axial resolution It is lower;Axial resolution is higher, and lateral resolution is lower.Therefore, how to improve lateral resolution and axial resolution is research The target of personnel's ongoing effort.There is important application valence if it can be achieved at the same time high lateral resolution and high axial resolution Value.
There is important research significance in the fields such as optical microscopy imaging using the vectorial property of linearly polarized light.For example, altogether Focusing microscope system is the point-to-point imaging of image conjugation, and the laser beam of focusing is scanned in sample surfaces, while photoelectric detector Part receives the fluorescence (or fluorescence of transmission) of sample reflection, and the variation of sample structure changes the fluorescence intensity of excitation, thus makes The output electric current of photoelectric detector changes, and by signal processing, simultaneous display is on the computer screen.Due to the linear polarization of irradiation Light by the lens focus of high-NA, generation be area very little ellipse light spot.If along ellipse light spot short-axis direction To Sample Scan, according to Rayleigh criterion, confocal microscope scanning step be twice of ellipse short shaft apart from when, photodetector The change for responding intensity of reflected light, that is, tell the difference of two o'clock, systemic resolution is very high.If along ellipse light spot long axis direction To Sample Scan, and less than twice transverse of scanning step apart from when, according to Rayleigh criterion, photodetector be will not respond to The change of intensity of reflected light can not tell the difference of two o'clock.Therefore the resolution ratio of system is decided by focal beam spot long axis size. In first technology, referring to " K.A.Serrels, E.Ramsay, R.J.Warburton and D.T.Reid, Nanoscale Optical microscopy in the vectorial focusing regime, nature photonics, vol.2, May2008,311-314 ", in order to improve resolution ratio, when scanning long axis direction mechanical insertion half wave plate change into The polarization direction of ray polarised light, but this can reduce the sweep speed and system resolution precision when system changes scanning direction, And since wherein a branch of incident light have passed through a half wave plate more, the incident power of this two beams crossed polarized light Difference will increase systematic error so that focus on light beam power be made to change, and system stability is not high.
Summary of the invention
In view of the deficiencies of the prior art, one of the objects of the present invention is to provide a kind of polyhedral prism and light beam frequency modulation are high High resolution speed imaging method and device, by a kind of effective optical texture, while it is orthogonal to construct two beam polarization directions Linearly polarized light illumination, and the elliptical light formed after two beam orhtogonal linear polarizaiton light focus is distinguished using the frequency modulation(PFM) of dual-beam The overlapping region of spot and other Non-overlapping Domains, while single-row, equidistant tool is generated using polyhedral prism and frequency modulator There is the multifocal of different modulating frequency, it is received multiple with different modulating frequency to distinguish detector using frequency demodulation algorithm Each self-excitation of focus fluorescence signal, thus reconstruct reflection sample message image.
The present invention provides one kind to be based on polyhedral prism and light beam frequency modulation high-resolution high-speed imaging device, has in this way Feature, including beam splitter, first frequency modulator, the first reflecting mirror, the second reflecting mirror, half wave plate, polarization spectro Prism, second frequency modulator, dichroscope, polyhedral prism, 2-D vibration mirror, the first lens unit, focusing objective len, second are thoroughly Mirror unit, pin hole component, detector, wherein beam splitter, the first reflecting mirror are set gradually along primary optic axis line, beam splitter, first Frequency modulator, the second reflecting mirror are set gradually along the second optical axis vertical with primary optic axis line, the first reflecting mirror, the first frequency Rate modulator, half wave plate and polarization splitting prism are set gradually along the third optical axis vertical with primary optic axis line, Third optical axis is parallel with the second optical axis, the second reflecting mirror, polarization splitting prism, second frequency modulator, dichroscope, more Face body prism and 2-D vibration mirror are set gradually along the 4th optical axis vertical with third optical axis, 2-D vibration mirror, the first lens Unit, focusing objective len are set gradually along the 5th optical axis vertical with the 4th optical axis, dichroscope, the second lens unit, needle Aperture member and detector are set gradually along the 6th optical axis vertical with the 4th optical axis, the first lens unit, the second lens Unit includes the combination of a lens or multiple lens.
Provided by the invention based in polyhedral prism and light beam frequency modulation high-resolution high-speed imaging device, feature exists In further including the third lens list being arranged on the second optical axis and between first frequency modulator and the second reflecting mirror Member, the third lens unit include the combination of a lens or multiple lens.
The present invention provides one kind to be existed based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, feature In including the following steps:
Step 1, beam splitter, the first reflecting mirror are set gradually along primary optic axis line;
Step 2, by beam splitter, first frequency modulator, the second reflecting mirror along second optical axis vertical with primary optic axis line Line is set gradually;
Step 3, by the first reflecting mirror, first frequency modulator, half wave plate and polarization splitting prism along with the The vertical third optical axis of one optical axis is set gradually, and third optical axis is parallel with the second optical axis;
Step 4, by the second reflecting mirror, polarization splitting prism, second frequency modulator, dichroscope, polyhedral prism with And 2-D vibration mirror is set gradually along the 4th optical axis vertical with third optical axis;
Step 5, by 2-D vibration mirror, the first lens unit, focusing objective len and sample along vertical with the 4th optical axis the Five optical axis are set gradually;
Step 6, by dichroscope, the second lens unit, pin hole component and detector are along vertical with the 4th optical axis 6th optical axis is set gradually, wherein the second lens unit faces the reflecting surface of dichroscope;
Step 7, a branch of to carry out incident first direction incident ray polarized light after beam splitter along primary optic axis line direction Export the first light beam and the second light beam that two beams have first direction linearly polarized light;
Step 8, the first light beam exports first after first frequency modulator applies carrier frequency f1 along the second optical axis direction Carrier frequency light beam, the first carrier frequency light beam are reflected along the 4th optical axis direction through the second reflecting mirror by polarization splitting prism transmission output First carrier frequency transmitted light beam, the second light beam along primary optic axis line direction by the first reflecting mirror reflection after, along third optical axis side To the second carrier frequency light beam is exported after first frequency modulator applies carrier frequency f2, the second carrier frequency light beam passes through half wave plate Second direction linearly polarized light is exported afterwards, and second direction linearly polarized light exports after polarization splitting prism reflects along the 4th optical axis The second carrier frequency the reflected beams in direction;
Step 9, the first carrier frequency transmitted light beam and the superposition synthesis output of the second carrier frequency the reflected beams have mixing first direction With the linear polarization carrier frequency mixed light beam of second direction;
Step 10, for linear polarization carrier frequency mixed light beam after second frequency modulator, output multi beam has different carrier frequency Mixing carrier frequency directional light;
Step 11, multi beam has the mixing carrier frequency directional light of different carrier frequency after dichroscope enters polyhedral prism, Export multi beam mixing carrier frequency deflecting light beams;
Step 12, multi beam mixing carrier frequency deflecting light beams are after 2-D vibration mirror, the first lens unit, focusing objective len in sample It is upper to generate multiple focal beam spots;
Step 13, multiple focal beam spot excitation samples generate fluorescence, form multiple fluorescence hot spots corresponding with focal beam spot;
Step 14, fluorescence hot spot passes through focusing objective len, the first lens unit, 2-D vibration mirror, polyhedral prism, dichroic After mirror, the second lens unit, a fluorescent foci hot spot is formed on pin hole component, and be received by a detector;
Step 15, using frequency demodulation algorithm, extract the received overlapping of detector array have carrier frequency f1 and f2 with And the fluorescent foci spot signal of other carrier frequency, and by analysis detector array on fluorescent foci hot spot intensity and with The scan variations of 2-D vibration mirror can distinguish the fluorescence signal of each self-excitation of focus with different modulating frequency, thus weight Structure goes out to reflect the two dimensional image of sample message.
Provided by the invention based in polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, can also have There is such feature:Wherein, first direction linearly polarized light is orthogonal with second direction linearly polarized light.
In addition, being gone back provided by the invention based in polyhedral prism and light beam frequency modulation high-resolution high speed imaging method It can have such feature:Wherein, polyhedral prism is the cylinder with bottom surface and multiple faceted pebbles, and cross section is polygon Shape, mixes direction and the plane perpendicular of carrier frequency directional light and mixes carrier frequency directional light and be introduced into bottom surface, and focal beam spot is one-dimensional battle array Column distribution, the quantity of focal beam spot and the quantity of faceted pebble are identical.
In addition, being gone back provided by the invention based in polyhedral prism and light beam frequency modulation high-resolution high speed imaging method It can have such feature:Wherein, the first lens unit is used for optical beam transformation, and the multi beam being emitted from polyhedral prism is mixed Carrier frequency deflecting light beams are full of the entrance pupil of focusing objective len always, realize the optimal imaging performance of focusing objective len, the first lens unit packet Include the combination of two optical beam transformation lens or multiple optical beam transformation lens, the angle of dichroscope and the 4th optical axis is 45 degree.
In addition, being gone back provided by the invention based in polyhedral prism and light beam frequency modulation high-resolution high speed imaging method It can have such feature:Wherein, when polyhedral prism be the cone prism with bottom surface and multiple faceted pebbles, the bottom surface of prism with It is vertical to mix the parallel light direction of carrier frequency, focal beam spot is two-dimensional array distribution, and the quantity of focal beam spot and the quantity of faceted pebble are identical.
In addition, being gone back provided by the invention based in polyhedral prism and light beam frequency modulation high-resolution high speed imaging method It can have such feature:It wherein, further include being arranged on the second optical axis and being located at first frequency modulator in step 2 With the second reflecting mirror for improving the third lens unit of axial resolution.
In addition, being gone back provided by the invention based in polyhedral prism and light beam frequency modulation high-resolution high speed imaging method It can have such feature:Wherein, the third lens unit includes the combination of a lens or multiple lens, for carrying first The focussing plane of frequency light beam and the focussing plane of the second carrier frequency light beam are separated by a distance.
In addition, being gone back provided by the invention based in polyhedral prism and light beam frequency modulation high-resolution high speed imaging method It can have such feature:Wherein, in step 15, using frequency demodulation algorithm, the tool of the received overlapping of detector is extracted There are the fluorescent foci spot signal of carrier frequency f1 and f2, and by the intensity of the fluorescent foci hot spot on analysis detector and with two The scan variations for tieing up galvanometer, can distinguish the fluorescence signal of each self-excitation of focus with different modulating frequency, to reconstruct Reflect the 3-D image of sample message out.
The action and effect of invention
The present invention distinguishes the ellipse light spot formed after two beam orhtogonal linear polarizaiton light focus using the frequency modulation(PFM) of dual-beam Overlapping region and other Non-overlapping Domains, using photodetector receive signal frequency demodulation it is inclined to extract two beam cross lines The corresponding useful signal in overlapping region for the ellipse light spot that vibration light is formed after focusing, to realize the mesh for improving two-dimensional resolution 's.And array focal beam spot is generated on the focusing surface of focusing objective len using polyhedral prism simultaneously, realize that multiple spot scans simultaneously Imaging significantly improves the frame speed of laser confocal scanning imaging, achievees the purpose that high speed imaging.
Alternatively, it is also possible to improve the pixel number of single-frame images in the case where frame speed is constant.Theoretically, compared to tradition Simple scan imaging, for a frame image of same pixel, the image taking speed of the multi-point scanning imaging of N number of focal beam spot can be with Improve N times;Identical for frame speed, the pixel number of a frame image can be improved N times.For example, single-point in current industry can be swashed The canonical parameter 512*32 pixel/frame of light confocal scanning imaging, frame fast 400 frames/second rise to 512*32 pixel/frame, frame speed 400N frame/second, or be 512*32N pixel/frame, frame fast 400 frames/second.
Detailed description of the invention
Fig. 1 is to improve transverse direction using the vectorial property of dual-beam linearly polarized light and frequency modulation(PFM) in the embodiment of the present invention The schematic illustration of resolution ratio;
Fig. 2 is the principle signal for improving axial resolution in the embodiment of the present invention using the frequency modulation(PFM) of dual-beam Figure;
Fig. 3 be in the embodiment of the present invention based on the warbled three-dimension high-resolution laser confocal scanning of dual-beam at As schematic diagram;And
Fig. 4 is to improve resolution ratio when scanning sample in the embodiment of the present invention while improving the schematic diagram of image taking speed;
Fig. 5 is the light beam schematic diagram of prism in the embodiment of the present invention two;
Fig. 6 is the focal beam spot distribution schematic diagram in the embodiment of the present invention two on sample;
Fig. 7 is prism section and focal beam spot distribution schematic diagram in the embodiment of the present invention three;
Fig. 8 is prism section and focal beam spot distribution schematic diagram in the embodiment of the present invention four;
Fig. 9 is prism section and focal beam spot distribution schematic diagram in the embodiment of the present invention five;And
Figure 10 is six neutral body prism of the embodiment of the present invention and focal beam spot distribution schematic diagram.
Specific embodiment
It is real below in order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention Example combination attached drawing is applied to have to of the invention based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method and apparatus work Body illustrates.
Embodiment one
If optical system uses linear polarization coherent light illumination, for high-NA optical system, on focussing plane Focal beam spot distribution significantly influenced by the polarization characteristic of illumination light.As shown in Figure 1, if using the direction y linear polarization coherent light It illuminates, the focal beam spot on focussing plane is ellipse distribution, and the direction of elliptical short axle and linearly polarized light (is at this time y Direction) vertically, i.e., elliptical short-axis direction is in the x-direction at this time.If focused flat using the direction x linear polarization coherent light illumination The short-axis direction of oval focal beam spot on face is in the y-direction.Optical system lateral resolution is decided by the size of focal beam spot.It is aobvious So, if using the direction y linear polarization coherent light illumination, the direction optical system x has higher lateral resolution;If using The direction x linear polarization coherent light illumination, then the direction optical system y has higher lateral resolution.Obviously, if it is possible to while benefit With the direction y linearly polarized light and x direction line polarized illumination, according to principle of stacking, then the focal beam spot on focussing plane is distributed as In Fig. 1 shown in right figure, it is clear that its shadow region has smaller sized fraction in the direction x and the direction y simultaneously, if using shadow region Domain then can significantly improve lateral resolution in the direction x and the direction y as effective focal beam spot.But, right figure in Fig. 1 at this time Shown non-hatched area can also deteriorate lateral resolution.In order to solve this problem, we are by the direction y linear polarization coherent light illumination Apply frequency be f1 carrier frequency (referred to as illumination light z1), the direction x linear polarization coherent light illumination apply frequency be f2 carrier frequency (referred to as Illumination light z2), then the carrier frequency of shadow region hot spot shown in right figure is f1+f2 in Fig. 1, the carrier frequency of other non-hatched areas is distinguished For f1 and f2.In this way carrier frequency be f1+f2 the fluorescence signal that is excited on sample of shadow region hot spot there is also carrier frequency f1+f2, By frequency demodulation algorithm, then the fluorescence signal of carrier frequency f1+f2 can be extracted.To reach the mesh for improving lateral resolution 's.
As in Fig. 1, the direction y linear polarization coherent light illumination is applied carrier frequency (the referred to as illumination light that frequency is f1 by we Z1), linear polarization coherent light illumination in the direction x applies frequency as the carrier frequency (referred to as illumination light z2) of f2, while increasing in illumination light z1 Add a lens, finely tune its focussing plane, the focussing plane of the focussing plane of illumination light z1 and illumination light z2 is made to separate a spacing From then as shown in Fig. 2, the focal beam spot of the focal beam spot (dashed region) of illumination light z1 and illumination light z2 are in axial direction (z-axis) side To being separated by a distance, the carrier frequency of shadow region hot spot is f1+f2 in figure, the carrier frequency of other non-hatched areas be respectively f1 and f2.There is also carrier frequency f1+f2 for the fluorescence signal that the shadow region hot spot that carrier frequency is f1+f2 in this way excites on sample, pass through frequency Rate demodulating algorithm can then extract the fluorescence signal of carrier frequency f1+f2.To achieve the purpose that improve axial resolution.
As shown in figure 3, the plane where Fig. 3 is the face yz, x-axis is perpendicular to the face yz.Based on the warbled three-dimensional of dual-beam High resolution imaging apparatus includes beam splitter 1, frequency modulator 2, lens unit 3, reflecting mirror 4, reflecting mirror 5, half wave Piece 6, polarization splitting prism 7, frequency modulator 8, dichroscope 9, polyhedral prism 10,2-D vibration mirror 11, lens 12, lens 13, focusing objective len 14, lens unit 16, pin hole component 17, detector 18.
Wherein, lens unit 3, lens unit 16 are the combination of a lens or multiple lens.Polyhedral prism 10 is Cylinder prism, cross section are the polygon with bottom edge and a plurality of seamed edge, and the effect of polyhedral prism is to generate multi beam and light Axis (y-axis) has the collimated light beam of certain angle.
In the present embodiment, optical beam transformation lens unit includes lens 12, lens 13, and lens unit 3,16 is simple lens, Polyhedral prism is prism 10 shown in fig. 5, and the direction of light beam g and base vertical and light beam g are introduced into bottom edge, focal beam spot Quantity it is identical as the quantity of seamed edge.
As shown in figure 3, beam splitter 1, reflecting mirror 5 are set gradually along primary optic axis line, beam splitter 1, frequency modulator 2, thoroughly Mirror 3, reflecting mirror 4 are set gradually along the second optical axis vertical with primary optic axis line, and reflecting mirror 5, frequency modulator 2, two/ One wave plate 6 and polarization splitting prism 7 are set gradually along the third optical axis vertical with primary optic axis line, third optical axis and Two optical axis are parallel, reflecting mirror 4, polarization splitting prism 7, frequency modulator 8, dichroscope 9, polyhedral prism 10 and two Dimension galvanometer 11 is set gradually along the 4th optical axis vertical with third optical axis, and 2-D vibration mirror 11, lens 13, focuses lens 12 Object lens 14, sample 15 are set gradually along the 5th optical axis vertical with the 4th optical axis, dichroscope 9, lens 16, pin hole component 17, detector 18 is set gradually along the 6th optical axis vertical with the 4th optical axis, and lens 16 face the reflection of dichroscope 9 Face.
The linearly polarized light in a branch of direction y is divided into II liang of beam polarised light of light beam I and light beam after beam splitter 1.Y direction line is inclined Vibration light light beam I has been applied carrier frequency f1 after frequency modulator 2, focuses by the second lens 3, the second reflecting mirror 4 reflects, warp It crosses after polarization splitting prism 7 transmits and exports the first carrier frequency transmitted light beam, the direction y linearly polarized light light beam II is reflected by reflecting mirror 5 After be frequency-modulated device 2 and be applied with carrier frequency f2, light beam II becomes the direction x linearly polarized light after half wave plate 6, by inclined Vibration Amici prism 7 is superimposed the line that synthesis output has mixing first direction and second direction with the first carrier frequency transmitted light beam after reflecting Polarization state carrier frequency mixed light beam, for the mixing carrier frequency light beam after frequency modulator 8, output multi beam has the mixing of different carrier frequency Carrier frequency directional light;In embodiment, polyhedral prism 10 is prism, which exports after passing through frequency modulator 8 With carrier frequency f3And f4Mixing carrier frequency directional light, the mixing carrier frequency directional light by dichroscope 9 enter polyhedral prism 10 Afterwards, multi beam mixing carrier frequency deflecting light beams are exported;Multi beam mixing carrier frequency deflecting light beams by 2-D vibration mirror 11, lens 12, lens 13, It is focused on after focusing objective len 14 on sample 15.
According to the reversible principle of optic path, multi beam mixing carrier frequency deflecting light beams pass through after focusing objective len 14 on sample 15 The fluorescence on the focal beam spot excitation sample 15 with different modulating frequency generated, forms fluorescence light corresponding with focal beam spot The fluorescence hot spot of spot, sending passes through focusing objective len 14, lens 13, lens 12,2-D vibration mirror 11, polyhedral prism 10, dichroic Mirror 9, lens 16 form a fluorescent foci hot spot on pin hole component 17, and are connect by the subsequent detector 18 of pin hole component 17 It receives.By the spot intensity on analysis detector 18 with the scan variations of 2-D vibration mirror 11, while frequency demodulation algorithm is utilized, The fluorescence signal of multiple each self-excitations of focus with different modulating frequency can be distinguished, to reconstruct reflection sample 15 The two dimensional image of information.
Frequency modulator 2 for applying different carrier frequency to light beam I and light beam II respectively.Frequency modulator 2 can be liquid Brilliant chopper, mechanical chopper or other schemes that can be modulated to incident beam real-time frequency.
Lens unit 3 can be simple lens, or lens group, effect are the focusing surface and light beam II for making light beam I Focusing surface in axial separation a certain distance, in two focal beam spots for being axially formed similar Fig. 2.The focusing light of illumination light z1 Spot (dashed region) and the focal beam spot of illumination light z2 are separated by a distance in axial direction (z-axis) direction, shadow region hot spot in figure Carrier frequency be f1+f2, the carrier frequency of other non-hatched areas is respectively f1 and f2.In this way carrier frequency be f1+f2 shadow region hot spot There is also carrier frequency f1+f2 for the fluorescence signal excited on sample, by frequency demodulation algorithm, then can extract carrier frequency f1+f2 Fluorescence signal.To achieve the purpose that improve axial resolution.
The effect of half wave plate 6 is to rotate the linear polarization of light beam II relative to the linear polarization direction of light beam I 90 °, to keep the linear polarization of the outgoing beam II of half wave plate 6 orthogonal with the linear polarization direction of light beam I.Therefore light Beam I and light beam II are equivalent to synthesize light beam after devating prism 7, orthogonal linear polarization that there are two light beam tools, Light beam I and light beam II are linearly polarized light, and in embodiment, the linear polarization of light beam I is orthogonal with the linear polarization of light beam II, and light Beam I and light beam II generate simultaneously, on the sample that the focal beam spot of the two focuses simultaneously.
Frequency modulator 8 is used to apply linear polarization carrier frequency mixed light beam different carrier frequency, is f in embodiment3And f4
Dichroscope 9 is placed between polyhedral prism 10 and frequency modulator 8, for reflexing to the fluorescence signal of return It is formed on detector 18 and the equal number of fluorescent foci hot spot of focal beam spot on sample 15 after lens 16;In embodiment, two Angle to Look mirror 9 and the 4th optical axis is 45 degree.
2-D vibration mirror 11 is mechanically fixed as quadrature arrangement mode by two one-dimensional galvanometers, and one-dimensional galvanometer can be Galvanometer galvanometer or resonance galvanometer, such as Cambridge Technology company Galvanometer Optical Scanner 6230H, with CRS 8kHz Resonant Scanner.
Lens 11, lens 12 form an optical beam transformation lens group, and effect has been optical beam transformation effect, make prism 8 Two beam oblique incidence directional lights of outgoing are full of the entrance pupil of focusing objective len 14 always, realize the optimal imaging performance of focusing objective len 14. In practical applications, the first lens unit that lens 11, lens 12 form might not be two first as shown in Fig. 3 schematic diagram Two lens compositions of lens unit, can form for more lens, to realize the effect of optical beam transformation.
Detector 18 is photodetector, including point type photodetector and array optical electric explorer, such as photomultiplier transit Pipe, avalanche diode, electron multiplication CCD etc..By combining frequency demodulation algorithm to detect while realizing multi-focus hot spot.
In embodiment, lens unit 3, lens unit 15 are simple lens, and frequency modulator 2 uses Liquid Crystal Chopper, and two Two one-dimensional galvanometers in dimension galvanometer 11 are all made of resonance galvanometer, and detector 18 uses photomultiplier tube.
By properly selecting the optical parameter of lens 3, lens 3 can make the focussing plane of light beam I and the focusing of light beam II Plane is separated by a distance, and forms focal beam spot shown in Fig. 2 in axial direction (z-axis direction).Since the light beam I with carrier frequency f1 is The direction y linear polarization, the light beam II with carrier frequency f2 is the direction x linear polarization, so light beam I is focused on sample 15 with light beam II Transverse focusing hot spot as shown in Fig. 1 right figure.The fluorescence letter that the shadow region hot spot that carrier frequency is f1+f2 in this way excites on sample Number there is also carrier frequency f1+f2, fluorescence signal is anti-by focusing objective len 14, lens 12, lens 11,2-D vibration mirror 11, dichroscope 9 It penetrates, then is focused on detector 18 by lens 15.By frequency demodulation algorithm, then the received load of detector 18 can be extracted The fluorescence signal of frequency f1+f2.Therefore, the direction the y linearly polarized light beam I with carrier frequency f1 and the direction the x linear polarization for having carrier frequency f2 (carrier frequency is the shadow region of f1+f2 to effective focal beam spot that three-dimensional (laterally and axially) of the light beam II on sample 15 is formed Domain hot spot) significantly less than do not use the invention patent when focal beam spot, so as to significantly improve dimensional resolution (laterally and It is axial).
Spot intensity by analyzing detector 18 is calculated with the scan variations of 2-D vibration mirror 11, while using frequency demodulation Method, so that it may distinguish partial enlarged view in the fluorescence signal such as Fig. 4 of multiple each self-excitations of focus with different modulating frequency D to reconstruct the 3-D image (assuming that scanning n row n arranges a point) of reflection sample message as shown in Figure 4, while being utilized more Face body prism generates array focal beam spot on the focusing surface of focusing objective len, realizes multiple spot scanning imagery simultaneously, significantly improves The frame speed of laser confocal scanning imaging, achievees the purpose that high speed imaging.
One kind being based on polyhedral prism and light beam frequency modulation high-resolution high-speed imaging device method, includes the following steps:
Step 1, beam splitter 1, the first reflecting mirror 5 are set gradually along primary optic axis line;
Step 2, by beam splitter 1, frequency modulator 2, lens unit 3, reflecting mirror 4 along vertical with primary optic axis line second Optical axis is set gradually;
Step 3, by reflecting mirror 5, frequency modulator 2, half wave plate 6 and polarization splitting prism 7 along with the first light The vertical third optical axis of axis is set gradually, and third optical axis is parallel with the second optical axis;
Step 4, by reflecting mirror 4, polarization splitting prism 7, frequency modulator 8, dichroscope 9, polyhedral prism 10 and 2-D vibration mirror 11 is set gradually along the 4th optical axis vertical with third optical axis;
Step 5,2-D vibration mirror 11, lens 12, lens 13, focusing objective len 14 and sample 15 are hung down along with the 4th optical axis The 5th straight optical axis is set gradually;
Step 6, dichroscope 9, lens unit 16, pin hole component 17 and detector 18 are along vertical with the 4th optical axis 6th optical axis is set gradually, wherein lens unit 16 faces the reflecting surface of dichroscope 9;
Step 7, a branch of to carry out incident first direction incident ray polarized light after beam splitter 1 along primary optic axis line direction Export the first light beam and the second light beam that two beams have first direction linearly polarized light;
Step 8, the first light beam exports the first carrier frequency after frequency modulator 2 applies carrier frequency f1 along the second optical axis direction Light beam, the first carrier frequency light beam is reflected through reflecting mirror 4 to be carried along the 4th optical axis direction by the transmission of polarization splitting prism 7 output first Frequency transmitted light beam,
Second light beam along primary optic axis line direction by reflecting mirror 5 reflection after, along third optical axis direction through overfrequency tune Device 2 processed exports the second carrier frequency light beam after applying carrier frequency f2, and the second carrier frequency light beam exports second direction after half wave plate 6 Linearly polarized light, the output after the reflection of polarization splitting prism 7 of second direction linearly polarized light are carried along the second of the 4th optical axis direction Frequency the reflected beams;
Step 9, the first carrier frequency transmitted light beam and the superposition synthesis output of the second carrier frequency the reflected beams have mixing first direction With the linear polarization carrier frequency mixed light beam of second direction;
Step 10, for linear polarization carrier frequency mixed light beam after frequency modulator 8, output multi beam has the mixed of different carrier frequency Close carrier frequency directional light;
Step 11, there is multi beam the mixing carrier frequency directional light of different carrier frequency to enter polyhedral prism 10 by dichroscope 9 Afterwards, multi beam mixing carrier frequency deflecting light beams are exported;
Step 12, multi beam mixing carrier frequency deflecting light beams are after 2-D vibration mirror 11, lens 12, lens 13, focusing objective len 14 Multiple focal beam spots are generated on sample 15;
Step 13, multiple focal beam spot excitation samples generate fluorescence, form multiple fluorescence hot spots corresponding with focal beam spot;
Step 14, fluorescence hot spot by focusing objective len 14, lens 13, lens 12,2-D vibration mirror 11, polyhedral prism 10, After dichroscope 9, lens unit 16, a fluorescent foci hot spot is formed on pin hole component 17, and received by detector 18;
Step 15, using frequency demodulation algorithm, extract the received overlapping of detector array have carrier frequency f1, f2 and The fluorescent foci spot signal of other carrier frequency, and by the intensity of the fluorescent foci hot spot on analysis detector array and with two The scan variations for tieing up galvanometer, can distinguish the fluorescence signal of each self-excitation of focus with different modulating frequency, to reconstruct Reflect the 3-D image of sample message out.
Embodiment two
As shown in figure 5, incident light g, refraction light is g1, g2, if the folder of two faceted pebble m1 and m2 and bottom surface of prism 8 Angle is respectively θ1And θ2, the refractive index of prism 9 is n, then the refraction light g1 of available faceted pebble m1 and primary optic axis line (z-axis) Angle is θ1'=asin (nsin θ11), as shown in fig. 6, distance h of the focal beam spot A to the 5th optical axis (x-axis)1=fsin [asin(nsinθ11)], wherein f is the focal length of focusing objective len 2.Similarly, distance h of the focal beam spot B to the 5th optical axis2= fsin[asin(nsinθ22)].It therefore, can be the angle of n, faceted pebble m1, m2 and z-axis by the refractive index of prism, and The focal length f of condenser lens 13 can accurately control the position of focal beam spot.If plane m1 and m2 be not right about primary optic axis line Claim, then focal beam spot A is different from the intensity of focal beam spot B.The intensity of focal beam spot A and focal beam spot B is decided by faceted pebble m1, m2 Area and the ratio between entire incident beam sectional area.
Embodiment three
The present embodiment is identical as other structures in embodiment one and setting, and only polyhedral prism changes into the present embodiment As shown in the left side in Fig. 7 polyhedral prism.The polyhedral prism is four sides cylinder prism, has bottom surface and three faceted pebbles, Three faceted pebbles are symmetrical arranged along primary optic axis line.
Focal beam spot is distributed as in the present embodiment:Three edges on x/y plane such as the right side in Fig. 7 are formed on sample 15 Y-axis arrangement focal beam spot.If faceted pebble is vertical with light beam g and is symmetrical arranged along primary optic axis line, obtained by the faceted pebble Focal beam spot on the origin of reference axis.
Further, it is assumed that cofocus scanning imaging will finally obtain the image of spoke n row n column, utilize the more of the present embodiment Face body prism generates the column distribution focus of 3 points, then need to only scan n/3 row, image taking speed can be imaged than existing single focus Speed improves 3 times.
Example IV
The present embodiment is identical as other structures in embodiment one and setting, and only polyhedral prism changes into the present embodiment As shown in the left side in Fig. 8 polyhedral prism.The polyhedral prism is five face cylinder prisms, has bottom surface and four faceted pebbles, Four faceted pebbles are symmetrical arranged along primary optic axis line.
Focal beam spot is distributed as in the present embodiment:Four edges on x/y plane such as the right side in Fig. 8 are formed on sample 15 Y-axis arrangement focal beam spot.
Further, it is assumed that cofocus scanning imaging will finally obtain the image of spoke n row n column, utilize the more of the present embodiment Face body prism generates the column distribution focus of four points, then need to only scan n/4 row, image taking speed can be imaged than existing single focus Speed improves 4 times.
Embodiment five
The present embodiment is identical as other structures in embodiment one and setting, and only polyhedral prism changes into the present embodiment As shown in the left side in Fig. 9 polyhedral prism.The polyhedral prism is six face cylinder prisms, has bottom surface and five faceted pebbles, Five faceted pebbles are symmetrical arranged along primary optic axis line.
Focal beam spot is distributed as in the present embodiment:Five edges on x/y plane such as the right side in Fig. 9 are formed on sample 15 Y-axis arrangement focal beam spot.
Further, it is assumed that cofocus scanning imaging will finally obtain the image of spoke n row n column, utilize the more of the present embodiment Face body prism generates the column distribution focus of five points, then need to only scan n/5 row, image taking speed can be imaged than existing single focus Speed improves 5 times.
Embodiment six
The present embodiment is identical as other structures in embodiment one and setting, and only polyhedral prism changes into the present embodiment As shown in the left side in Figure 10 polyhedral prism.The polyhedral prism is cone prism, has bottom surface and four faceted pebbles, four ribs Face then generates the focal beam spot of the two-dimensional array about z-axis rotation distribution along axis y rotary setting.
Focal beam spot is distributed as in the present embodiment:Four edges on x/y plane such as the right side in Figure 10 are formed on sample 15 The focal beam spot that uniformly arranges of x, y-axis.
If the face of polyhedral prism is changed along two dimensions, the focal beam spot of two-dimensional array is obtained.
The action and effect of embodiment
Polyhedral prism and light beam frequency modulation high-resolution high-speed imaging device are based on according to involved in the present embodiment, including Beam splitter, first frequency modulator, the first reflecting mirror, the second reflecting mirror, half wave plate, polarization splitting prism, the second frequency Rate modulator, dichroscope, polyhedral prism, 2-D vibration mirror, the first lens unit, focusing objective len, the second lens unit, pin hole Component, detector.
The present embodiment distinguishes the elliptical light formed after two beam orhtogonal linear polarizaiton light focus using the frequency modulation(PFM) of dual-beam The overlapping region of spot and other Non-overlapping Domains receive the frequency demodulation of signal using photodetector to extract two beam cross lines The corresponding useful signal in overlapping region for the ellipse light spot that polarised light is formed after focusing, to realize the mesh for improving dimensional resolution 's.And array focal beam spot is generated on the focusing surface of focusing objective len using polyhedral prism simultaneously, realize that multiple spot scans simultaneously Imaging significantly improves the frame speed of laser confocal scanning imaging, achievees the purpose that high speed imaging.
Alternatively, it is also possible to improve the pixel number of single-frame images in the case where frame speed is constant.Theoretically, compared to tradition Simple scan imaging, for a frame image of same pixel, the image taking speed of the multi-point scanning imaging of N number of focal beam spot can be with Improve N times;Identical for frame speed, the pixel number of a frame image can be improved N times.For example, single-point in current industry can be swashed The canonical parameter 512*32 pixel/frame of light confocal scanning imaging, frame fast 400 frames/second rise to 512*32 pixel/frame, frame speed 400N frame/second, or be 512*32N pixel/frame, frame fast 400 frames/second.
It further, further include being arranged on the second optical axis and being located at frequency modulator and the second reflecting mirror in embodiment For improving the third lens unit of axial resolution.It is that the focusing surface of light beam I and the focusing surface of light beam II is made to exist that it, which is acted on, Axial separation a certain distance is being axially formed two focal beam spots, is improving axial resolution.It is differentiated to realize that raising is three-dimensional The purpose of rate.
Further, when polyhedral prism is prism, if generating the column distribution focus of 2 points using prism, N/2 row then need to be only scanned, image taking speed can improve 1 times than existing image taking speed.
Further, when polyhedral prism is four prism, if generating the column distribution focus of 3 points using four prisms, N/3 row then need to be only scanned, image taking speed can improve 3 times than existing image taking speed.
It is further possible to improve the pixel number of single-frame images in the case where frame speed is constant.Theoretically, compared to Traditional simple scan imaging, for a frame image of same pixel, the image taking speed of the multi-point scanning imaging of N number of focal beam spot It can be improved N times;Identical for frame speed, the pixel number of a frame image can be improved N times.For example, can will be single in current industry The canonical parameter 512*32 pixel/frame of dot laser confocal scanning imaging, frame fast 400 frames/second, rise to 512*32 pixel/frame, Frame speed 400N frame/second, or be 512*32N pixel/frame, frame fast 400 frames/second.
Above embodiment is preferred case of the invention, the protection scope being not intended to limit the invention.

Claims (10)

1. one kind is based on polyhedral prism and light beam frequency modulation high-resolution high-speed imaging device, which is characterized in that including:Beam splitting Mirror, first frequency modulator, the first reflecting mirror, the second reflecting mirror, half wave plate, polarization splitting prism, second frequency tune Device processed, dichroscope, polyhedral prism, 2-D vibration mirror, the first lens unit, focusing objective len, the second lens unit, pin hole group Part, detector,
Wherein, the beam splitter, first reflecting mirror are set gradually along primary optic axis line,
The beam splitter, the first frequency modulator, second reflecting mirror are along vertical with the primary optic axis line second Optical axis is set gradually,
First reflecting mirror, the first frequency modulator, the half wave plate and the polarization splitting prism edge The third optical axis vertical with the primary optic axis line is set gradually, and the third optical axis is parallel with second optical axis,
Second reflecting mirror, the polarization splitting prism, the second frequency modulator, the dichroscope, the multi-panel Body prism and the 2-D vibration mirror are set gradually along the 4th optical axis vertical with the third optical axis,
The 2-D vibration mirror, first lens unit, the focusing objective len are along fiveth light vertical with the 4th optical axis Axis is set gradually,
The dichroscope, second lens unit, the pin hole component and detector edge and the 4th optical axis The 6th vertical optical axis of line is set gradually,
First lens unit, second lens unit include the combination of a lens or multiple lens.
2. according to claim 1 be based on polyhedral prism and light beam frequency modulation high-resolution high-speed imaging device, feature It is, further includes:
Third on second optical axis and between the first frequency modulator and second reflecting mirror is set Lens unit,
The third lens unit includes the combination of a lens or multiple lens.
3. one kind is based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, base described in claim 1 is utilized In polyhedral prism and light beam frequency modulation high-resolution high-speed imaging device, which is characterized in that include the following steps:
Step 1, the beam splitter, first reflecting mirror are set gradually along primary optic axis line;
Step 2, the beam splitter, the first frequency modulator, second reflecting mirror edge are hung down with the primary optic axis line The second straight optical axis is set gradually;
Step 3, by first reflecting mirror, the first frequency modulator, the half wave plate and the polarization point Light prism is set gradually along the third optical axis vertical with the primary optic axis line, the third optical axis and second optical axis Line is parallel;
Step 4, by the second reflecting mirror, polarization splitting prism, second frequency modulator, the dichroscope, described Polyhedral prism and the 2-D vibration mirror are set gradually along the 4th optical axis vertical with the third optical axis;
Step 5, by the 2-D vibration mirror, first lens unit, the focusing objective len and sample edge and the 4th light The 5th vertical optical axis of axis is set gradually;
Step 6, by the dichroscope, second lens unit, the pin hole component and the detector along with it is described The 6th vertical optical axis of 4th optical axis is set gradually, wherein second lens unit is anti-in face of the dichroscope Penetrate face;
Step 7, a branch of to carry out incident first direction incident ray polarized light by the beam splitting along primary optic axis line direction The first light beam and the second light beam that two beams have first direction linearly polarized light are exported after mirror;
Step 8, first light beam is along second optical axis direction after the first frequency modulator applies carrier frequency f1 The first carrier frequency light beam is exported, the first carrier frequency light beam is reflected through second reflecting mirror to be passed through along the 4th optical axis direction The polarization splitting prism transmission first carrier frequency transmitted light beam of output,
Second light beam along primary optic axis line direction by first reflecting mirror reflection after, along the third optical axis Direction exports the second carrier frequency light beam after the first frequency modulator applies carrier frequency f2, and the second carrier frequency light beam passes through institute Second direction linearly polarized light is exported after stating half wave plate, the second direction linearly polarized light passes through the polarization splitting prism The second carrier frequency the reflected beams along the 4th optical axis direction are exported after reflection;
Step 9, the first carrier frequency transmitted light beam and the second carrier frequency the reflected beams superposition synthesis output have mixing first The linear polarization carrier frequency mixed light beam in direction and second direction;
Step 10, for the linear polarization carrier frequency mixed light beam after the second frequency modulator, output multi beam has difference The mixing carrier frequency directional light of carrier frequency;
Step 11, there is multi beam the mixing carrier frequency directional light of different carrier frequency to enter the polyhedron by the dichroscope After prism, multi beam mixing carrier frequency deflecting light beams are exported;
Step 12, mixing carrier frequency deflecting light beams described in multi beam pass through the 2-D vibration mirror, first lens unit, the focusing Multiple focal beam spots are generated after object lens on to the sample;
Step 13, the multiple focal beam spot excites the sample to generate fluorescence, is formed multiple corresponding with the focal beam spot Fluorescence hot spot;
Step 14, the fluorescence hot spot is by the focusing objective len, first lens unit, the 2-D vibration mirror, described more After face body prism, the dichroscope, second lens unit, a fluorescent foci light is formed on the pin hole component Spot, and received by the detector;
Step 15, using frequency demodulation algorithm, extract the received overlapping of the detector has carrier frequency f1, f2 and other The fluorescent foci spot signal of carrier frequency, and the intensity by analyzing the fluorescent foci hot spot on the detector and with The scan variations of the 2-D vibration mirror, the fluorescence signal of each self-excitation of focus with different modulating frequency can be distinguished, To reconstruct the two dimensional image of reflection sample message.
4. according to claim 3 be based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, feature It is:
Wherein, the first direction linearly polarized light is orthogonal with the second direction linearly polarized light.
5. according to claim 3 be based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, feature It is:
Wherein, the polyhedral prism is the cylinder with bottom surface and multiple faceted pebbles, and cross section is polygon, and the mixing carries The direction of frequency directional light is with the plane perpendicular and the carrier frequency directional light that mixes is introduced into the bottom surface, and the focal beam spot is One-dimensional array distribution, the quantity of the focal beam spot are identical as the quantity of the faceted pebble.
6. according to claim 3 be based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, feature It is:
Wherein, first lens unit is used for optical beam transformation, carries mixing described in the multi beam being emitted from the polyhedral prism Frequency deflecting light beams are full of the entrance pupil of the focusing objective len always, realize the optimal imaging performance of the focusing objective len,
First lens unit includes the combination of two optical beam transformation lens or multiple optical beam transformation lens,
The angle of the dichroscope and the 4th optical axis is 45 degree.
7. according to claim 3 be based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, feature It is:
Wherein, when the polyhedral prism is the cone prism with bottom surface and multiple faceted pebbles, the bottom surface of the prism is mixed with described It is vertical to close the parallel light direction of carrier frequency, the focal beam spot is two-dimensional array distribution, the quantity of the focal beam spot and the faceted pebble Quantity it is identical.
8. according to claim 3 be based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, feature It is:
It wherein, further include being arranged on second optical axis and being located at the first frequency modulator and institute in the step 2 State the third lens unit for being used to improve axial resolution of the second reflecting mirror.
9. according to claim 8 be based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, feature It is:
Wherein, the third lens unit includes the combination of a lens or multiple lens, is used for the first carrier frequency light beam The focussing plane of focussing plane and the second carrier frequency light beam be separated by a distance.
10. according to claim 8 or claim 9 based on polyhedral prism and light beam frequency modulation high-resolution high speed imaging method, It is characterized in that:
Wherein, in step 15, using frequency demodulation algorithm, extract the received overlapping of the detector has carrier frequency f1, f2 And the fluorescent foci spot signal of other carrier frequency, and by analyzing the fluorescent foci hot spot on the detector Intensity and with the scan variations of the 2-D vibration mirror, can distinguish the glimmering of each self-excitation of focus with different modulating frequency Optical signal, to reconstruct the 3-D image of reflection sample message.
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