CN110824681A - Non-scanning high super-resolution optical three-dimensional microscopic imaging method - Google Patents
Non-scanning high super-resolution optical three-dimensional microscopic imaging method Download PDFInfo
- Publication number
- CN110824681A CN110824681A CN201911064422.4A CN201911064422A CN110824681A CN 110824681 A CN110824681 A CN 110824681A CN 201911064422 A CN201911064422 A CN 201911064422A CN 110824681 A CN110824681 A CN 110824681A
- Authority
- CN
- China
- Prior art keywords
- lens
- image
- imaging
- light modulator
- spatial light
- 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
Images
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
-
- 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
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Multimedia (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention belongs to the technical field of optical microscopic imaging, relates to a non-scanning high super-resolution optical three-dimensional microscopic imaging method, and particularly relates to a method for realizing high-resolution three-dimensional volume imaging without mechanical scanning by utilizing the self-bending propagation effect of a non-diffraction light beam. The method comprises the steps of forming an enlarged image of a sample at an image surface of an inverted fluorescence microscope system, imaging the enlarged image by a CCD camera through a first lens, a reflective or transmissive spatial light modulator and a second lens, generating a variable modulation pattern by using the spatial light modulator to obtain a series of projection images, and reconstructing the projection images by using a reconstruction algorithm to obtain three-dimensional structure information of an original object. The invention can realize the non-scanning high-super-resolution three-dimensional microscopic imaging and has higher imaging speed.
Description
Technical Field
The invention belongs to the technical field of optical microscopic imaging, and particularly relates to a non-scanning high-super-resolution three-dimensional microscopic imaging method.
Background
The optical microscope is a precise instrument which has been used for more than 300 years, has the characteristics of non-contact and low toxicity, and is very suitable for imaging living biological tissues and cells. In order to obtain high-contrast and clear images in biological tissues, fluorescence microscopy becomes an important branch of the development of optical microscopes, and becomes a main research direction in the field of biological high-resolution microscopy. The development of fluorescence microscopy technology is always pursuing performance improvement in three aspects: resolution, imaging speed (dynamic imaging) and imaging depth of field (volume imaging). Far-field optical imaging is limited in imaging resolution by abbe diffraction limit theory, with a conventional maximum lateral resolution of about half the wavelength of light. To break through the limitation, three types of technologies are mainly developed at present: 1) single molecule localization microscopy such as random optical reconstruction microscopy (STORM) and light activated localization microscopy (PALM). The technology combines single-molecule fluorescence excitation, data processing and reconstruction to obtain a super-resolution image, but the image reconstruction time is long, and real-time imaging and three-dimensional imaging are difficult to realize. (2) Stimulated radiation depletion microscopy (STED). The technology combines optical excitation and optical loss, can realize high-resolution imaging, but has poor real-time performance and three-dimensional imaging performance. (3) Structured light apparent micro technology (SIM). The technology is based on a frequency domain information processing method, and has high resolution, low real-time performance and poor three-dimensional imaging capability. In terms of imaging speed, as resolution increases, data processing amount increases, resulting in a decrease in imaging speed, and thus breakthrough is required in terms of device performance and subsequent data algorithms. In terms of three-dimensional information acquisition, currently mainly based on the z-scan mode, it is necessary to mechanically move the sample or the objective lens along the z-axis, further affecting the imaging speed.
Disclosure of Invention
Based on the defects, the invention aims to provide a non-scanning high-super-resolution three-dimensional microscopic imaging method, which is a high-super-resolution three-dimensional microscopic imaging method capable of realizing a certain depth without mechanical scanning, and is suitable for fluorescent microscopic imaging and a microscopic imaging system with a transparent structure and a self-luminous sample.
The technology adopted by the invention is as follows: a non-scanning high super-resolution optical three-dimensional microscopic imaging method comprises the following steps:
(1) light path structure
An amplified image formed by a sample on an image surface of an inverted fluorescence microscope system is imaged by a CCD camera through a first lens, a reflective or transmissive spatial light modulator and a second lens, wherein the first lens and the second lens are the same, and the distances between a diaphragm and the first lens, between the first lens and the spatial light modulator, between the spatial light modulator and the second lens and between the second lens and the CCD camera are the focal length f of the first lens;
(2) light field modulation pattern design
(2.1) generating a main pattern on the spatial light modulator using the formula:
K(x,y)＝exp{iβ[(x+y)3+(x-y)3]}
in the formula, x and y are plane coordinates, the range of x and y is determined by the pixel number of the spatial light modulator, β is a modulation coefficient, and the value is a (0, 1) interval according to different imaging depths β;
(2.2) covering both side portions of the spatial light modulator pattern with a step grating pattern to form a stripe-shaped central main pattern;
(2.3) rotating the central strip pattern for 360 degrees at intervals of an integer for one circle, and acquiring an image on a CCD camera to form a projection image sequence for image reconstruction after rotating for one angle;
(3) image reconstruction
Carrying out data reconstruction on CCD projection images obtained by different modulation images so as to obtain a three-dimensional image of an original object, wherein the reconstruction steps are as follows:
(3.1) performing radon transform on each CCD projection image and defining a uniform coordinate system (u, v, w) according to the formula:
wherein, (x ', y ', z ') is the coordinate of each projection plane, theta is the included angle between the projection direction and the principal axis of the optical path,is the angle of rotation of each projected image;
(3.2) Fourier transform is performed on the distribution of each projection in the unified coordinate system to obtain three-dimensional distribution (x) in the image spaceT,yT,zT) The formula according to is:
(3.3) correcting the spatial coordinates of the image to obtain the real coordinate distribution (x, y, z) of the object space
Wherein M is the microscope magnification and is determined by the objective lens; k is 2 pi/lambda, and lambda is the wavelength of imaging light wave; and R is the diameter of the main lobe of the Airy light spot of the system point spread function and is measured by experiments.
In step 2.3, the number of the rotation interval acquisition times is not less than 6.
The invention has the advantages and beneficial effects that: the invention is based on frequency domain regulation, can control a light field to capture z-axis information, is a new imaging mechanism, combines the self-bending propagation characteristic and the non-diffraction characteristic of a light beam, and can realize non-scanning high-super-resolution three-dimensional microscopic imaging through projected image reconstruction. The light path of the invention can be built based on a traditional inverted fluorescence microscope system, thus being compatible with the traditional microscope technology. The invention is applicable to the field of fluorescence microscopy imaging, including biological microscopy applications and transparent crystal self-luminous microscopy applications. Because mechanical scanning is not needed, the imaging speed is high, the three-dimensional imaging speed can reach 5Hz and the imaging depth can reach more than 10 microns according to the current commercial spatial light modulator 60Hz technology.
Drawings
FIG. 1 is a diagram of a reflective imaging optical path;
FIG. 2 is a diagram of a transmission imaging optical path;
FIG. 3 is a modulation image;
FIG. 4 is the modulated image of FIG. 3 rotated 45;
FIG. 5 is a graph of the result of imaging bulk quantum dots with a thickness of 8 microns;
FIG. 6 is an image of a 10 micron thick kidney tube of a mouse;
FIG. 7 is a graph of resolution measurement data in three directions, x, y, and z;
the device comprises a sample 1, a sample 2, an objective lens 3, a light source 4, a dichroic mirror 5, a tube mirror 6, a reflector 7, a diaphragm 8, a CCD camera 9, a second lens 10, a first lens 11, a reflective spatial light modulator 12 and a transmissive spatial light modulator.
Detailed Description
The invention is further illustrated by way of example in the accompanying drawings of the specification:
example 1
A non-scanning high super-resolution optical three-dimensional microscopic imaging method comprises the following steps:
(1) light path structure
As shown in fig. 1-2, a sample is imaged by an inverted fluorescence microscope system, and then passes through a reflective imaging optical path structure or a transmissive imaging optical path structure, where the optical path structure is specifically as follows: the light source excites a luminescent substance of a sample through an objective lens system after being reflected by a dichroic mirror, the sample luminescent substance passes through the objective lens, the dichroic mirror and a tube lens to form a magnified image of the sample at a diaphragm, the magnification of the image is determined by the selected objective lens multiple, and the magnified image is imaged by a CCD camera through a first lens, a reflective or transmissive spatial light modulator and a second lens, wherein the first lens and the second lens are the same, and the distances between the diaphragm and the first lens, between the first lens and the spatial light modulator, between the spatial light modulator and the second lens and between the second lens and the CCD camera are the focal length f of the first lens;
(2) the light field modulation pattern design and the realization of the invention need to change the pattern of the spatial light modulator for a plurality of times to form a CCD image sequence, and then a three-dimensional image of an original object is obtained through data reconstruction. The light field modulation pattern is generated as follows:
(2.1) generating a master pattern on a spatial light modulator using the formula:
K(x,y)＝exp{iβ[(x+y)3+(x-y)3]}
in the formula, x and y are plane coordinates of the spatial light modulator, the range of x and y is determined by the number of pixels of the spatial light modulator, β is a modulation coefficient, and the value is (0, 1) interval according to different imaging depths β;
(2.2) covering both side portions of the spatial light modulator pattern with a step grating pattern to form a stripe-shaped center main pattern, as shown in fig. 3; the tail of the Airy beam can be reduced, the main maximum distribution with concentrated energy is obtained, and the point spread function is easier to calculate during data reconstruction. The magnitude of modulation pattern compression in practical applications can be set according to imaging intensity and resolution requirements. The circled portion in fig. 3 is an illumination area.
(2.3) rotating the central strip pattern for 360 degrees at intervals of an integer for one circle, and acquiring an image on a CCD camera to form a projection image sequence for image reconstruction after rotating for one angle; the interval angle is selected according to the effect of reconstructing the three-dimensional image, the number of rotation times which can be divided by 360 is usually selected, and according to experimental experience, the number of rotation interval acquisition times for obtaining a better reconstruction effect is not less than 6, as shown in fig. 4, the result is that the rotation interval acquisition times are rotated by 45 degrees, that is, the number of interval acquisition times of one week is 8.
(3) Image reconstruction
Carrying out data reconstruction on CCD projection images obtained by different modulation images so as to obtain a three-dimensional image of an original object, wherein the reconstruction steps are as follows:
(3.1) performing radon transform on each CCD projection image and defining a uniform coordinate system (u, v, w) according to the formula:
wherein the components (x ', y',z') is the coordinate of each projection plane, theta is the included angle between the projection direction and the principal axis of the optical path,
is the angle of rotation of each projected image;
(3.2) performing inverse Fourier transform on the distribution of each projection in the unified coordinate system to obtain three-dimensional distribution (x) of the image spaceT,yT,zT) The formula according to is:
(3.3) correcting the spatial coordinates of the image to obtain the real coordinate distribution (x, y, z) of the object space
Wherein M is the microscope magnification and is determined by the objective lens; k is 2 pi/lambda, and lambda is the wavelength of imaging light wave; and R is the diameter of the main lobe of the Airy light spot of the system point spread function and is measured by experiments.
Example 2
In this embodiment, the reflective optical path of fig. 1 is used, based on a laser light source with a 647 waveband, a quantum dot with a volume structure and a mouse kidney tube with a thickness of 8 micrometers are respectively imaged, a modulated image is generated by using formula (1), the β value is 0.5, the rotation angle is 30 °, namely, the rotation angle is 12 times in a circle, the modulation pattern in the middle left after shielding in fig. 3-4 accounts for one third of the linear degree of the direction, and a reconstructed image is obtained according to the above image reconstruction algorithm, as shown in fig. 5 and fig. 6, as shown in fig. 7, the resolution in the x and y directions is about 0.5 micrometer and is close to the limit of the conventional optical resolution, and the resolution in the z direction is about 1.5 micrometer and exceeds the limit of the conventional optical resolution.
Claims (2)
1. A non-scanning high super-resolution optical three-dimensional microscopic imaging method is characterized by comprising the following steps:
(1) light path structure
An amplified image formed by a sample on an image surface of an inverted fluorescence microscope system is imaged by a CCD camera through a first lens, a reflective or transmissive spatial light modulator and a second lens, wherein the first lens and the second lens are the same, and the distances between a diaphragm and the first lens, between the first lens and the spatial light modulator, between the spatial light modulator and the second lens and between the second lens and the CCD camera are the focal length f of the first lens;
(2) light field modulation pattern design
(2.1) generating a main pattern on the spatial light modulator using the formula:
K(x,y)＝exp{iβ[(x+y)3+(x-y)3]}
in the formula, x and y are plane coordinates, the range of x and y is determined by the pixel number of the spatial light modulator, β is a modulation coefficient, and the value is a (0, 1) interval according to different imaging depths β;
(2.2) covering both side portions of the spatial light modulator pattern with a step grating pattern to form a stripe-shaped central main pattern;
(2.3) rotating the central strip pattern for 360 degrees at intervals of an integer for one circle, and acquiring an image on a CCD camera to form a projection image sequence for image reconstruction after rotating for one angle;
(3) image reconstruction
Carrying out data reconstruction on CCD projection images obtained by different modulation images so as to obtain a three-dimensional image of an original object, wherein the reconstruction steps are as follows:
(3.1) performing radon transform on each CCD projection image and defining a uniform coordinate system (u, v, w) according to the formula:
wherein, (x ', y ', z ') is the coordinate of each projection plane, theta is the included angle between the projection direction and the principal axis of the optical path,
is the angle of rotation of each projected image;
(3.2) Fourier transform is performed on the distribution of each projection in the unified coordinate system to obtain three-dimensional distribution (x) in the image spaceT,yT,zT) The formula according to is:
(3.3) correcting the spatial coordinates of the image to obtain the real coordinate distribution (x, y, z) of the object space
Wherein M is the magnification of the microscope and is determined by the objective lens; k is 2 pi/lambda, and lambda is the wavelength of imaging light wave; and R is the diameter of the main lobe of the Airy light spot of the system point spread function.
2. The non-scanning high super-resolution optical three-dimensional microscopic imaging method according to claim 1, characterized in that: in step 2.3, the number of the rotation interval acquisition times is not less than 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911064422.4A CN110824681B (en) | 2019-11-04 | 2019-11-04 | Non-scanning high super-resolution optical three-dimensional microscopic imaging method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911064422.4A CN110824681B (en) | 2019-11-04 | 2019-11-04 | Non-scanning high super-resolution optical three-dimensional microscopic imaging method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110824681A true CN110824681A (en) | 2020-02-21 |
| CN110824681B CN110824681B (en) | 2021-10-19 |
Family
ID=69552237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911064422.4A Active CN110824681B (en) | 2019-11-04 | 2019-11-04 | Non-scanning high super-resolution optical three-dimensional microscopic imaging method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110824681B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474699A (en) * | 2020-04-09 | 2020-07-31 | 浙江未来技术研究院(嘉兴) | Operation microscope with programmable aperture |
| CN113647904A (en) * | 2021-07-12 | 2021-11-16 | 中国科学院深圳先进技术研究院 | Rotation control module, method and system |
| CN114967092A (en) * | 2022-06-16 | 2022-08-30 | 华东师范大学 | Ultra-high-speed structured light illumination super-resolution microscopic imaging device based on compressed sensing |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7154598B2 (en) * | 2002-07-12 | 2006-12-26 | Decision Biomarkers, Inc. | Excitation and imaging of fluorescent arrays |
| CN103148800A (en) * | 2013-01-28 | 2013-06-12 | 浙江大学 | Label-free three-dimensional microscope method based on light filed propagation and device |
| CN103413748A (en) * | 2013-08-13 | 2013-11-27 | 中国科学院化学研究所 | Multi-channel optical imaging laser ionization source |
| CN105547144A (en) * | 2015-11-30 | 2016-05-04 | 哈尔滨工业大学 | Super-resolution structure detection array confocal coherent imaging device and imaging method thereof |
| CN109870441A (en) * | 2019-03-27 | 2019-06-11 | 浙江大学 | Three-dimensional super-resolution light slice fluorescent microscopic imaging method and device based on shift frequency |
| CN110389121A (en) * | 2019-07-24 | 2019-10-29 | 中国科学院半导体研究所 | Multifocal Structured Illumination fluoroscopic imaging systems |
-
2019
- 2019-11-04 CN CN201911064422.4A patent/CN110824681B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7154598B2 (en) * | 2002-07-12 | 2006-12-26 | Decision Biomarkers, Inc. | Excitation and imaging of fluorescent arrays |
| CN103148800A (en) * | 2013-01-28 | 2013-06-12 | 浙江大学 | Label-free three-dimensional microscope method based on light filed propagation and device |
| CN103413748A (en) * | 2013-08-13 | 2013-11-27 | 中国科学院化学研究所 | Multi-channel optical imaging laser ionization source |
| CN105547144A (en) * | 2015-11-30 | 2016-05-04 | 哈尔滨工业大学 | Super-resolution structure detection array confocal coherent imaging device and imaging method thereof |
| CN109870441A (en) * | 2019-03-27 | 2019-06-11 | 浙江大学 | Three-dimensional super-resolution light slice fluorescent microscopic imaging method and device based on shift frequency |
| CN110389121A (en) * | 2019-07-24 | 2019-10-29 | 中国科学院半导体研究所 | Multifocal Structured Illumination fluoroscopic imaging systems |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474699A (en) * | 2020-04-09 | 2020-07-31 | 浙江未来技术研究院(嘉兴) | Operation microscope with programmable aperture |
| CN113647904A (en) * | 2021-07-12 | 2021-11-16 | 中国科学院深圳先进技术研究院 | Rotation control module, method and system |
| CN114967092A (en) * | 2022-06-16 | 2022-08-30 | 华东师范大学 | Ultra-high-speed structured light illumination super-resolution microscopic imaging device based on compressed sensing |
| CN114967092B (en) * | 2022-06-16 | 2023-06-23 | 华东师范大学 | Super-high-speed structured light illumination super-resolution microscopic imaging device based on compressed sensing |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110824681B (en) | 2021-10-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110824681B (en) | Non-scanning high super-resolution optical three-dimensional microscopic imaging method | |
| CN108982452B (en) | Multi-focus scanning three-dimensional imaging method and system based on double-spiral point spread function | |
| CN108680544B (en) | Structured illumination light slice fluorescence microscopic imaging method and device | |
| US8019136B2 (en) | Optical sectioning microscopy | |
| US20090219607A1 (en) | Method and apparatus for enhanced resolution microscopy of living biological nanostructures | |
| CN110954521B (en) | Wide-field super-resolution microscopic imaging method and system thereof | |
| CN109712072B (en) | Fringe illumination Fourier domain iterative updating super-resolution microscopic imaging method based on total internal reflection | |
| CN112557359B (en) | Two-photon multi-focus microscopic imaging system and method based on spatial light modulator | |
| CN106885796B (en) | Super-resolution fluorescence digital holographic tomography microscopic imaging system and method | |
| CN113670878B (en) | Super-resolution structured light illumination microscopic imaging method based on space-frequency domain hybrid reconstruction | |
| JP2018055082A (en) | Converter, illuminator, and light sheet fluorescence microscope | |
| WO2010085775A2 (en) | System, method and computer-accessible medium for providing wide-field superresolution microscopy | |
| CN114113019B (en) | Array scanning super-resolution microscopic imaging device, method, equipment and storage medium based on multiple signal classification algorithm | |
| JP5085608B2 (en) | Wide-field super-resolution optical microscope using a spatial light modulator | |
| CN108181235B (en) | STED parallel microscopic imaging system based on uniform structured light illumination | |
| CN116183568B (en) | High-fidelity reconstruction method and device for three-dimensional structured light illumination super-resolution microscopic imaging | |
| CN110954522A (en) | Multifocal super-resolution optical microscopic imaging method and device | |
| Shao et al. | Multifocal multiphoton microscopy based on a spatial light modulator | |
| CN111610150B (en) | Full-field structured light coherence coding tomography device and method | |
| CN111024664B (en) | Imaging method of structured light illumination super-resolution microscopic chip | |
| US11947098B2 (en) | Multi-focal light-sheet structured illumination fluorescence microscopy system | |
| KR102082747B1 (en) | Focal-length adjustable 3D imaging device and method based LED array | |
| Shao et al. | Addressable multiregional and multifocal multiphoton microscopy based on a spatial light modulator | |
| CN108982455A (en) | A kind of multifocal light slice fluorescent microscopic imaging method and device | |
| CN114740008A (en) | Super-resolution wafer defect detection system |
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 |