CN108956562A - A kind of light slice fluorescent microscopic imaging method and device based on reorientation - Google Patents
A kind of light slice fluorescent microscopic imaging method and device based on reorientation Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention discloses a kind of, and the light based on reorientation is sliced fluorescent microscopic imaging method and device, belong to optical image technology field, including the laser, cylindrical mirror and the sample stage for carrying fluorescent samples being sequentially arranged along optical path, and collect the detection system that fluorescent samples issue fluorescence；Detection system includes the first detector, beam splitter, the second detector and third detector, further includes the processor connecting with detection system and sample stage, and control sample stage is moved with fixed step-length along Z axis, and to fluorescent image I1With fluorescent image I2It is compared, obtains various pieces fluorescence in figure in the location information of Z axis, according to location information, to fluorescent image I0In fluorescence information carry out relocate and three-dimensionalreconstruction, obtain the three-dimensional imaging result of fluorescent samples.It may be implemented in and do not reduce imaging viewing field range, do not increase the photobleaching to sample, the axial resolution of imaging is improved in the case where not reducing image taking speed.
Description
Technical field
The present invention relates to optical image technology fields, specifically, it is aobvious to be related to a kind of light slice fluorescence based on reorientation
Micro- imaging method and device.
Background technique
With the development of science and technology, it is proposed in terms of people are for differentiating micro-structure and biological function micro-imaging
Increasingly higher demands.Other than high-resolution requirement, when detecting between and detection range on similarly need faster
It is wider.
Fluorescent microscopic imaging is right for a long time as a kind of Observations Means to the specific events occurred in microcosmos
It is of great significance in modern life science research.Especially need in Developmental Biology, regenerative medicine, organizational project etc. to more
In the field that cell sample observed, is analyzed, three dimensionality, comprehensive imaging are carried out for analyzing the structure and biology of sample
It is extremely important to learn characteristic.However, either inverted microscope or Laser Scanning Confocal Microscope, it can not be under low range, big visual field
There is provided axial resolution 3-D image good enough.The slice light micro imaging system that new development in recent years comes out provides one kind
The obtaining means of novel three-dimensional microscopic image, are different from the micro- means of traditional optical path, and slice light micro imaging system increases
Additional illumination path is sliced optical illumination to provide the very thin line focus of thickness, when stained specimens are placed in laser light-piece focus
When near place, it will be cut through just like by a virtual laser blade, while only illuminated every time by the laser light-piece transillumination
Thinly-sliced face can emit fluorescence, form image.Traditional light slice fluorescent microscopic imaging device is as shown in Figure 1.
Light, which is sliced fluorescence microscopy, has good 4D imaging capability, and compared to traditional 4D imaging technique (wide field
Microtechnic, Confocal laser endomicroscopy, multiphoton excitation microtechnic etc.), light, which is sliced fluorescence microscopy, has many advantages,
Such as: illumination is limited near focal plane by it, can be minimized photobleaching and light injury；The fluorescence that can be minimized in sample is full
With it is also ensured that high s/n ratio under higher frame per second.Light is sliced these advantages of fluorescence microscopy, gives birth to it in development
The fields such as object are widely used.But the axial resolution of traditional light slice fluorescence microscopy is about cross
To 2~3 times of resolution ratio, this greatly limits the isotropism of imaging.
The axial resolution for mentioning light slice fluorescence microscopy, is always the problem that researchers extremely pay close attention to.
Up to the present, in this respect, considerable exploration has been carried out.For example, one sufficiently thin excitation mating plate of production, makes
Axial resolution is no longer dependent on plus the numerical aperture (NA) of survey object lens, but this relatively thin mating plate, far from beam waist
Position can occur to spread faster, limit the field range of imaging；Excitation beam can also be modulated to bessel beam,
Avoid the reduction of visual field, but this can introduce irradiation outside focal plane again, increase the photobleaching to sample；It can also pass through
Sample is rotated, sample is repeatedly imaged in different detection angles, then multiple views are merged using computer,
But this method not only reduces image taking speed, but also reduces lateral resolution.
Therefore, all there is respective ask in the improvement method of several pairs of above-mentioned light slice fluorescence microscopy axial resolution
Topic.
Summary of the invention
It is an object of the present invention to provide a kind of, and the light based on reorientation is sliced fluorescent microscopic imaging method, can using this method
To effectively improve the axial resolution of traditional light slice fluorescence microscopy, and the field range of imaging will not be reduced, it will not
Increase the photobleaching to sample, image taking speed will not be reduced.
Another object of the present invention is to provide a kind of light slice fluorescent microscopic imaging device based on reorientation, which can
It realizes above-mentioned imaging method, is guaranteeing not reducing imaging viewing field range, do not increasing the photobleaching to sample, do not reduce image taking speed
In the case where improve the axial resolution of imaging.
To achieve the goals above, the light provided by the invention based on method for relocating is sliced fluorescent microscopic imaging method packet
It includes:
Laser beam focusing is to project to excite fluorescence on fluorescent samples after light is sliced, and collects fluorescence and obtains corresponding fluorescence
Image；
The first fluorescence that fluorescent samples issue downwards along Z axis is collected, fluorescent image I is obtained0；To fluorescent samples along Z
The second fluorescence issued in axial direction is split and is collected, and obtains fluorescent image I1With fluorescent image I2；
The fluorescent samples are moved along Z axis with fixed step-length, acquire fluorescence and obtain fluorescent samples in each Z axis position
The fluorescent image I at place0, fluorescent image I1With fluorescent image I2, to fluorescent image I1With fluorescent image I2It is compared, obtains in figure
Various pieces fluorescence Z axis location information, according to the location information, to the fluorescent image I at each Z axis position0In it is glimmering
Optical information is relocated, and determines position of the fluorescence information on Z axis；
To all fluorescent image I after reorientation0Three-dimensionalreconstruction is carried out, the three-dimensional imaging result of fluorescent samples is obtained.
In above-mentioned technical proposal, by obtaining image I of the fluorescent samples at Z axis position0、I1、I2, and pass through image I1
And I2It is compared, to figure I0It is relocated, it can be from figure I after processing0Two dimensional image in obtain the third dimension depth letter
Breath, to improve the axial resolution of imaging.It can effectively improve the axis that traditional light is sliced fluorescence microscopy by this method
To resolution ratio, and the field range of imaging will not be reduced, not will increase the photobleaching to sample, imaging speed will not be reduced
Degree.
Specific scheme is Z axis to perpendicular to the laser beam for being incident on fluorescent samples.
Another specific scheme is figure I0For the image on focal plane, I is schemed1For with a negative defocus-Δ z
Image, scheme I2For with positive defocus+Δ z image.
More specific scheme is to fluorescent image I1With fluorescent image I2It is compared, obtains in figure various pieces fluorescence in Z
The location information of axis includes:
Using following formula to figure I1With figure I2It is compared:
Wherein, I1(x, y) indicates figure I1Coordinate in the two-dimensional surface perpendicular to Z axis, I2(x, y) indicates figure I2It is hanging down
Directly in the coordinate in the two-dimensional surface of Z axis；
Assuming that the center of light slice is z0If f (x, y) < 0, I is indicated1(x, y) < I2(x, y), i.e., at this point,
Fluorescence intensity with negative defocusing amount image is lower than the fluorescence intensity with positive out of focus spirogram picture, illustrates the fluorescence being collected at this
With a upward defocusing amount, then at this fluorescence the position of Z axis be z0+Δz；If f (x, y)=0, illustrate have it is positive and negative from
Coke amount fluorescence intensity it is identical, then at this fluorescence the position of Z axis be z0；If f (x, y) > 0, indicate that there is negative defocusing amount image
Fluorescence intensity be lower than with positive out of focus spirogram picture fluorescence intensity, illustrate the fluorescence have a downward defocusing amount, then should
Locating fluorescence in the position of Z axis is z0-Δz。
In this way, two dimensional image being obtained in Z axis position and being divided into three width pictures, axial position is respectively z0+Δ
z、z0And z0-Δz。
In order to achieve the above-mentioned another object, the light provided by the invention based on reorientation is sliced fluorescent microscopic imaging device packet
Laser, cylindrical mirror and the sample stage for carrying fluorescent samples being sequentially arranged along optical path are included, and collects fluorescent samples and issues
The detection system of fluorescence；Detection system includes:
First detector collects the first fluorescence that fluorescent samples issue downwards along Z axis, obtains fluorescent image I0；
Beam splitter is split fluorescent samples along the second fluorescence that Z axis issues upwards；
Second detector and third detector obtain the fluorescence being emitted by beam splitter respectively, obtain fluorescent image I1With it is glimmering
Light image I2；
It further include the processor being connect with detection system and sample stage, control sample stage is moved with fixed step-length along Z axis,
And to fluorescent image I1With fluorescent image I2It is compared, obtains various pieces fluorescence in figure in the location information of Z axis, according to position
Confidence breath, to fluorescent image I0In fluorescence information carry out relocate and three-dimensionalreconstruction, obtain the three-dimensional imaging of fluorescent samples
As a result.
Above-mentioned apparatus improves the light path of traditional light slice fluorescence microscope, can obtain three width figures simultaneously
As I0、I1、I2；Pass through image I1And I2It is compared, to figure I0It is relocated, then will scheme I0In image handled,
It can be from figure I0Two dimensional image in obtain the depth information of the third dimension, to improve the axial resolution of imaging, and guarantee not
Reduce imaging viewing field range, does not increase the photobleaching to sample, do not reduce image taking speed.
Specific scheme be the first detector include the first objective lens set gradually downwards along Z axis, the first optical filter,
First pipe lens and the first CCD camera, the fluorescence downward along Z axis are collected by the first objective lens, are filtered out by the first optical filter
After stray light, by obtaining image I on the first pipe lens focus to the first CCD camera0。
More specific scheme be the second detector include along the second fluorescence a wherein beam splitting optical path be sequentially arranged second
Pipe lens and the second CCD camera focus in the second CCD camera after the second pipe lens through the light beam of semi-transparent semi-reflecting lens,
Obtain image I1；Third detector includes the third pipe lens being sequentially arranged along another beam splitting optical path of the second fluorescence and third
CCD camera is focused in third CCD camera after third pipe lens by the light beam that semi-transparent semi-reflecting lens reflect, obtains image I2。
Further more specifically scheme is that the imaging surface position of the first CCD camera and the center of light slice are conjugated；2nd CCD
The position of the imaging surface of camera has forward defocusing amount-φ compared with the conjugate planes in light centre of slice face, therefore collects and obtain
Fluorescent image also have a forward defocus；The position of the imaging surface of third CCD camera and the conjugate planes in light centre of slice face
It compares, there is defocusing amount+φ backward, therefore collect obtained fluorescent image also to have a defocus backward.
Obtained figure I0It is identical as the image that traditional light slice fluorescence microscope detects；Obtained figure I1It is width tool
There is certain negative defocus (image of-Δ z)；Obtained figure I2It is defocusing amount and figure I1Identical but contrary (+Δ z).By from
Coke measures identical, the contrary image I of defocus1And I2, can determine and point each in sample is positioned.
The principle of the program are as follows: fluorescent samples are sliced by light and excite, and emit fluorescence around.Along the fluorescence that Z axis is downward,
It is identical as traditional light slice fluorescence microscopy, it is collected by the first objective lens, the first optical filter filters, the first pipe lens
It focuses in the first CCD camera, obtains sample in Z axis and be distributed I to the fluorescence intensity of position0.Along the upward fluorescence of Z axis, quilt
Beam splitter is divided into two beams, is collected respectively, obtains fluorescent image I1、I2, this two width fluorescent image, compared to the center of light slice
There is certain defocus in position, and the distance of defocus is identical, contrary.By comparing the two fluorescent images, figure is obtained
As the position in axial direction of upper each point, to I0The fluorescence of middle various pieces relocates, and the image of a width Z axis is divided into three
Width, to improve the Z-direction resolution ratio of last reconstruction result.
Another more specific scheme is that the second detector includes being arranged in a wherein beam splitting optical path for the second fluorescence
Galvanometer, and along the pin hole I and photoelectric tube I being sequentially arranged by the optical path of the light beam after vibration mirror reflected, processor controls galvanometer rotation
Turn, photoelectric tube I is made to be collected into the image I of I conjugate planes of pin hole1；Third detector includes another point for being arranged in the second fluorescence
Pin hole II and photoelectric tube II on beam optical path, processor control semi-transparent semi-reflecting lens rotation, so that photoelectric tube II is collected into pin hole II total
The image I of yoke plane2。
Further more specifically scheme is that the position that pin hole I is conjugated is more on the lower side than the center that light is sliced, and pin hole II is total to
The position of yoke is more on the upper side than light centre of slice position.
Preferably, beam splitter is semi-transparent semi-reflecting lens.
Compared with prior art, the invention has the benefit that
The be excited fluorescence of sending of fluorescent samples is towards all directions, and traditional light slice fluorescence microscopy is only
The fluorescence in some direction is had collected, the fluorescence of the both direction up and down of fluorescent samples sending is had collected in the present invention, was both improved
Collection efficiency, and can successfully realize in the case where not reducing visual field, not reducing image taking speed, improve the axial direction to sample
Resolution ratio.
Detailed description of the invention
Fig. 1 is the structural schematic diagram that traditional light is sliced fluorescent microscopic imaging device；
Fig. 2 is that the light based on reorientation of the embodiment of the present invention 1 is sliced the structural schematic diagram of fluorescent microscopic imaging device；
Fig. 3 be in the embodiment of the present invention 1 fluorescent samples in the imaging schematic diagram of YZ plane: wherein (a) is imaging center position
Areas imaging when focal plane；It (b) is the areas imaging with negative defocus, and the areas imaging with positive out of focus；(c) attach most importance to
After positioning, the equivalent areas imaging of three images；
Fig. 4 is that the light based on reorientation of the embodiment of the present invention 2 is sliced the structural schematic diagram of fluorescent microscopic imaging device；
Fig. 5 is the position view that the aperture of the embodiment of the present invention 2 is conjugated.
Each label in figure are as follows:
1, laser；2, single mode optical fiber；3, collimation lens；4, convex lens；5, convex lens；6, cylindrical mirror；7, the first relaying
Lens；8, reflecting mirror；9, the second relay lens；10, third relay lens；11, illumination objective lens；12, fluorescent samples；13, first
Objective lens；14, the first optical filter；15, the first pipe lens；16, the first CCD camera；17, the second objective lens；18, the second filter
Mating plate；19, semi-transparent semi-reflecting lens；20, the second pipe lens；21, the second CCD camera；22, third pipe lens；23, third CCD camera；
24, computer；25, semi-transparent semi-reflecting lens；26, galvanometer；27, pin hole I；28, photoelectric tube I；29, pin hole II；30, photoelectric tube II.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, with reference to embodiments and its attached drawing is to this hair
It is bright to be described further.
Embodiment 1
Referring to fig. 2, the light slice fluorescent microscopic imaging device based on reorientation of the present embodiment includes laser 1, single mode
Optical fiber 2, collimation lens 3, the telescope being made of convex lens 4 and convex lens 5, cylindrical mirror 6, the first relay lens 7, reflecting mirror 8,
Second relay lens 9, third relay lens 10, illumination objective lens 11, fluorescent samples 12, the first objective lens 13, the first optical filter
14, the first pipe lens 15, the first CCD camera 16, the second objective lens 17, the second optical filter 18, semi-transparent semi-reflecting lens 19, second are managed
Lens 20, the second CCD camera 21, third pipe lens 22, third CCD camera 23, computer 24.
Using imaging device shown in Fig. 2, the specific step of the light slice fluorescent microscopic imaging method based on reorientation is realized
It is rapid as follows:
(1) laser that laser 1 issues filters by single mode optical fiber 2, carries out expanding standard to laser using collimation lens 2
Directly, then pass through adjustment of the telescope to laser beam size, then the slice of light required for being collected as by cylindrical mirror 6 passes through
Relay lens group (the first relay lens 7, the second relay lens 9, third relay lens 10) is expanded, is focused, exciting light slice
Final illuminated object lens 11 focus on fluorescent samples 12.
(2) fluorescent samples 12 are sliced excitation by light and issue fluorescence, and downward fluorescence is collected by the first objective lens 13, pass through
After first optical filter 14 filters out stray light, is focused in the first CCD camera 16 by the first pipe lens 15, obtain image I0。
(3) upward fluorescence is collected by the second objective lens 17, then filters out stray light by the second optical filter 18, then
Divided by semi-transparent semi-reflecting lens 19 for transmitted light beam and the reflected beams.
(4) it is focused through the transmitted light beam of semi-transparent semi-reflecting lens 19 by the second pipe lens 20, by with negative defocus forward
Second CCD camera 21 is collected, and obtains the fluorescence intensity profile with a negative defocus as I1。
(5) the reflected beams reflected by semi-transparent semi-reflecting lens 19 pass through third pipe lens 22 focusing, by have backward just from
Burnt third CCD camera 23 is collected, and obtains the fluorescence intensity profile with a positive out of focus as I2。
(6) computer 24 is used, to figure I1With figure I2It is compared, it is as follows to compare formula:
Wherein, (x, y) indicates the coordinate perpendicular to the certain point on detection lens axis direction in two-dimensional surface, I1(x,
Y) figure I is indicated1Coordinate in the two-dimensional surface perpendicular to Z axis, I2(x, y) indicates figure I2In the two-dimensional surface perpendicular to Z axis
Coordinate；
If f (x, the y) < 0 acquired, I is indicated1(x, y) < I2(x, y), that is, at this point, there is negative defocus spirogram
The fluorescence intensity of picture is lower than the fluorescence intensity with positive out of focus spirogram picture, illustrate this go out fluorescence for being collected into have one it is upward
The position of defocusing amount, three parts as shown in Fig. 3 (b), the fluorescence is in undermost part；If f (x, y)=0, explanation
Fluorescence intensity with positive and negative defocusing amount is identical, three parts as shown in Fig. 3 (b), and the position of the fluorescence is in middle section；
If f (x, y) > 0, indicate that the fluorescence intensity with negative defocusing amount image is lower than the fluorescence intensity with positive out of focus spirogram picture, such as
The position of three parts shown in Fig. 3 (b), the fluorescence is in top layer.
(7) according in step (6), calculating to various pieces fluorescent places, to I0The fluorescence signal weight of middle various pieces
New definition, as shown in figure 3, Fig. 3 (a) is the imaging results of script, imaging results that Fig. 3 (c) is that treated.
(8) along Z axis mobile example platform, the different axial positions of fluorescent samples is imaged, calculating is then passed through
Fluorescent samples 12 are reconstructed in machine 24, obtain the 3-D image of fluorescent samples 12.
The principle of the present embodiment is as follows:
Fluorescent samples are sliced by light and excite, and emit fluorescence around.Along the fluorescence that Z axis is downward, it is sliced with traditional light
Fluorescence microscopy is identical, collects by the first objective lens, the first optical filter filters, the first pipe lens focus to the first CCD
On camera, sample is obtained in Z axis and is distributed I to the fluorescence intensity of position0.Along the upward fluorescence of Z axis, it is divided by semi-transparent semi-reflecting lens
Two beams, are collected respectively, obtain fluorescent image I1、I2, this two width fluorescent image, the center being sliced compared to light has one
Fixed defocus, and the distance of defocus is identical, contrary.By comparing the two fluorescent images, each point on image is obtained
In axial position, to I0The fluorescence of middle various pieces relocates, and the image of a width Z axis is divided into three width, to improve most
The Z-direction resolution ratio of reconstruction result afterwards.
Embodiment 2
Referring to fig. 4, the light slice fluorescent microscopic imaging device based on reorientation of the present embodiment includes laser 1, single mode
Optical fiber 2, collimation lens 3, the telescope being made of convex lens 4 and convex lens 5, cylindrical mirror 6, the first relay lens 7, reflecting mirror 8,
Second relay lens 9, third relay lens 10, illumination objective lens 11, fluorescent samples 12, the first objective lens 13, the first optical filter
14, the first pipe lens 15, the first CCD camera 16, the second objective lens 17, semi-transparent semi-reflecting lens 25, galvanometer 26, pin hole I 27, photoelectricity
Pipe I 28, pin hole II 29, photoelectric tube II 30, computer 24.
Using imaging device shown in Fig. 4, the specific mistake of the light slice fluorescent microscopic imaging method based on reorientation is realized
Journey is as follows:
(1) laser that laser 1 issues filters by single mode optical fiber 2, carries out expanding standard to laser using collimation lens 2
Directly, then pass through adjustment of the telescope to laser beam size, then the slice of light required for being collected as by cylindrical mirror 6 passes through
Relay lens group (the first relay lens 7, the second relay lens 9, third relay lens 10) is expanded, is focused, exciting light slice
Final illuminated object lens 11 focus on fluorescent samples 12.
(2) fluorescent samples 12 are sliced excitation by light and issue fluorescence, and downward fluorescence is collected by the first objective lens 13, pass through
After first optical filter 14 filters out stray light, is focused in the first CCD camera 16 by the first pipe lens 15, obtain image I0。
(3) upward fluorescence is collected by the second objective lens 17, is then divided by semi-transparent semi-reflecting lens 25 for transmitted light beam and anti-
Irradiating light beam.
(4) transmitted light beam for penetrating semi-transparent semi-reflecting lens 25, is reflected by galvanometer 26, then filters through needle passing hole I 27, such as Fig. 5
It is shown, it is collected by the photoelectric tube I 28 that the photon of pin hole I 27 is had negative defocus forward, computer 24 controls galvanometer 26 and rotates,
To which each point of the axial direction be imaged, two-dimensional fluoroscopic distributed image I is obtained1。
(5) the reflected beams reflected by semi-transparent semi-reflecting lens 25 filter through needle passing hole II 29, as shown in figure 5, passing through pin hole II
The photoelectric tube II 30 that 29 photon is had positive out of focus backward is collected, and computer 24 controls semi-transparent semi-reflecting lens 25 and rotates, thus right
Each point of the axial direction is imaged, and two-dimensional fluoroscopic distributed image I is obtained2。
(6) computer 24 is used, to figure I1With figure I2It is compared, as follows compared with formula:
Wherein, (x, y) indicates the coordinate perpendicular to the certain point on detection lens axis direction in two-dimensional surface, I1(x,
Y) figure I is indicated1Coordinate in the two-dimensional surface perpendicular to Z axis, I2(x, y) indicates figure I2In the two-dimensional surface perpendicular to Z axis
Coordinate；
If f (x, the y) < 0 acquired, I is indicated1(x, y) < I2(x, y), that is, at this point, there is negative defocus spirogram
The fluorescence intensity of picture is lower than the fluorescence intensity with positive out of focus spirogram picture, illustrate this go out fluorescence for being collected into have one it is upward
The position of defocusing amount, three parts as shown in Fig. 3 (b), the fluorescence is in undermost part；If f (x, y)=0, explanation
Fluorescence intensity with positive and negative defocusing amount is identical, three parts as shown in Fig. 3 (b), and the position of the fluorescence is in middle section；
If f (x, y) > 0, indicate that the fluorescence intensity with negative defocusing amount image is lower than the fluorescence intensity with positive out of focus spirogram picture, such as
The position of three parts shown in Fig. 3 (b), the fluorescence is in top layer.
(7) according to the calculating in step (6) to various pieces fluorescent places, to image I0The fluorescence signal of middle various pieces
It relocates, as shown in figure 3, Fig. 3 (a) is the imaging results of script, imaging results that Fig. 3 (c) is that treated.
(8) along Z axis mobile example, the different axial positions of sample are imaged, it is then right by computer 24
Sample 12 is reconstructed, and obtains the 3-D image of sample 12.
Embodiment 3
Light slice fluorescent microscopic imaging method provided in this embodiment based on reorientation is already contained in embodiment 1 or real
It applies in example 2, details are not described herein again.
Claims (10)
1. a kind of light based on reorientation is sliced fluorescent microscopic imaging method, laser beam focusing is to project fluorescence after light is sliced
Fluorescence is excited on sample, is collected fluorescence and is obtained corresponding fluorescent image；It is characterized by:
The first fluorescence that fluorescent samples issue downwards along Z axis is collected, fluorescent image I is obtained0；It is upward along Z axis to fluorescent samples
The second fluorescence issued is split and is collected, and obtains fluorescent image I1With fluorescent image I2；
The fluorescent samples are moved along Z axis with fixed step-length, acquire fluorescence and to obtain fluorescent samples glimmering at each Z axis position
Light image I0, fluorescent image I1With fluorescent image I2, by fluorescent image I1With fluorescent image I2It is compared, obtains each portion in figure
Divide fluorescence in the location information of Z axis, according to the location information, to the fluorescent image I at each Z axis position0Middle fluorescence information
It is relocated, determines position of the fluorescence information on Z axis；
To all fluorescent image I after reorientation0Three-dimensionalreconstruction is carried out, the three-dimensional imaging result of fluorescent samples is obtained.
2. the light according to claim 1 based on reorientation is sliced fluorescent microscopic imaging method, it is characterised in that:
The Z axis is to perpendicular to the laser beam for being incident on fluorescent samples.
3. light according to claim 1 is sliced fluorescent microscopic imaging method, it is characterised in that:
Scheme I0For the image on focal plane, I is schemed1To scheme I with negative defocus-Δ z image2For with one just
Defocus+Δ z image.
4. light according to claim 3 is sliced fluorescent microscopic imaging method, which is characterized in that described to fluorescent image I1
With fluorescent image I2It is compared, obtaining in figure various pieces fluorescence in the location information of Z axis includes:
Using following formula to figure I1With figure I2It is compared:
Wherein, I1(x, y) indicates figure I1Coordinate in the two-dimensional surface perpendicular to Z axis, I2(x, y) indicates figure I2Perpendicular to Z
Coordinate in the two-dimensional surface of axis；
Assuming that the center of light slice is z0If f (x, y) < 0, fluorescence is z in the position of Z axis at this0+Δz；If f (x,
Y)=0, then at this fluorescence the position of Z axis be z0；If f (x, y) > 0, fluorescence is z in the position of Z axis at this0-Δz。
5. a kind of light based on reorientation is sliced fluorescent microscopic imaging device, including laser, the cylinder being sequentially arranged along optical path
Mirror and the sample stage for carrying fluorescent samples, and collect the detection system that fluorescent samples issue fluorescence；It is characterized by:
The detection system includes:
First detector collects the first fluorescence that fluorescent samples issue downwards along Z axis, obtains fluorescent image I0；
Beam splitter is split fluorescent samples along the second fluorescence that Z axis issues upwards；
Second detector and third detector, obtain the fluorescence being emitted by the beam splitter respectively, obtain fluorescent image I1And fluorescence
Image I2；
It further include the processor being connect with the detection system and sample stage, control sample stage is moved with fixed step-length along Z axis,
And to fluorescent image I1With fluorescent image I2It is compared, obtains various pieces fluorescence in figure in the location information of Z axis, according to institute
The location information stated, to fluorescent image I0In fluorescence information carry out relocate and three-dimensionalreconstruction, obtain the three of fluorescent samples
Tie up imaging results.
6. light according to claim 5 is sliced fluorescent microscopic imaging device, it is characterised in that:
First detector includes the first objective lens set gradually downwards along Z axis, the first optical filter, the first pipe lens
With the first CCD camera, the fluorescence downward along Z axis is collected by the first objective lens, after the first optical filter filters out stray light, warp
It crosses on the first pipe lens focus to the first CCD camera, obtains image I0。
7. light according to claim 5 or 6 is sliced fluorescent microscopic imaging device, it is characterised in that:
Second detector includes the second pipe lens and second being sequentially arranged along a wherein beam splitting optical path for the second fluorescence
CCD camera, light beam focus in the second CCD camera after the second pipe lens, obtain image I1；
The third detector includes the third pipe lens and the 3rd CCD being sequentially arranged along another beam splitting optical path of the second fluorescence
Camera, light beam focus in third CCD camera after third pipe lens, obtain image I2。
8. light according to claim 7 is sliced fluorescent microscopic imaging device, it is characterised in that:
The imaging surface position of first CCD camera and the center of light slice are conjugated；The imaging surface of second CCD camera
Position compared with the conjugate planes in light centre of slice face, have forward defocusing amount-φ；The imaging of the third CCD camera
The position in face has defocusing amount+φ backward compared with the conjugate planes in light centre of slice face.
9. light according to claim 5 or 6 is sliced fluorescent microscopic imaging device, it is characterised in that:
Second detector includes the galvanometer being arranged in a wherein beam splitting optical path for the second fluorescence, and along by the vibration
The pin hole I and photoelectric tube I that the optical path of light beam after mirror reflection is sequentially arranged, the processor control the galvanometer rotation, make light
Fulgurite I is collected into the image I of I conjugate planes of pin hole1；
The third detector includes the pin hole II and photoelectric tube II being arranged in another beam splitting optical path of the second fluorescence, described
Processor controls the semi-transparent semi-reflecting lens rotation, and photoelectric tube II is made to be collected into the image I of II conjugate planes of pin hole2。
10. light according to claim 9 is sliced fluorescent microscopic imaging device, it is characterised in that:
The position that the pin hole I is conjugated is more on the lower side than the center that light is sliced, and light is compared in the position of the conjugation of the pin hole II
Centre of slice position is on the upper side.
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