CN108982455A - A kind of multifocal light slice fluorescent microscopic imaging method and device - Google Patents
A kind of multifocal light slice fluorescent microscopic imaging method and device Download PDFInfo
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- CN108982455A CN108982455A CN201810857170.XA CN201810857170A CN108982455A CN 108982455 A CN108982455 A CN 108982455A CN 201810857170 A CN201810857170 A CN 201810857170A CN 108982455 A CN108982455 A CN 108982455A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
Abstract
The invention discloses a kind of multifocal light slice fluorescent microscopic imaging method and systems, belong to optical image technology field, detection system and processor including the fluorescence that the lighting system for forming illumination light slice, the sample stage for carrying fluorescent samples, detection sample issue.The laser beam that laser issues becomes the light beam with multiple focuses after phase-modulation.It is rotated by galvanometer, scans light beam along X-direction；By the change of the varifocal Concave Mirrors Focus of electric drive, it is moved forward and backward light beam along Y direction.Movement of the multifocal light beam Jing Guo X and Y both direction is capable of forming a kind of virtual light slice.Compared to the Gauss light slice in traditional light slice fluorescence microscope, this light slice is very weak in the diffusion along lighting optical axis direction, and in focusing range, bias light is weaker, fluorescent samples can be imaged with lower ambient noise in bigger field range.
Description
Technical field
The present invention relates to optical image technology fields, specifically, being related to a kind of multifocal light slice fluorescent microscopic imaging
Method and apparatus.
Background technique
In latter stage in 16th century, first optical microscopy is born in the world, it is greatly expanded people to microcosmos
Understanding, in several centuries later, optical microscopy is continuously improved, and image taking speed, resolution ratio, sensitivity etc. are all
Significant progress has been obtained, has played increasingly important role in bio-imaging field.And as the high speed of biotechnology is sent out
Exhibition needs to have more deep understanding to physiology course, and also to 3D Real Time Imaging Technology, more stringent requirements are proposed for this.But
It is that the development of 3D Real Time Imaging Technology is also faced with many challenges --- successful 3D Real Time Imaging Technology needs to reach simultaneously
High spatial resolution, high image taking speed, good smooth sectioning capability, low light loss wound and photobleaching ability.
There are the technology of many 3D real time imageries, such as wide field microtechnic, Confocal laser endomicroscopy, double light at present
Sub- fluorescence microscopy and light are sliced fluorescence microscopy.Wherein, light is sliced fluorescence microscopy, due to its high speed, low light
Bleachability and imaging the advantages such as non-invasive have obtained extremely rapidly development in this is several years.
Since light is sliced the non-invasive of fluorescence microscopy, it has highly important status in bio-imaging field.
But due to the diffusion property of illumination light slice, the field range that light slice fluorescence microscopy can be imaged is extremely limited, this
Limit its application in large sample imaging.In order to solve this problem, it is thus proposed that be sliced field stitching technology and light
Fluorescence microscopy be combined with each other, although can successfully extend the areas imaging of light slice fluorescence microscope in this way, this needs
Increase electricity driving displacement platform in systems, which increase the cost of realization and difficulty, and need that sample is repeatedly imaged
After splice, this greatly reduces image taking speeds, can not observe the physiology course of some fast speeds.
In contrast, shaping is modulated to light beam, the shape for changing light slice can guarantee to slow down its diffusion
In the case where image taking speed, extended field of view.The application of bessel beam and Ai Li light beam in light slice fluorescence microscopy, just
It is the successful implementation to this scheme, but the light slice that both light beams are formed, more serious secondary lobe can be generated, is caused tight
The ambient noise of weight, needs using related algorithm, carries out recovery processing to obtained image.
Summary of the invention
It is an object of the present invention to provide a kind of multifocal light to be sliced fluorescent microscopic imaging method, can be not using this method
In the case where increasing ambient noise, expand the field range of light slice fluorescence microscope.
Another object of the present invention is to provide a kind of light slice fluorescent microscopic imaging device for realizing the above method, the device
It can be used for realizing the above method, phase-modulation carried out to incident light beam by phase mask plate, generating one has multiple cokes
The light beam of point, the spacing between these focuses are of substantially equal.Then, by vibration mirror scanning, discontinuous light slice is formed.With
Afterwards, by the adjusting varifocal concavees lens of electric drive, move back and forth the focus point of light beam before and after illumination objective lens optical axis direction,
Movement in this way, the light slice for forming vibration mirror scanning interconnect, are formed along lighting optical axis direction, spread slower
Virtual light slice.The light to be formed slice is set not only to have biggish field range, but also weaker in the location context light of focusing, it is right
When sample is imaged, ambient noise can be preferably eliminated.
To achieve the goals above, multifocal light slice fluorescent microscopic imaging method provided by the invention includes following step
It is rapid:
1) laser is formed a series of along lighting optical axis direction and with the multifocal of identical spacing after phase-modulation
Light beam；
2) along X-direction scanning light beam, and it is moved forward and backward light beam along Y direction, obtain one has along the Y direction
The light slice of certain areas imaging；
3) along Z-direction, the fluorescence that fluorescent samples issue is collected, obtains sample in the two-dimensional light intensity of the axial position
Signal pattern.
4) it along Z axis mobile example, repeats step 3) and obtains several two-dimentional light intensity signal images, to several two-dimentional light intensity letters
Number image carries out three-dimensionalreconstruction, obtains the three-dimensional imaging information of fluorescent samples.
In above-mentioned technical proposal, X-direction be perpendicular to lighting optical axis and detect optical axis direction, Y direction be along
The direction of lighting optical axis, Z-direction are along the direction of detection optical axis, these three directions are vertical two-by-two, constitute three-dimensional right angle
Coordinate system.In step 2), light beam is scanned along X-direction, and light beam is made to form discontinuous light slice.Pass through step 1) and step
2) a virtual light slice can be generated, compared to the light slice that traditional Gauss light beam is formed, this light slice is along illumination light
Axis direction spreads slower, and in the range of focusing, bias light is also weaker, therefore, can in bigger field range,
Obtain the lower image of ambient noise.
Specific scheme is in order to obtain with the light beam of multiple focus points, to need first to be converted to laser in step 1)
Then radial polarisation light carries out phase-modulation to the radial polarisation light again.
Another specific scheme is phase modulation function used in step 1) are as follows:
Wherein, (r, θ) indicates that the polar coordinates of certain point on light beam, r are the normalized cumulant of the point and optical axis, and θ hangs down for light beam
Directly in the angle of the polar coordinate vectors of optical axis section and laser emitting optical axis.
Another specific scheme is, in step 2), is moved forward and backward light beam, makes focus move to form light along Y-axis, light
The range of Shu Yidong is the distance between any two focus of multifocal light beam.
In order to achieve the above-mentioned another object, multifocal light provided by the invention is sliced fluorescent microscopic imaging device, including shape
It is issued at the detection system and one of fluorescence at the lighting system of light slice, the sample stage of carrying fluorescent samples, detection fluorescent samples
Device is managed, lighting system includes being sequentially arranged along optical path: laser；Laser beam is changed into the radial polarisation of radial polarisation light
Converter；Phase-modulation is carried out to radial polarisation light and it is made to be converted into the phase mask plate of multifocal light beam；Make to be irradiated to glimmering
The uniaxial galvanometer that light beam on light sample is scanned along X-direction；And the electric drive for making focal length that consecutive variations occur is varifocal
Concavees lens；Consecutive variations and control sample stage occur for the focal length that processor is used to control the varifocal concavees lens of electric drive with fixed step
Length is moved along Z-direction, and several two-dimentional light intensity signal images that detection system is collected are reconstructed, and obtains fluorescent samples
Three-dimensional imaging information.
In above-mentioned technical proposal, laser beam will form multiple focus points after phase-modulation, these focus points along
X-axis scanning, and be moved forward and backward along Y-axis, to form a kind of virtual big visual field light slice.Fluorescent samples are sliced by this light to swash
Hair generates fluorescence, is detected examining system and collects, obtains the two dimensional image that a width contains fluorescent samples information on X/Y plane.Then,
Sample stage is controlled by processor, is moved along Z axis, and two-dimensional images are obtained, and reconstruction obtains the three-dimensional imaging knot of fluorescent samples
Fruit.It is sliced fluorescence microscope compared to traditional light, the light slice that the device that this programme provides generates is not allowed more in Y direction
Easily occur diffusion and it is weaker in the bias light of focusing range, bigger visual field and reasons for its use noise can be generated more
It is weak.
Specific scheme is, in order to obtain the light beam with multiple focuses, on phase mask plate used in the present invention
Modulation function are as follows:
Wherein, (r, θ) indicates that the polar coordinates of certain point on light beam, r are the normalized cumulant of the point and optical axis, and θ hangs down for light beam
Directly in the angle of the polar coordinate vectors of optical axis section and laser emitting optical axis.
Another specific scheme is that detection system includes the objective lens being sequentially arranged along Z-direction, optical filter, pipe
Lens and CCD camera.Wherein, for objective lens for collecting the fluorescence that fluorescent samples are inspired, optical filter is spuious for filtering out
Light, pipe lens are used for fluorescent foci to CCD camera, and CCD camera is used to record the fluorescence signal on photosurface, and by fluorescence
Signal passes to processor.
Another specific scheme is by being equipped with the laser issued to laser between laser and radial polarisation converter
The extender lens that light beam is expanded；It is equipped between phase mask plate and the uniaxial galvanometer for adjusting laser beam size
The first convex lens group；The second convex lens group and illumination objective lens are successively arranged between the varifocal concavees lens of electric drive and sample stage.
Another specific scheme is that the varifocal Concave Mirrors Focus variation range of electric drive is any the two of multifocal light beam
The distance between a focus.By the variation of focal length, realizes that light beam is moved forward and backward in the Y-axis direction, obtain a field range
Broader light slice.The varifocal concavees lens of electric drive are controlled by processor, realize the consecutive variations of focal length.
The principle of the present invention is as follows:
In traditional light slice fluorescence microscopy, focusing or galvanometer using common Gaussian beam by cylindrical mirror
Scanning form light slice, light that this mode is formed slice can spread very fast in the Y-axis direction, and only one in focusing is small
Part can effectively excite the fluorescence of sample, and field range is extremely limited.
In the present invention, by the modulation of phase mask plate, multifocal light beam is formed, it then can by electric drive focal length
The focal length for becoming concavees lens changes, and is moved forward and backward light beam in the Y-axis direction, during fast moving, several focuses are mutually connected
It connects, forms the light slice that usable range is bigger in a Y direction.The light slice formed by this method, very one section long
It is good apart from upper all focusing, greatly expand the field range of light slice fluorescence microscope, and the light of this well focussed
Slice, can also inhibit ambient noise well.
Compared with prior art, the invention has the benefit that
Present invention greatly enhances the field ranges of light slice fluorescence microscopy, and the background for reducing imaging is made an uproar
Sound.
Detailed description of the invention
Fig. 1 is that the light of the embodiment of the present invention is sliced the structural schematic diagram of fluorescent microscopic imaging device；
Fig. 2 is the phase distribution schematic diagram of the phase mask plate of the embodiment of the present invention；
Fig. 3 is the multifocal light beam that is formed after phase mask plate of laser beam of the embodiment of the present invention in YZ plane
Light distribution；
Fig. 4 (a) is that the finally obtained light of the embodiment of the present invention is sliced the surface of intensity distribution in YZ plane；It (b) is tradition
Gauss light be sliced the surface of intensity distribution in YZ plane；
Fig. 5 is that the embodiment of the present invention and traditional light are sliced the system point spread function of fluorescence microscopy along Y-axis normalizing
Change the comparison diagram of the curve of light distribution.
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.
Installation practice
Referring to Fig. 1, the multifocal light slice fluorescent microscopic imaging device of the present embodiment includes laser 1, extender lens 2,
Radial polarisation converter 3, phase mask plate 4, the first convex lens group (convex lens 5, convex lens 6), uniaxial galvanometer 7, electric drive can
Zoom concavees lens 8, the second convex lens group (convex lens 9, convex lens 10), illumination objective lens 11, sample stage 12, objective lens 13, filter
Mating plate 14, pipe lens 15, CCD camera 16, computer 17.
The device of the invention embodiment can be divided into four parts: generate lighting system, the carrying sample of illumination light slice
Sample stage 12, the test sample detection system and processor of the fluorescence that issue, the processor of the present embodiment is computer 17.
Wherein, lighting system includes being sequentially arranged along optical path: laser 1, extender lens 2, radial polarisation converter 3,
Phase mask plate 4, convex lens 5, convex lens 6, uniaxial galvanometer 7, the varifocal concavees lens 8 of electric drive, convex lens 9, convex lens 10 with
And illumination objective lens 11.
Laser 1 issues laser；Extender lens 2 carries out beam-expanding collimation to laser；Radial polarisation converter 3 is by incident light
Beam is converted to the light beam of radial polarisation；Phase mask plate 4 carries out phase-modulation to the light beam of radial polarisation, forms it into more
The light beam of a focus, the phase distribution on phase mask plate 4 is as shown in Fig. 2, its modulation function are as follows:
Wherein, (r, θ) indicates that the polar coordinates of certain point on light beam, r are the normalized cumulant of the point and optical axis, and θ hangs down for light beam
Directly in the angle of the polar coordinate vectors of optical axis section and laser emitting optical axis.
Beam size is adjusted in convex lens 5 and convex lens 6；Uniaxial 7 the reflected beams of galvanometer can be made by rotation
Light beam scans at sample along X-direction；The varifocal concavees lens 8 of electric drive are controlled by computer 17, and focal length changes, and makes
The light beam being radiated on sample is moved forward and backward along Y direction；Illumination objective lens 11 project excitation beam on fluorescent samples.
The sample stage 12 for carrying sample can be controlled by computer 17 to be moved along Z-direction with fixed step-length.
Detection system includes being sequentially arranged along Z-direction: objective lens 13, optical filter 14, pipe lens 15 and CCD camera
16。
Objective lens 13 are for collecting the fluorescence that fluorescent samples are issued by laser excitation；Optical filter 14 is collected for filtering out
The stray light arrived；Pipe lens 15 are used to the fluorescence being collected into gathering CCD camera 16；CCD camera 16 is for recording fluorescence
Signal, and pass the signal along on computer 17.
Several two-dimensional fluoroscopic signals that CCD camera 16 records are reconstructed in 17 one side of computer, reconstruct three-dimensional
The imaging results of fluorescent samples；On the other hand the sample stage 12 of control carrying fluorescent samples, makes it with fixed step-length along Z axis
Mobile, another further aspect controls the varifocal concavees lens 8 of electric drive, makes its focal length continuous transformation, the range of transformation is multifocal
In point light beam, the distance between two focuses.
The process for carrying out three-dimensional imaging to fluorescent samples using above-mentioned apparatus is as follows:
The laser beam issued in laser 1 is converted by extender lens 2 by after beam-expanding collimation by radial polarisation
Device 3 is converted into radial polarisation light, then carries out phase-modulation by phase mask plate 4 again, the phase point on phase mask plate 4
For cloth as shown in Fig. 2, after the phase-modulation, light beam becomes multifocal focus on light beam.
The multifocal light beam is irradiated to uniaxial galvanometer 7 after convex lens 5 and convex lens 6 are to the size adjustment of light beam
On, it is reflected by uniaxial galvanometer 7.Light beam after reflection, by the scattering of the varifocal concavees lens 8 of electric drive, then again by convex lens 9
It is expanded with the focusing of convex lens 10, illuminated object lens 11 project fluorescent samples (sample stage 12 for being placed in carrying fluorescent samples)
On.
Light distribution of the multifocal excitation beam on the section YZ by the uniaxial rotation of galvanometer 7 and electricity as shown in figure 3, driven
The zooming transform for moving varifocal concavees lens 8, is scanned in X-axis and Y-axis respectively, obtains virtual big visual field light slice, which cuts
Shown in such as Fig. 4 (a) of the light distribution on the section YZ of piece.
Fluorescent samples are sliced by above-mentioned light and excite, and issue fluorescence, are detected object lens 13 and collect, filter using optical filter 14
Then light is focused in CCD camera 16 by pipe lens 15, the two-dimensional fluoroscopic signal of record is transmitted to computer by CCD camera 16
On 17.
Computer 17, which controls, to be carried the sample stages 12 of fluorescent samples and is moved along Z-direction with fixed step-length, every
A axial position all obtains the image that a width has fluorescent samples 2D signal.Multiple image is rebuild, the three-dimensional of sample is obtained
Imaging results.
In order to verify expansion of the method used in the present embodiment to light slice fluorescence microscopy field range, to this implementation
The slice of light obtained in example and traditional Gauss light are sliced, and the light distribution on the section YZ compares, as shown in figure 4, (a)
For the light slice that the present invention obtains, (b) it is sliced for traditional Gauss light, by comparing it is found that the present invention obtains in Y direction
Light slice diffusion want much slower.
By the way that the curve comparison of light distribution will be normalized in the Y-axis direction in Fig. 5, it can be seen that used in the present invention
Method greatly expands light slice fluorescence microscopy method, is calculated by full width at half maximum (FWHM) in curve, and the present invention is by light
It is octuple to be sliced the field expander of fluorescence microscopy.
Embodiment of the method
The multifocal light slice fluorescent microscopic imaging method of the present embodiment is glimmering based on the light slice in above-mentioned apparatus embodiment
Light microscopic imaging device is realized comprising following steps:
1) laser is formed a series of along lighting optical axis direction and with the multifocal of identical spacing after phase-modulation
Light beam；
2) along X-direction scanning light beam, and it is moved forward and backward light beam along Y direction, obtain one has along the Y direction
The light slice of certain areas imaging；
3) along Z-direction, the fluorescence that fluorescent samples issue is collected, obtains sample in the two-dimensional light intensity of the axial position
Signal pattern.
4) it along Z axis mobile example, repeats step 3) and obtains several two-dimentional light intensity signal images, to several two-dimentional light intensity letters
Number image carries out three-dimensionalreconstruction, obtains the three-dimensional imaging information of fluorescent samples.
In the present embodiment, X-direction is perpendicular to lighting optical axis and detects the direction of optical axis, and Y direction is along illumination
The direction of optical axis, Z-direction are the directions along detection optical axis.These three directions, are mutually perpendicular to two-by-two, constitute three-dimensional right angle and sit
Mark system.
In step 1) modulation function of phase-modulation be wherein,
(r, θ) indicates that the polar coordinates of certain point on light beam, r are the normalized cumulant of the point and optical axis, and θ is beam orthogonal in light
The polar coordinate vectors of axis section and the angle of laser emitting optical axis.
Claims (9)
1. a kind of multifocal light is sliced fluorescent microscopic imaging method, which comprises the following steps:
1) laser is formed a series of along lighting optical axis direction and with the multifocal light of identical spacing after phase-modulation
Beam；
2) along X-direction scanning light beam, and it is moved forward and backward light beam along Y direction, obtain one has centainly along the Y direction
The light of areas imaging is sliced；
3) along Z-direction, the fluorescence that fluorescent samples issue is collected, obtains sample in the two-dimensional light intensity signal of the axial position
Image.
4) it along Z axis mobile example, repeats step 3) and obtains several two-dimentional light intensity signal images, to several two-dimentional light intensity signal figures
As carrying out three-dimensionalreconstruction, the three-dimensional imaging information of fluorescent samples is obtained.
2. multifocal light according to claim 1 is sliced fluorescent microscopic imaging method, it is characterised in that:
In step 1), laser is first set to be converted to radial polarisation light before carrying out phase-modulation to laser, then again to the radial direction
Polarised light carries out phase-modulation.
3. multifocal light according to claim 1 is sliced fluorescent microscopic imaging method, it is characterised in that:
The modulation function of phase-modulation described in step 1) are as follows:
Wherein, (r, θ) indicate light beam on certain point polar coordinates, r be the point and optical axis normalized cumulant, θ be beam orthogonal in
The polar coordinate vectors of optical axis section and the angle of laser emitting optical axis.
4. multifocal light according to claim 1 is sliced fluorescent microscopic imaging method, it is characterised in that:
In step 2), the back-and-forth motion light beam makes focus move to form light along Y-axis, and the mobile range of light beam is more
The distance between any two focus of focus light beam.
5. a kind of multifocal light is sliced fluorescent microscopic imaging device, lighting system, carrying fluorescent samples including forming light slice
Sample stage, detection fluorescent samples issue fluorescence detection system and a processor, it is characterised in that:
The lighting system includes being sequentially arranged along optical path: laser；Laser beam is changed into the radial direction of radial polarisation light
Polarization converter；Phase-modulation is carried out to radial polarisation light and it is made to be converted into the phase mask plate of multifocal light beam；Make to irradiate
The uniaxial galvanometer that light beam on to fluorescent samples is scanned along X-direction；And make the electric drive of focal length generation consecutive variations can
Zoom concavees lens；
Consecutive variations and control sample stage occur for the focal length that the processor is used to control the varifocal concavees lens of electric drive with fixation
Step-length is moved along Z-direction, and several two-dimentional light intensity signal images that detection system is collected are reconstructed, and obtains fluorescent samples
Three-dimensional imaging information.
6. multifocal light according to claim 5 is sliced fluorescent microscopic imaging device, it is characterised in that:
The phase mask plate carries out the modulation function of phase-modulation to radial polarisation light are as follows:
Wherein, (r, θ) indicate light beam on certain point polar coordinates, r be the point and optical axis normalized cumulant, θ be beam orthogonal in
The polar coordinate vectors of optical axis section and the angle of laser emitting optical axis.
7. multifocal light according to claim 5 is sliced fluorescent microscopic imaging device, it is characterised in that:
The detection system includes the objective lens being sequentially arranged along Z-direction, optical filter, pipe lens and CCD camera.
8. multifocal light according to claim 5 is sliced fluorescent microscopic imaging device, it is characterised in that:
The laser beam for issuing laser is equipped between the laser and the radial polarisation converter to expand
The extender lens of beam；The for adjusting laser beam size is equipped between the phase mask plate and the uniaxial galvanometer
One convex lens group；
It is successively arranged to be focused laser beam between the varifocal concavees lens of the electric drive and the sample stage and expand
The second convex lens group and illumination objective lens.
9. multifocal light according to claim 5 is sliced fluorescent microscopic imaging device, it is characterised in that:
The focal-distance tuning range of the varifocal concavees lens of the electric drive be multifocal light beam any two focus between away from
From.
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| CN110220875A (en) * | 2019-06-10 | 2019-09-10 | 浙江大学 | A kind of lattice light slice fluorescent microscopic imaging equipment and method based on fluorescence calculus of finite differences |
| CN110836877A (en) * | 2019-10-16 | 2020-02-25 | 浙江大学 | Light section microscopic imaging method and device based on liquid crystal zoom lens |
| CN114217055A (en) * | 2021-12-02 | 2022-03-22 | 极瞳生命科技(苏州)有限公司 | Portable fluorescence scanning detection device and method |
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