CN108680544A - A kind of the light slice fluorescent microscopic imaging method and device of structured lighting - Google Patents
A kind of the light slice fluorescent microscopic imaging method and device of structured lighting Download PDFInfo
- Publication number
- CN108680544A CN108680544A CN201810367723.3A CN201810367723A CN108680544A CN 108680544 A CN108680544 A CN 108680544A CN 201810367723 A CN201810367723 A CN 201810367723A CN 108680544 A CN108680544 A CN 108680544A
- Authority
- CN
- China
- Prior art keywords
- light
- light beam
- fluorescent
- sliced
- optical axis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
-
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Multimedia (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a kind of light of structured lighting slice fluorescent microscopic imaging method and systems, belong to optical image technology field, including light source, the sample stage of carrying fluorescent samples and detection fluorescent samples send out the detecting system of fluorescence；It is equipped between light source and sample stage and is sequentially arranged along light path：Light beam is become to the polarizing film and radial polarisation converter of radial polarisation light；The phase mask plate of phase-modulation is carried out to radial polarisation light；The diffraction grating of diffraction is carried out to modulated light beam；And move the galvanometer of lighting pattern on fluorescent samples；The light beam being emitted by galvanometer is irradiated to from Y-direction on fluorescent samples, and detecting system collects fluorescence from Z-direction；Further include a processor, lighting pattern and control sample stage of the control galvanometer from X to portable lighting are moved in Z-direction, and the light distribution image collected to detecting system is handled and reconstructed, and the 3-D view of sample is generated.The light beam formed by the device and method, with larger visual field while with smaller main lobe.
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 of structured lighting
Micro- imaging method and device.
Background technology
The high speed development of biotechnology in recent years needs us to have more deep understanding to physiology course, this is also right
More stringent requirements are proposed for 3D Real Time Imaging Technologies.But the development of 3D Real Time Imaging Technologies, also it is faced with many challenges ---
Successful 3D Real Time Imaging Technologies need to reach simultaneously high spatial resolution, high image taking speed, good smooth sectioning capability, low
Light injury and photobleaching ability.And these characteristics are often conflicting, such as, if it is desired to obtaining has higher space
The image of resolution ratio, it is possible to which needs take more time, this just sacrifices image taking speed；And it needs that higher pump is added
Pu energy, this can improve light injury and photobleaching ability to sample.
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.
Such as the Chinese patent literature that publication No. is CN103743714A discloses a kind of inclination wide field light slice and is scanned into
As microscopic system and its imaging method, by the laser light incident two-dimensional scanning mirrors of laser beam emitting device transmitting, collimation lens set and
The laser scanning light path of first microcobjective composition carries out dip sweeping to the sample on sample stage；Second microcobjective, field lens,
Detector constitutes imaging detection light path, and optical axis is vertical with laser scanning light path light axis, and imaging display control unit passes through number
Control the synchronization action and sample of two-dimensional scanning mirrors, the second microcobjective, detector respectively according to capture card, sample stage control device
The automatic displacement of sample platform forms sample wide field three-dimensional image information to the detector image-forming data processing of acquisition.The invention can be real
Existing wide field scanning imagery, but resolution ratio is not high.
In order to improve the resolution ratio that light is sliced fluorescence microscopy, researchers are made that many trials, by stimulated emission
It consumes the super resolution technologies such as microtechnic, Structured Illumination technology, unimolecule location technology and is sliced fluorescence microscopy phase with light
It mutually combines, it is intended to improve the resolution capability of light slice fluorescence microscopy, but effect is all not fully up to expectations.
Invention content
The purpose of the present invention is to provide a kind of light slice fluorescent microscopic imaging method of structured lighting, can using this method
It obtains resolution ratio to be improved, visual field obtains widened image.
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
Phase-modulation is carried out to radial polarisation light using phase mask plate, generates the center spot light narrower than normal Gaussian beam
Spot.Then, which forms a series of repeat patterns by diffraction grating, then is moved by galvanometer, is shot in each plane
Number pictures are handled these images, most by the conventional micro- processing method to original image of wide field structured illumination
After obtain resolution ratio and be improved, visual field obtains widened image.
To achieve the goals above, structured lighting provided by the invention light slice fluorescent microscopic imaging method include with
Lower step：
1) laser is converted to radial polarisation light after beam-expanding collimation, and radial polarisation light passes through phase-modulation and diffraction shape
At one group along X to the lighting pattern of arrangement, and projected along Y-direction and excite fluorescence on fluorescent samples；
2) fluorescence is collected from Z-direction, obtains the light distribution image of Z-direction position；
3) it along X to portable lighting pattern, in moving process, collects fluorescence from Z-direction and obtains several light distribution images, locate
The two dimensional image of Z-direction is obtained after reason；
4) fluorescent samples are moved along Z-direction with fixed step-length, repeats step 3) and obtains one of sample at each Z-direction position
Group light distribution image, handles multigroup light distribution image to obtain the two dimensional image at multiple Z-direction positions；
5) three-dimensionalreconstruction is carried out to the two dimensional image at multiple Z-direction positions, realizes the three-dimensional imaging to sample.
In above-mentioned technical proposal, for X to being perpendicular to lighting optical axis and detecting the direction of optical axis, Y-direction is along lighting optical axis
Direction, Z-direction be along detection optical axis direction.In step 1), light beam will produce a center light after phase-modulation
The spot hot spot smaller than Gaussian beam, in point spread function, the secondary lobe of hot spot can by select suitable illuminating lens and
The NA values of lens are detected to eliminate, therefore, mainly the main lobe of illuminating bundle is imaged sample, and fluorescence microscopy skill is sliced according to light
The principle of art, main lobe is smaller, and the axial resolution for being ultimately imaged result is higher.The light beam formed by this method, with
While smaller main lobe, it may have larger visual field.
Specific scheme is Z-direction, X is mutually perpendicular to two-by-two to Y-direction, constitutes three-dimensional cartesian coordinate system.
Another specific scheme is that the modulation function of phase-modulation in step 1) is
Wherein, (r, θ) indicate light beam on certain point polar coordinates, r be the point at a distance from optical axis, θ be beam orthogonal in light
The angle of axis profile position polar coordinate vectors and laser emitting optical axis；r2/r1=0.45.
Another specific scheme is at least to collect three light distribution images in step 3)；For most of sample,
The image of shooting is 7 or 9.
Another specific scheme is the two-dimensional imaging of each axial position in step 3) as a result, being N (N >=3) width original
Beginning image obtains after digital processing, and processing formula is
Wherein, i indicates imaginary unit；J=0,1 ..., N-1；IjIt indicates to collect the primary light that fluorescence obtains in Z-direction position
Strong distribution map；N is the quantity for the original image collected in the Z-direction position.
In order to realize that above-mentioned another object, the light of structured lighting provided by the invention are sliced fluorescent microscopic imaging device,
Including light source, carries the sample stage of fluorescent samples and detect the detecting system that fluorescent samples send out fluorescence；Between light source and sample stage
Equipped with what is be sequentially arranged along light path：Light beam is become to the polarizing film and radial polarisation converter of radial polarisation light；To radial polarisation
Light carries out the phase mask plate of phase-modulation；The diffraction grating of diffraction is carried out to modulated light beam；And mobile fluorescent samples
The galvanometer of upper lighting pattern；The light beam being emitted by galvanometer is irradiated to from Y-direction on fluorescent samples, and detecting system collects fluorescence from Z-direction；
Further include a processor, is moved in Z-direction for controlling lighting pattern of the galvanometer from X to portable lighting and control sample stage, and right
The light distribution image that detecting system is collected is handled and is reconstructed, and the 3-D view of sample is generated.
In above-mentioned technical proposal, the light beam after phase-modulation, by diffraction grating, can obtain along X to one
(for X to being perpendicular to lighting optical axis and detecting the direction of optical axis, Y-direction is the direction along lighting optical axis, Z-direction to serial repeat patterns
It is the direction along detection optical axis), it is moved by galvanometer, hot spot is made, to moving, in the same axial position, to be obtained more along X
Width two dimensional image, then again by method identical with traditional wide field Structured Illumination microtechnic, at these images
Reason, obtains X to the higher image of resolution ratio.The light beam formed by the device, while with smaller main lobe, it may have
Larger visual field.
Specific scheme is that the modulation function that phase mask plate carries out radial polarisation light phase-modulation is
Wherein, (r, θ) indicate light beam on certain point polar coordinates, r be the point at a distance from optical axis, θ be beam orthogonal in light
The angle of axis profile position polar coordinate vectors and laser emitting optical axis；r2/r1=0.45.
Another specific scheme is equipped between light source and polarizing film laser beam being filtered single mode optical fiber and right
Light beam carries out the collimation lens of beam-expanding collimation；First be focused to the light beam after diffraction is equipped between diffraction grating and galvanometer
Convex lens and the second convex lens that light beam is collimated.
Another specific scheme is equipped between galvanometer and sample stage and is focused to the light beam after vibration mirror reflected successively
Scanning lens, field lens that light beam is collimated and the illumination objective lens close to sample stage；Detecting system include along Z-direction successively
Objective lens, optical filter, pipe lens and the CCD camera of arrangement.
Objective lens are used to filter out the stray light in signal for collecting the fluorescence that fluorescent samples are sent out, filter plate, and pipe is saturating
Mirror is used to the fluorescence signal after filtering focusing on CCD camera, and CCD camera is for recording fluorescence intensity signals.
It is 0.3 that further specific scheme, which is the aperture NA of illumination objective lens,；The aperture NA of objective lens is 1.1.To eliminate
The secondary lobe of hot spot.
The principle of the present invention is as follows：
It is sliced in fluorescence microscopy in light, the axial resolution of acquired image and the thickness of mating plate are closely related, light
Piece is thinner, and the axial resolution of obtained image is higher, therefore a relatively thin mating plate can be obtained by certain method, comes
Improve the axial resolution of light slice fluorescence microscopy.
In the present invention, by the phase-modulation of phase mask, one is modulated the light beam into perpendicular to lighting optical axis
In section, the center spot light beam smaller than Gaussian spot, because in point spread function, the secondary lobe of hot spot can pass through selection
The NA values of suitable illuminating lens and detection lens are eliminated, and therefore, mainly the main lobe of illuminating bundle is imaged sample, according to
Light is sliced the principle of fluorescence microscopy, and main lobe is smaller, and the axial resolution for being ultimately imaged result is higher.Shape by this method
At light beam, while with smaller main lobe, it may have larger visual field.
Light beam after phase-modulation can obtain that (X is to being to a series of repeat patterns along X by diffraction grating
Perpendicular to the direction of lighting optical axis and detection optical axis, Y-direction is the direction along lighting optical axis, and Z-direction is the side along detection optical axis
To), it can be moved by galvanometer, hot spot is made, to moving, in the same axial position, to obtain two-dimensional images, then along X
Again by method identical with traditional wide field Structured Illumination microtechnic, these images are handled, obtain X to resolution ratio
Higher image.
Compared with prior art, beneficial effects of the present invention are：
Apparatus of the present invention are simple, and it is convenient that method is realized, improve the X of light slice fluorescence microscopy to the resolution with Z-direction
Rate expands the visual field of light slice fluorescence microscopy.
Description of the drawings
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 (a) is for light beam in the light distribution on laser emitting optical axial plane after phase mask plate；(b)
Be light beam after diffraction grating in the light distribution on laser emitting optical axial plane；
Fig. 4 is pair that the mating plate that the embodiment of the present invention obtains and traditional Gauss mating plate normalize the curve of light distribution in an axial direction
Than figure；
Fig. 5 is that the system point spread function that the embodiment of the present invention is sliced fluorescence microscopy with traditional light normalizes in an axial direction
The comparison diagram of the curve of light distribution；
Fig. 6 (a) is intensity distribution of the traditional Gauss mating plate in YZ planes；(b) it is that mating plate of the embodiment of the present invention is flat in YZ
Intensity distribution on face；(c) it is pair of Gauss mating plate and mating plate of the embodiment of the present invention along the normalization curve of light distribution of Y-direction
Than figure, X is to being perpendicular to lighting optical axis and detecting the direction of optical axis, and Y-direction is the direction along lighting optical axis, and Z-direction is along inspection
The direction of photometer axis.
Specific implementation mode
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.
Device embodiment
Referring to Fig. 1, the light slice fluorescent microscopic imaging device of the structured lighting of the present embodiment includes that may make up three-dimensional seat
The laser emitting optical axis, lighting optical axis and detection optical axis for marking system, galvanometer is equipped in laser emitting optical axis and lighting optical axis intersection
10, it is equipped with sample stage 14 in lighting optical axis and detection optical axis intersection.X to be perpendicular to lighting optical axis and detect optical axis side
To Y-direction is the direction along lighting optical axis, and Z-direction is the direction along detection optical axis.
On the laser emitting optical axis of the present embodiment successively be equipped with laser 1, single mode optical fiber 2, collimation lens 3, polarizing film 4,
Radial polarisation converter 5, phase mask plate 6, diffraction grating 7 and convex lens group, convex lens group include to the light beam after diffraction into
First convex lens 8 of line focusing and the second convex lens 9 that the light beam after diffraction is collimated.Single mode optical fiber 2 is used for laser
Light beam is filtered, and collimation lens 3 is used to collimate beam expander, and polarizing film 4 and radial polarisation converter 5 are for becoming light beam
For radial polarisation light, phase mask plate 6 is used to carry out phase-modulation to radial polarisation light, and diffraction grating 7 is used for modulated
Light beam carries out diffraction.Galvanometer 10 is used for portable lighting mating plate.Wherein, phase mask plate 6 carries out phase-modulation to radial polarisation light
Modulation function be
Wherein, (r, θ) indicate light beam on certain point polar coordinates, r be the point at a distance from optical axis, θ be beam orthogonal in light
The angle of axis profile position polar coordinate vectors and laser emitting optical axis；r2/r1=0.45.
The lighting optical axis of the present embodiment is that sample is excited the optical axis of the fluorescence sent out, is equipped with scanning lens successively thereon
11, field lens 12 and illumination objective lens 13, lighting optical axis are overlapped with the focal plane of objective lens 15, and the aperture NA of illumination objective lens 13 is
0.3.For being focused to the light beam after vibration mirror reflected, field lens 12 is used to carry out the light beam after vibration mirror reflected scanning lens 11
Collimation.
The detection optical axis of the present embodiment is equipped with detection device, which includes the inspection being sequentially arranged along detection optical axis
Object lens 15, filter plate 16, pipe lens 17 and CCD camera 18 are surveyed, the aperture NA of objective lens 15 is 1.1.Inspection in detection device
It surveys object lens 15 and is used to filter out the stray light in signal for collecting the fluorescence that fluorescent samples are sent out, filter plate 16, pipe lens 17 are used
It is focused in CCD camera 18 in by the fluorescence signal after filtering, CCD camera 18 is for recording fluorescence intensity signals.
Above-mentioned galvanometer 10, sample stage 14 and CCD camera 18 are electrically connected to processor 19, and processor 19 controls galvanometer 10 and revolves
Turn；Sample stage 14 is controlled in an axial direction with a fixed step size and frequency shifts；To obtaining several original images on each axial position
It is handled, obtains the image of resolution ratio raising；To treated, two dimensional image is reconstructed, and obtains the graphics of sample.
Galvanometer 10 can rotate a certain angle under the control of processor 19, go out along laser to control lighting pattern
The axial movement of optical axis is penetrated, and sample stage 14 is moved in the axial direction by the control of processor 19 with certain step-length, to determine
The axial position of imaging；The image that CCD camera 18 obtains, is transferred to processor 19, and processor 19 is to these images and into line number
Word processing and three-dimensionalreconstruction.
The process that using above-mentioned apparatus fluorescent samples are carried out with three-dimensional imaging is as follows：
The laser beam sent out from laser 1, is first introduced into single mode optical fiber 2 and is filtered, then using collimation
Lens 3 complete collimation, and passing through polarizing film 4 later becomes linearly polarized light, and radial polarisation is converted to using radial polarisation converter 5
Then light carries out phase-modulation by phase mask plate 6 again, the phase distribution on phase mask plate 6 is as shown in Fig. 2, by phase
Shown in the modulated light distribution on laser emitting optical axial plane of bit mask plate 6 such as Fig. 3 (a), light beam continues to lead to
Diffraction grating 7 is crossed, in the plane perpendicular to lighting optical axis, is formed and repeats to scheme along a series of of laser emitting optical axis direction
Case, as shown in Fig. 3 (b).
The light beam being emitted after the modulation of diffraction grating 7 carries out light beam tune using the first convex lens 8, the second convex lens 9
It after whole, is reflected, is focused using scanning lens 11, after the collimation of field lens 12, illuminated object lens 13 focus on sample stage by galvanometer 10
On fluorescent samples on 14, fluorescent samples are irradiated with a laser, and send out fluorescence, are detected object lens 15 and are collected, then pass through optical filter
16 filter out spurious signal, are focused in CCD camera 18 by pipe lens 17, and CCD camera 18 passes to image on processor 19.
Galvanometer 10 rotates certain angle by the control of computer 19, irradiation to the mating plate on fluorescent samples also along X to
Certain offset occurs, the fluorescence that fluorescent samples are excited is after objective lens 15 and pipe lens 17, the weight in CCD camera 18
New imaging, is then passed on processor 19.
By the movement of galvanometer 10, N width images are obtained in same axial position, processor 19 carries out these images
Digital processing：
Wherein, i indicates imaginary unit；J=0,1 ..., N-1；IjIt indicates to collect the primary light that fluorescence obtains in Z-direction position
Strong distribution map；N is the quantity for the original image collected in the Z-direction position.
Then, processor 19 is controlled sample and is axially moved with fixed step-length, and each axial position obtains N width figures
Picture obtains the final two-dimensional imaging of the axial position as a result, then by these X-Y schemes after the digital processing of processor 19
As being reconstructed, required graphics is obtained.
The promotion of fluorescence microscopy axial resolution is sliced to verify the method used in the present invention to light, while to this
The axial intensity distribution for the mating plate that the mating plate and traditional light used in invention is sliced fluorescence microscopy compares, such as Fig. 4
Shown, the mating plate that the mating plate that the present invention obtains is sliced fluorescence microscopy than traditional light is thin.
The point spread function for illuminating mating plate is multiplied with the point spread function of the NA objective lens for being 1.1 can obtain entirely
The point spread function of system, the present invention and traditional light are sliced the distribution of the system point spread function of fluorescence microscopy axially
As shown in figure 5, the full width at half maximum (FWHM) according to curve in figure is calculated, traditional light can be sliced fluorescence microscopy by the present invention
The axial resolution of technology improves 1.3 times.
Fig. 6 is the light distribution of the mating plate (b) that generates on the sections YZ in traditional Gauss mating plate (a) and the present invention, from Fig. 6
(c) mating plate in can be seen that the method used in the present invention in the normalization light intensity curve of Y-direction and traditional light be sliced fluorescence microscopy
The field range of technology expands about one times.
Embodiment of the method
The light of the structured lighting of the present embodiment is sliced fluorescent microscopic imaging method based on the light in above-mentioned apparatus embodiment
It is sliced fluorescent microscopic imaging device to realize comprising following steps：
1) laser is converted to radial polarisation light after beam-expanding collimation, and radial polarisation light passes through phase-modulation and diffraction shape
At one group along X to the lighting pattern of arrangement, and projected along Y-direction and excite fluorescence on fluorescent samples；
2) fluorescence is collected from Z-direction, obtains the light distribution image of Z-direction position；
3) it along X to portable lighting pattern, in moving process, collects fluorescence from Z-direction and obtains several light distribution images, locate
The two dimensional image of Z-direction is obtained after reason；
4) fluorescent samples are moved along Z-direction with fixed step-length, repeats step 3) and obtains one of sample at each Z-direction position
Group light distribution image, handles multigroup light distribution image to obtain the two dimensional image at multiple Z-direction positions；
5) three-dimensionalreconstruction is carried out to the two dimensional image at multiple Z-direction positions, realizes the three-dimensional imaging to sample.
In the present embodiment, Z-direction, X are mutually perpendicular to two-by-two to Y-direction, constitute three-dimensional cartesian coordinate system.
The modulation function of phase-modulation is in step 1)
Wherein, (r, θ) indicate light beam on certain point polar coordinates, r be the point at a distance from optical axis, θ be beam orthogonal in light
The angle of axis profile position polar coordinate vectors and laser emitting optical axis；r2/r1=0.45.
N light distribution images are collected in step 3).The two-dimensional imaging of each axial position is as a result, be N (N >=3) width
What original image obtained after digital processing, processing formula is
Wherein, i indicates imaginary unit；J=0,1 ..., N-1；IjIt indicates to collect the primary light that fluorescence obtains in Z-direction position
Strong distribution map；N is the quantity for the original image collected in the Z-direction position.
Claims (10)
1. a kind of light of structured lighting is sliced fluorescent microscopic imaging method, which is characterized in that include the following steps：
1) laser is converted to radial polarisation light after beam-expanding collimation, and the radial polarisation light passes through phase-modulation and diffraction shape
At one group along X to the lighting pattern of arrangement, and projected along Y-direction and excite fluorescence on fluorescent samples；
2) fluorescence is collected from Z-direction, obtains the light distribution image of Z-direction position；
3) it in moving process, collects fluorescence to the lighting pattern is moved along X from Z-direction and obtains several light distribution images, locate
The two dimensional image of Z-direction is obtained after reason；
4) fluorescent samples are moved along Z-direction with fixed step-length, repeats step 3) and obtains one of sample at each Z-direction position
Group light distribution image, handles multigroup light distribution image to obtain the two dimensional image at multiple Z-direction positions；
5) three-dimensionalreconstruction is carried out to the two dimensional image at multiple Z-direction positions, realizes the three-dimensional imaging to sample.
2. light according to claim 1 is sliced fluorescent microscopic imaging method, it is characterised in that：
The Z-direction, X are mutually perpendicular to two-by-two to Y-direction, constitute three-dimensional cartesian coordinate system.
3. 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) is
Wherein, (r, θ) indicate light beam on certain point polar coordinates, r be the point at a distance from optical axis, θ be beam orthogonal cutd open in optical axis
The angle of face position polar coordinate vectors and laser emitting optical axis；r2/r1=0.45.
4. light according to claim 1 is sliced fluorescent microscopic imaging method, it is characterised in that：
Three light distribution images are at least collected in step 3).
5. light according to claim 4 is sliced fluorescent microscopic imaging method, it is characterised in that：
The two-dimensional imaging of each axial position is as a result, be that N (N >=3) width original image obtains after digital processing in step 3)
It arrives, processing formula is
Wherein, i indicates imaginary unit；J=0,1 ..., N-1；IjIt indicates to collect the original light distribution that fluorescence obtains in Z-direction position
Figure；N is the quantity for the original image collected in the Z-direction position.
6. the light of structured lighting a kind of is sliced fluorescent microscopic imaging device, including light source, carry fluorescent samples sample stage and
Detection fluorescent samples send out the detecting system of fluorescence；It is characterized in that：
It is equipped between the light source and sample stage and is sequentially arranged along light path：Light beam is become to the polarizing film and radial direction of radial polarisation light
Polarization converter；The phase mask plate of phase-modulation is carried out to radial polarisation light；The diffraction of diffraction is carried out to modulated light beam
Grating；And move the galvanometer of lighting pattern on fluorescent samples；
The light beam being emitted by the galvanometer is irradiated to from Y-direction on fluorescent samples, and the detecting system collects fluorescence from Z-direction；
Further include a processor, is moved from X to the movement lighting pattern and control sample stage in Z-direction for controlling the galvanometer
It is dynamic, and the light distribution image collected to the detecting system is handled and is reconstructed, and the 3-D view of sample is generated.
7. light according to claim 6 is sliced fluorescent microscopic imaging device, it is characterised in that：
The phase mask plate to radial polarisation light carry out phase-modulation modulation function be
Wherein, (r, θ) indicate light beam on certain point polar coordinates, r be the point at a distance from optical axis, θ be beam orthogonal cutd open in optical axis
The angle of face position polar coordinate vectors and laser emitting optical axis；r2/r1=0.45.
8. light according to claim 6 is sliced fluorescent microscopic imaging device, it is characterised in that：
It is equipped between the light source and the polarizing film and laser beam is filtered single mode optical fiber and light beam is expanded
The collimation lens of beam collimation；
Be equipped between the diffraction grating and the galvanometer the first convex lens that the light beam after diffraction is focused and
The second convex lens that light beam is collimated.
9. light according to claim 6 is sliced fluorescent microscopic imaging device, it is characterised in that：
Be equipped with successively between the galvanometer and the sample stage scanning lens that the light beam after vibration mirror reflected is focused,
The field lens collimated to light beam and the illumination objective lens close to the sample stage；
The detecting system includes objective lens, optical filter, pipe lens and the CCD camera being sequentially arranged along Z-direction.
10. light according to claim 9 is sliced fluorescent microscopic imaging device, it is characterised in that：
The aperture NA of the illumination objective lens is 0.3；The aperture NA of the objective lens is 1.1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810367723.3A CN108680544B (en) | 2018-04-23 | 2018-04-23 | Structured illumination light slice fluorescence microscopic imaging method and device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810367723.3A CN108680544B (en) | 2018-04-23 | 2018-04-23 | Structured illumination light slice fluorescence microscopic imaging method and device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108680544A true CN108680544A (en) | 2018-10-19 |
| CN108680544B CN108680544B (en) | 2021-04-06 |
Family
ID=63801377
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810367723.3A Active CN108680544B (en) | 2018-04-23 | 2018-04-23 | Structured illumination light slice fluorescence microscopic imaging method and device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108680544B (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110007453A (en) * | 2019-05-13 | 2019-07-12 | 中国科学院生物物理研究所 | The fluorescence signal measuring device and its measurement method of a kind of more light illumination modes and application |
| 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 |
| CN111221072A (en) * | 2020-03-30 | 2020-06-02 | 南京聚科光电技术有限公司 | Device and method for writing fiber grating by femtosecond laser |
| CN111381357A (en) * | 2018-12-29 | 2020-07-07 | 中国科学院深圳先进技术研究院 | Image three-dimensional information extraction method, object imaging method, device and system |
| US10795168B2 (en) | 2017-08-31 | 2020-10-06 | Metalenz, Inc. | Transmissive metasurface lens integration |
| CN112268879A (en) * | 2020-09-17 | 2021-01-26 | 中国科学院生物物理研究所 | Fluorescence microscopic imaging system |
| CN112969910A (en) * | 2018-11-14 | 2021-06-15 | 科磊股份有限公司 | Radial polarizer for particle detection |
| CN112986195A (en) * | 2021-02-06 | 2021-06-18 | 清华大学 | Microscopic tomography method and device |
| CN113296253A (en) * | 2021-07-23 | 2021-08-24 | 清华大学 | Multi-light-sheet light field fluorescence microscopic imaging device |
| CN113496463A (en) * | 2020-03-19 | 2021-10-12 | 深圳华大生命科学研究院 | Image reconstruction method, system, electronic device and storage medium |
| CN114166515A (en) * | 2021-12-02 | 2022-03-11 | 哈尔滨工业大学 | Device and method for realizing SLIPI-3DLIF measurement of engine combustion chamber |
| US11906698B2 (en) | 2017-05-24 | 2024-02-20 | The Trustees Of Columbia University In The City Of New York | Broadband achromatic flat optical components by dispersion-engineered dielectric metasurfaces |
| US11927769B2 (en) | 2022-03-31 | 2024-03-12 | Metalenz, Inc. | Polarization sorting metasurface microlens array device |
| US11978752B2 (en) | 2019-07-26 | 2024-05-07 | Metalenz, Inc. | Aperture-metasurface and hybrid refractive-metasurface imaging systems |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203705345U (en) * | 2014-01-14 | 2014-07-09 | 苏州大猫单分子仪器研发有限公司 | Inclined wide field optical section scanning imaging microscope system |
| US9256202B2 (en) * | 2011-05-20 | 2016-02-09 | Massachusetts Institute Of Technology | System, method and apparatus for phase-coded multi-plane microscopy |
| CN106970055A (en) * | 2017-04-28 | 2017-07-21 | 浙江大学 | A kind of three-dimensional fluorescence difference super-resolution microscopic method and device |
| CN107329245A (en) * | 2017-07-06 | 2017-11-07 | 中国科学院西安光学精密机械研究所 | Interference type structured light illumination microscope system and method based on radial polarization modulation |
-
2018
- 2018-04-23 CN CN201810367723.3A patent/CN108680544B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9256202B2 (en) * | 2011-05-20 | 2016-02-09 | Massachusetts Institute Of Technology | System, method and apparatus for phase-coded multi-plane microscopy |
| CN203705345U (en) * | 2014-01-14 | 2014-07-09 | 苏州大猫单分子仪器研发有限公司 | Inclined wide field optical section scanning imaging microscope system |
| CN106970055A (en) * | 2017-04-28 | 2017-07-21 | 浙江大学 | A kind of three-dimensional fluorescence difference super-resolution microscopic method and device |
| CN107329245A (en) * | 2017-07-06 | 2017-11-07 | 中国科学院西安光学精密机械研究所 | Interference type structured light illumination microscope system and method based on radial polarization modulation |
Non-Patent Citations (2)
| Title |
|---|
| 吴美瑞 等: "结构光照明荧光显微镜突破衍射极限的原理和在生命科学中的应用", 《激光与光电子学进展》 * |
| 徐豪 等: "基于波前相位调制的宽视场光片显微镜研究", 《光学学报》 * |
Cited By (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11906698B2 (en) | 2017-05-24 | 2024-02-20 | The Trustees Of Columbia University In The City Of New York | Broadband achromatic flat optical components by dispersion-engineered dielectric metasurfaces |
| US11579456B2 (en) | 2017-08-31 | 2023-02-14 | Metalenz, Inc. | Transmissive metasurface lens integration |
| US11988844B2 (en) | 2017-08-31 | 2024-05-21 | Metalenz, Inc. | Transmissive metasurface lens integration |
| US10795168B2 (en) | 2017-08-31 | 2020-10-06 | Metalenz, Inc. | Transmissive metasurface lens integration |
| CN112969910B (en) * | 2018-11-14 | 2022-06-17 | 科磊股份有限公司 | System and method for particle detection |
| CN112969910A (en) * | 2018-11-14 | 2021-06-15 | 科磊股份有限公司 | Radial polarizer for particle detection |
| CN111381357A (en) * | 2018-12-29 | 2020-07-07 | 中国科学院深圳先进技术研究院 | Image three-dimensional information extraction method, object imaging method, device and system |
| CN111381357B (en) * | 2018-12-29 | 2021-07-20 | 中国科学院深圳先进技术研究院 | Image three-dimensional information extraction method, object imaging method, device and system |
| CN110007453B (en) * | 2019-05-13 | 2023-11-21 | 中国科学院生物物理研究所 | Multi-illumination-mode fluorescent signal measuring device and measuring method and application thereof |
| CN110007453A (en) * | 2019-05-13 | 2019-07-12 | 中国科学院生物物理研究所 | The fluorescence signal measuring device and its measurement method of a kind of more light illumination modes and application |
| 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 |
| US11978752B2 (en) | 2019-07-26 | 2024-05-07 | Metalenz, Inc. | Aperture-metasurface and hybrid refractive-metasurface imaging systems |
| 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 |
| CN113496463B (en) * | 2020-03-19 | 2024-05-31 | 深圳华大生命科学研究院 | Image reconstruction method, system, electronic device and storage medium |
| CN113496463A (en) * | 2020-03-19 | 2021-10-12 | 深圳华大生命科学研究院 | Image reconstruction method, system, electronic device and storage medium |
| CN111221072A (en) * | 2020-03-30 | 2020-06-02 | 南京聚科光电技术有限公司 | Device and method for writing fiber grating by femtosecond laser |
| CN112268879A (en) * | 2020-09-17 | 2021-01-26 | 中国科学院生物物理研究所 | Fluorescence microscopic imaging system |
| CN112268879B (en) * | 2020-09-17 | 2022-08-26 | 中国科学院生物物理研究所 | Fluorescence microscopic imaging system |
| CN112986195A (en) * | 2021-02-06 | 2021-06-18 | 清华大学 | Microscopic tomography method and device |
| CN113296253A (en) * | 2021-07-23 | 2021-08-24 | 清华大学 | Multi-light-sheet light field fluorescence microscopic imaging device |
| CN114166515B (en) * | 2021-12-02 | 2023-03-10 | 哈尔滨工业大学 | Device and method for realizing SLIPI-3DLIF measurement of engine combustion chamber |
| CN114166515A (en) * | 2021-12-02 | 2022-03-11 | 哈尔滨工业大学 | Device and method for realizing SLIPI-3DLIF measurement of engine combustion chamber |
| US11927769B2 (en) | 2022-03-31 | 2024-03-12 | Metalenz, Inc. | Polarization sorting metasurface microlens array device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108680544B (en) | 2021-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108680544A (en) | A kind of the light slice fluorescent microscopic imaging method and device of structured lighting | |
| CN108982452B (en) | Multi-focus scanning three-dimensional imaging method and system based on double-spiral point spread function | |
| CN105807412B (en) | A kind of total internal reflection microscopic method and device based on free form surface shaping | |
| CN105659143B (en) | Scan the imaging of Fourier overlapping associations in aperture | |
| CN105784653B (en) | A kind of wide field super-resolution fluorescence microscopic imaging device | |
| US20080073524A1 (en) | Method and apparatus for reviewing defects | |
| CN106226895B (en) | A kind of the rotation total internal reflection microscopic method and device of band feedback | |
| CN110082900A (en) | Variable illumination Fourier overlapping associations imaging device, system and method | |
| CN111257227B (en) | Dark field confocal microscopic measurement device and method based on polarization autocorrelation | |
| JP2005181312A (en) | Coordinate conversion method from scanner image coordinates of rare cell into microscope coordinates using reticle mark on sample medium | |
| CN101893755B (en) | Fluorescence microscopy method and apparatus using a quadrangular pyramid mirror to produce structured illumination | |
| CN106841136B (en) | A kind of high-precision axially position to ultra-thin cell and imaging method and device | |
| CN110220875B (en) | Lattice light section fluorescence microscopic imaging equipment and method based on fluorescence difference method | |
| CN109712072A (en) | Striped illumination Fourier iteration based on total internal reflection updates super-resolution micro imaging method | |
| CN104155274A (en) | Double beam plate lighting microscan imaging method and microscope | |
| CN103852458A (en) | Microscopic method based on wide field stimulated emission difference and microscopic device based on wide field stimulated emission difference | |
| CN116183568B (en) | High-fidelity reconstruction method and device for three-dimensional structured light illumination super-resolution microscopic imaging | |
| CN111257226A (en) | Dark field confocal microscopic measurement device and method based on polarization autocorrelation | |
| CN108982455B (en) | Multi-focus light section fluorescence microscopic imaging method and device | |
| CN109870441A (en) | Three-dimensional super-resolution light slice fluorescent microscopic imaging method and device based on shift frequency | |
| CN106556570A (en) | For the apparatus and method of the three-dimensional infrared imaging to surface | |
| CN107678151A (en) | The burnt parallel microscopic imaging apparatus of copolymerization based on interference array light field | |
| CN113358611B (en) | Embedded three-dimensional photoelectric correlation imaging device and method | |
| CN115656129A (en) | Fluorescence emission ratio super-resolution imaging method | |
| CN109085137B (en) | Three-dimensional imaging device based on K space transformation and imaging method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |