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 PDFInfo
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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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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0036—Scanning details, e.g. scanning stages
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0036—Scanning details, e.g. scanning stages
- G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
- G02B26/08—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
- G02B26/10—Scanning systems
- G02B26/12—Scanning systems using multifaceted mirrors
- G02B26/123—Multibeam scanners, e.g. using multiple light sources or beam splitters
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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
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 θ1-θ1), as shown in fig. 6, distance h of the focal beam spot A to the 5th optical axis (x-axis)1=fsin
[asin(nsinθ1-θ1)], 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(nsinθ2-θ2)].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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