CN113433681B - Structured light illumination microscopic imaging system and method - Google Patents
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Abstract
The application relates to a structured light illumination microscopic imaging system and a structured light illumination microscopic imaging method, and relates to the technical field of structured light illumination microscopic imaging. In the structured light illumination microscopic imaging system, an illumination system is used for emitting light wave signals to a diffraction optical system. The diffraction optical system is used for modulating the received light wave signals, generating two-dimensional sinusoidal periodic dot matrix light spots, and scanning the target sample by using the two-dimensional sinusoidal periodic dot matrix light spots. And the displacement platform is used for moving the target sample so that the two-dimensional sinusoidal periodic lattice light spot performs phase-shift scanning on the target sample. The microscopic imaging system is used for acquiring a scanning image of the target sample after the displacement table moves the target sample every time, and performing super-resolution reconstruction based on the acquired multiple scanning images. The structured light illumination microscopic imaging system has a relatively simple illumination light path, and only a diffraction optical element needs to be added in the illumination light path, so that the structural complexity of the structured light illumination microscopic imaging system is simplified.
Description
Technical Field
The application relates to the technical field of structured light illumination microscopic imaging, in particular to a structured light illumination microscopic imaging system and a structured light illumination microscopic imaging method.
Background
Structured light microscope (SIM) is a super-resolution technique that uses periodic Structured light patterns to illuminate a sample to improve imaging resolution.
The current SIM system usually needs to generate periodic structured Light to illuminate a sample by interference or projection, and the working process includes many links such as periodic modulation of a Light field, spatial filtering, polarization control, and coupling with a microscope Light path, and each link needs to be completed based on a specific Device, such as a Spatial Light Modulator (SLM), a Digital Micromirror Device (DMD), and the like.
The SIM system is bulky due to its complex structure, and the sizes of the Nikon microscope SIM system currently in commercial use are: 550mm 300mm 350mm, which is not suitable for Point-of-care testing (POCT), so that the SIM system cannot be popularized and applied well.
Disclosure of Invention
In view of the above, it is necessary to provide a structured light illumination microscopy imaging system and method.
The application provides a structured light illumination microscopic imaging system, includes:
an illumination system for emitting a lightwave signal onto the diffractive optical system;
the diffraction optical system is used for modulating the received light wave signals to generate two-dimensional sinusoidal periodic lattice light spots and scanning a target sample by using the two-dimensional sinusoidal periodic lattice light spots;
the displacement platform is used for moving the target sample so as to enable the two-dimensional sinusoidal periodic dot matrix light spot to perform phase-shifting scanning on the target sample;
and the microscopic imaging system is used for acquiring a scanning image of the target sample after the displacement table moves the target sample every time, and performing super-resolution reconstruction based on the acquired multiple scanning images.
In one embodiment, a lighting system includes:
an illumination assembly for emitting a lightwave signal;
and the collimation and beam expansion assembly is used for collimating and expanding the light wave signals so as to adapt the spot size of the light wave signals to the size of a receiving surface of the diffraction optical system.
In one embodiment, the illumination assembly is a laser or LED emitter, but may be other non-coherent light sources.
In one embodiment, a diffractive optical system comprises a diffractive optical element and an illumination objective, wherein:
the diffractive optical element is attached to the entrance pupil position of the illumination objective lens or is positioned at a preset position in front of the entrance pupil position of the illumination objective lens, the aperture size of the diffractive optical element is matched with the entrance pupil size of the illumination objective lens, and the target sample is positioned on the back focal plane of the illumination objective lens;
a diffractive optical element for modulating an optical field distribution of the received lightwave signal;
and the illumination objective lens is used for being matched with the diffractive optical element to generate a two-dimensional sinusoidal periodic lattice light spot on the back focal plane of the illumination objective lens.
In one embodiment, the diffractive optical element is any one or any combination of a binary optical element, a holographic optical element, a micro-nano optical element, a super-structured surface and a spatial light modulator.
In one embodiment, the system further comprises a control system,
and the control system is used for controlling the displacement table to move and controlling the microscopic imaging system to acquire a scanning image of the target sample.
In one embodiment, a microscopic imaging system comprises:
the imaging objective lens is used for imaging a target sample scanned by the two-dimensional sinusoidal periodic dot matrix light spots to obtain an initial image;
the fluorescence color filter is used for filtering the initial image;
a tube lens for performing aberration correction and magnification matching on an initial image;
and the image sensor is used for acquiring the scanning images of the target sample and performing super-resolution reconstruction according to the plurality of scanning images.
In one embodiment, the two-dimensional sinusoidal periodic lattice spot is a lattice spot generated by adding or multiplying two or more one-dimensional sinusoidal distribution patterns in different directions in a plane.
In one embodiment, the mathematical model of the two-dimensional sinusoidal periodic lattice spot is:
or ,
wherein ,
for a two-dimensional structured light pattern light intensity distributionN is the number of the structured light illumination pattern, and n is usually [1,5 ] in two different patterns]And [1,9 ]]Integer of interval->
Is a position vector in the null field>
The spatial frequency phi corresponding to the period of the two-dimensional structured light pattern in the x and y directions xn 、φ yn The phase parameters of the original image of the nth frame in the x direction and the y direction are respectively.
In a second aspect:
the application provides a structured light illumination microscopic imaging method, which comprises the following steps:
generating a two-dimensional sinusoidal periodic lattice light spot by using a diffractive optical element, and imaging the two-dimensional sinusoidal periodic lattice light spot on a target sample of a structured light illumination microscope imaging system;
and performing phase shift scanning on the target sample by using the two-dimensional sinusoidal periodic dot matrix light spot to obtain a plurality of scanning images, and performing super-resolution reconstruction according to the plurality of scanning images.
The structured light illumination microscopic imaging system and method provided by the embodiment of the application have the advantages of simple structure, small size, high speed, good imaging quality and the like. In the structured light illumination microscopic imaging system, an illumination system is used for emitting light wave signals to a diffraction optical system, the diffraction optical system is used for modulating the received light wave signals to generate two-dimensional sinusoidal periodic dot matrix light spots, and the two-dimensional sinusoidal periodic dot matrix light spots are used for scanning a target sample. And the displacement platform is used for moving the target sample so that the two-dimensional sinusoidal periodic lattice light spot performs phase-shift scanning on the target sample. The microscopic imaging system is used for acquiring a scanning image of the target sample after the displacement table moves the target sample every time, and performing super-resolution reconstruction based on the acquired multiple scanning images. The illumination light path of the structured light illumination microscopic imaging system provided by the embodiment of the application is relatively simple, and can be realized only by adding the diffractive optical element in the illumination light path or imaging the diffractive optical element at or near the entrance pupil of the illumination objective lens through the imaging system; the structure complexity of the structured light illumination microscopic imaging system is simplified, and the cost of the structured light illumination microscopic imaging system is reduced.
Drawings
FIG. 1 is a schematic structural diagram of a structured light illumination microscopy imaging system according to an embodiment of the present disclosure;
fig. 2 is a schematic view of an illumination system provided in an embodiment of the present application;
FIG. 3 is a schematic diagram of a diffractive optical system provided in an embodiment of the present application;
FIG. 4 is a schematic diagram of a microscopy imaging system provided by an embodiment of the present application;
FIG. 5 is a schematic diagram of a control system provided in an embodiment of the present application;
FIG. 6 is a basic flow chart of a design of a diffractive optical element provided by an embodiment of the present application;
fig. 7 is a schematic distribution diagram of two-dimensional sinusoidal periodic lattice light spots provided in the embodiment of the present application;
FIG. 8 is an original image of a two-dimensional sinusoidal periodic lattice spot illuminated onto a target sample;
FIG. 9 is a schematic diagram of experimental results of a structured light illuminated microscopy imaging system for imaging microspheres as provided in an example of the present application;
fig. 10 is a schematic diagram of the result of imaging the microtubule of the cell by the structured light illumination microscopic imaging system provided in the embodiment of the present application.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Structured light microscope (SIM) is a super-resolution technique that uses periodic Structured light patterns to illuminate a sample to improve imaging resolution.
Typically, microscope imaging can be considered as a low-pass filtering process for the spatial spectrum of the sample, and the high-frequency spectrum representing the details of the sample is lost during the filtering process, thereby causing the resolution of the sample image to be limited.
In the prior art, a structured light illumination microscope SIM illuminates a sample by using a periodic structured light pattern in a space domain, shifts super-resolution spectrum information which cannot be transmitted by an optical imaging system originally into a system detectable region, and performs detection by aliasing in a space spectrum of an original image. The range of the sample space spectrum can be expanded only by calculating and separating the super-resolution spectrums, so that the resolution of the corresponding space domain image is improved.
In practical applications, since the structured light pattern needs to be generated through the objective lens, the spatial frequency of the structured light pattern is also limited by the diffraction limit, so that the spatial spectrum range (diameter) can be extended by 1 time at most, that is, the imaging resolution is improved by 1 time, and the imaging resolution of the structured light illumination microscope in the visible light band is generally about 100 nm. Because the original images need to be acquired by SIM super-resolution imaging is few, compared with other technologies, the SIM super-resolution imaging has a significant imaging speed advantage, and even though the resolution is inferior to that of Single Molecule positioning super-resolution Microscopy (SMLM) and Stimulated Emission Depletion (STED) microscopes, the SIM still becomes one of several super-resolution technologies widely used today.
At present, the demand for a Point-of-care testing (POCT) application is rapidly growing in the field of modern medical research. In the past, patients usually need to go to a large medical institution to carry out precise medical detection or to be sent to the large medical institution to carry out precise medical detection after sampling the patients, and the appearance of POCT enables the patients to carry out sampling and detection nearby in communities or own homes, so that the interval time from sampling to sample testing is remarkably shortened, and the sample change caused by long-time transportation or environmental change is avoided, and the detection result is influenced. The characteristics of real-time detection, anytime and anywhere, and wide use determine that the used detection equipment has the advantages of compact volume, portability, simple and flexible use and low cost.
SIM, one of the most suitable techniques for in vivo imaging in super-resolution technology, is bound to develop miniaturization research to meet the application requirements of POCT in the future. However, the current SIM is a set of microscope systems with large volume and high price, and is far from meeting the requirements of POCT application for portability and cost reduction of the device. The fundamental reason is that the current SIM usually needs to generate periodic structured Light to illuminate a sample through an interference or projection method, and the system has many links such as periodic modulation, spatial filtering, polarization control of a Light field, and coupling with a microscope Light path, so that the SIM system is bulky (the size of the current commercial Nikon microscope SIM system is 550mm × 300mm × 350 mm), and Spatial Light Modulators (SLM) and Digital micro-mirror devices (DMD) used therein are also expensive, which brings many obstacles to further popularization and application of the SIM.
The embodiment of the application provides a structured light illumination super-resolution microscopic imaging system based on a diffractive optical element 1021, which can reduce the volume of an SIM (subscriber identity module) and reduce the cost of the SIM.
Referring to fig. 1, fig. 1 is a schematic structural diagram of a structured light illumination microscopy imaging system according to an embodiment of the present disclosure, where the structured light illumination microscopy imaging system includes an illumination system 101, a diffractive optical system 102, a displacement stage (not shown in the figure), and a microscopy imaging system 103.
The illumination system 101 is used to emit a light wave signal to the diffractive optical system 102. Optionally, in the embodiment of the present application, the light wave signal may be a collimated wave, a spherical wave, a gaussian beam, or an illumination light wave with any distribution.
The diffractive optical system 102 is configured to modulate the received light wave signal to generate a two-dimensional sinusoidal periodic lattice light spot, and scan the target sample with the two-dimensional sinusoidal periodic lattice light spot.
And the displacement platform is used for moving the target sample so that the two-dimensional sinusoidal periodic lattice light spot performs phase-shift scanning on the target sample.
The microscopic imaging system 103 is configured to obtain a scanning image of the target sample after the displacement stage moves the target sample each time, and perform super-resolution reconstruction based on the obtained multiple scanning images.
Optionally, in the structured light illumination microscopic imaging system provided by the application, due to feedback control of the displacement table itself, after each step of phase-shifting scanning is performed, the displacement table needs to be waited for a certain stabilization time, and generally, after each displacement is finished, shooting is started after the displacement table needs to be waited for stabilization. In practical use, in order to simplify the control flow, the trigger signal of the displacement table can be used to directly control the image sensor to shoot, and the shooting of the sample image can be started immediately after the movement of the displacement table is completed.
It can be understood that, in the embodiment of the present invention, the illumination system 101 is used to emit a light wave signal to the diffractive optical system 102, the diffractive optical system 102 modulates the light wave signal, generates a two-dimensional sinusoidal periodic lattice spot in a spatial domain as a structured light, illuminates a target sample with the structured light (the two-dimensional sinusoidal periodic lattice spot), and moves the target sample through the displacement stage to implement phase shift detection and reconstruct super-resolution information. The microscopic imaging system 103 is used for recording sample information of the target sample at different positions and performing super-resolution reconstruction. The method is characterized in that a diffraction optical system 102 is utilized to generate two-dimensional sinusoidal periodic lattice (array) light spots as structured light for illumination, and the two-dimensional sinusoidal periodic lattice (array) light spots are matched with a common computer for data processing, so that the method can be realized, has the advantages of simple structure, small volume, high speed, good imaging quality and the like, and is suitable for the application requirements of medical science and biology for instant detection.
The structured light illumination microscopic imaging system provided by the embodiment of the application combines the advantages of super-resolution of the structured light illumination microscope and the advantage of miniaturization of the diffraction optical element, has the advantages of simple structure, small volume, high speed, good imaging quality and the like, and provides possibility for application requirements such as instant detection and the like; furthermore, the target sample is illuminated by the two-dimensional sinusoidal periodic dot matrix light spots by using a diffraction method based on a diffraction optical system, so that the imaging efficiency and the energy utilization rate are improved; the illumination light path of the structured light illumination microscopic imaging system provided by the embodiment of the application is relatively simple, and can be realized only by adding the diffractive optical element in the illumination light path or imaging the diffractive optical element at or near the entrance pupil of the illumination objective lens through the imaging system; the structure complexity of the structured light illumination microscopic imaging system is simplified, and the cost of the structured light illumination microscopic imaging system is reduced.
In an embodiment, the present application further provides a structured light illumination microscopic imaging method, which is applied to the structured light illumination microscopic imaging system provided in the present application, and the method includes generating a two-dimensional sinusoidal periodic lattice spot by using a diffractive optical element, where the two-dimensional sinusoidal periodic lattice spot is imaged on a target sample of the structured light illumination microscopic imaging system; and performing phase-shift scanning on the target sample by using the two-dimensional sinusoidal periodic dot matrix light spot to obtain a plurality of scanning images, and performing super-resolution reconstruction according to the plurality of scanning images.
The light wave signal sequentially passes through the illumination system and the diffraction optical system, and a two-dimensional sinusoidal periodic lattice light spot is generated on a back focal plane of an illumination objective lens of the diffraction optical system, namely a sample plane generates a two-dimensional sinusoidal periodic lattice light spot to illuminate a target sample. Performing multi-step phase-shifting detection on a target sample by using a displacement table; after the information of the target sample sequentially passes through the imaging objective lens, the fluorescent color filter, the tube lens and the image sensor, the scanning images of the target sample at different positions are processed by the image sensor, and a super-resolution image is reconstructed.
In one embodiment, as shown in FIG. 2, the illumination system 101 includes an illumination assembly 1011 and a collimated beam expanding assembly 1012.
Wherein the illumination assembly 1011 is configured to emit the lightwave signal. In the embodiment of the present application, the light wave signal may be a monochromatic laser signal, an LED light signal, or an incoherent light signal, and the light source may be a laser, an LED illuminator, or other incoherent light sources.
The collimating and beam expanding assembly 1012 is used to collimate and expand the light wave signal to adapt the spot size of the light wave signal to the size of the receiving surface of the diffractive optical system 102.
The collimating and beam expanding assembly 1012 comprises a collimator and a beam expander, wherein the collimator is used for converting light waves emitted by the light source into collimated light beams, and the beam expander is used for expanding light wave signals and enlarging the aperture of the light source. And uniform light waves with preset sizes can be obtained through collimation and beam expansion. The predetermined size is the size of the receiving surface of the diffractive optical system 102.
The adaptation of the spot size of the collimated and expanded light wave signal to the size of the receiving surface of the diffractive optical system 102 may mean that the spot size of the collimated and expanded light wave signal is equal to the size of the receiving surface of the diffractive optical system 102. The light spots, which may also refer to the collimated and expanded light wave signals, can all be directed onto the receiving surface of the diffractive optical system 102.
The resolution of the microscopic imaging system can be improved by maximally injecting the lightwave signal into the diffractive optical system 102 by adapting the spot size of the lightwave signal to the size of the receiving surface of the diffractive optical system 102.
In one embodiment, as shown in FIG. 3, the Diffractive Optical system 102 includes a Diffractive Optical Element 1021 (DOE for short) and an illumination objective 1022, wherein: the diffractive optical element 1021 may be attached to the entrance pupil position of the illumination objective 1022, or may be located at a predetermined position in front of the entrance pupil position of the illumination objective 1022, where the predetermined position is a position where the distance to the entrance pupil surface of the illumination objective 1022 is a predetermined distance. And the aperture size of the diffractive optical element 1021 is adapted to the entrance pupil size of the illumination objective 1022, and the target sample is located on the back focal plane of the illumination objective 1022.
In practical applications, if the aperture size of the diffractive optical element 1021 is larger than the entrance pupil size of the illumination objective 1022, the formed two-dimensional sinusoidal periodic lattice spot is deformed, and the super-resolution reconstruction effect is affected. If the aperture size of the diffractive optical element 1021 is smaller than the entrance pupil size of the illumination objective 1022, the resolution of the microscopic imaging system will be reduced. The aperture size of the diffractive optical element 1021 is equal to the entrance pupil size of the illumination objective 1022, so that the best resolution effect can be achieved.
The diffractive optical element 1021 is used to modulate the optical field distribution of the received lightwave signal.
The illumination objective 1022 is used in conjunction with the diffractive optical element 1021 to generate a two-dimensional sinusoidal periodic lattice spot on the back focal plane of the illumination objective 1022.
Optionally, the diffractive optical element 1021 may be a binary optical element, or may be one or a combination of various elements that implement optical field phase modulation and/or amplitude modulation, such as a holographic optical element, a micro-nano optical element, a super-structured surface (english: metassurface), a spatial light modulator, and the like.
Optionally, in the embodiment of the present application, the diffractive optical element 1021 may be used to adjust the period, number, and other elements of the two-dimensional sinusoidal periodic lattice light spot on the back focal plane of the illumination objective 1022.
Alternatively, the two-dimensional sinusoidal periodic lattice spot may be a lattice spot generated by adding or multiplying two or more one-dimensional sinusoidal distribution patterns in different directions in a plane.
It should be noted that the collimation and beam expansion assembly is used to collimate and expand the lightwave signal so as to adapt the spot size of the lightwave signal to the size of the receiving surface of the diffractive optical system 102, which is essential to adapt the spot size of the lightwave signal to the size of the receiving surface of the diffractive optical element 1021.
It will be appreciated that the diffractive optical element 1021 may modulate the optical field distribution of the collimated and expanded lightwave signals such that a specific pattern of spots, i.e., a two-dimensional sinusoidal periodic lattice of spots, is formed in the spatial domain after modulation. The illumination objective 1022 is used for cooperating with the diffractive optical element 1021 to present the two-dimensional sinusoidal periodic lattice spot on the back focal plane of the illumination objective 1022. The target sample is located on the back focal plane of the illumination objective 1022, so that the two-dimensional sinusoidal periodic lattice light spot can be exactly irradiated on the target sample, and the purpose of scanning the target sample is achieved.
In the embodiment of the application, the diffraction optical system 102 is used for generating the two-dimensional sinusoidal periodic dot matrix light spot, and the two-dimensional sinusoidal periodic dot matrix light spot is used for scanning the target sample, so that more information on the target sample can be acquired.
In one embodiment, as shown in fig. 4, microscopic imaging system 103 includes imaging objective 1031, fluorescence filter 1032, tube lens 1033, and image sensor 1034, wherein:
the imaging objective lens 1031 is used for imaging the target sample scanned by the two-dimensional sinusoidal periodic dot matrix light spot to obtain an initial image. The fluorescence filter 1032 is used to filter the original image. The tube lens 1033 is used for aberration correction and magnification matching of the initial image. The image sensor 1034 is configured to acquire scan images of a target sample and perform super-resolution reconstruction according to the plurality of scan images.
Optionally, in this embodiment of the application, the image sensor 1034 includes a data processing module, and the data processing module may process the scan images of the target sample at different positions to reconstruct a super-resolution image.
As can be understood, the imaging objective 1031 images the target sample illuminated by the two-dimensional sinusoidal periodic lattice light spots; the fluorescence filter 1032 can improve the signal-to-noise ratio of the microscopic imaging system, enhance the fluorescence signal of the excitation wave band and minimize unnecessary radiation; the tube lens 1033 is used for matching and correcting magnification and aberration of the microscopic imaging system; the image sensor 1034 is used to obtain information of the target sample under the illumination of the two-dimensional sinusoidal periodic lattice light spots.
Optionally, the image sensor may be various devices for realizing image information acquisition, such as a CMOS (Complementary Metal Oxide Semiconductor transistor, chinese), an sCMOS (Complementary Metal Oxide Semiconductor transistor, chinese), a CCD (Charge-coupled Device, chinese), and an EMCCD (Electron-Multiplying Charge-coupled Device, chinese).
In one embodiment, as shown in fig. 5, the structured light illumination microscopy imaging system provided in the embodiment of the present application further includes a control system 105, where the control system 105 is configured to control the displacement stage 104 to move and control the microscopy imaging system 103 to acquire a scanning image of the target sample.
Optionally, the control system may be further connected to the illumination system 101 and the diffractive optical system 102 to control the illumination system 101 and the diffractive optical system 102 to start, where after the illumination system 101 and the diffractive optical system 102 start, the illumination system 101 emits a light wave signal to the diffractive optical element, the diffractive optical element modulates the light wave signal to form a two-dimensional sinusoidal periodic lattice spot on a back focal plane of the illumination objective lens, and meanwhile, the control system controls the displacement stage 104 to move, and after each movement, controls the microscopic imaging system 103 to acquire a scanning image of the target sample, so as to acquire a plurality of scanning images, and then performs super-resolution reconstruction based on the plurality of scanning images.
It can be understood that, as can be understood from the principle of the structured light illumination microscopy, the spatial frequency of the two-dimensional structured light illumination fringes is a main factor determining the resolution improvement of the structured light illumination microscopy imaging system. As long as the two-dimensional structured light pattern meeting the requirements is adopted for sample illumination, the structured light illumination microscope can realize corresponding linear/nonlinear super-resolution imaging. Accurate generation of the desired two-dimensional structured light illumination pattern is therefore critical for super-resolution microscopy imaging.
In the structured light illumination microscopic imaging system provided by the embodiment of the application, the two-dimensional structured light illumination pattern is a two-dimensional sine periodic dot matrix light spot. The following explains the process of designing a two-dimensional sinusoidal periodic dot matrix light spot according to the present application:
in the embodiment of the application, the mathematical model of the two-dimensional sinusoidal periodic lattice light spot includes the following two types, which are respectively:
and ,
wherein ,
is the light intensity distribution of two-dimensional sinusoidal periodic lattice light spots, n is the serial number of the two-dimensional sinusoidal periodic lattice light spots, and n is usually [1,5 ] under two different patterns]And [1,9 ]]Integer of interval->
Is a position vector in the null field>
Respectively is the space frequency phi corresponding to the periods of the two-dimensional sinusoidal periodic lattice faculae in the x direction and the y direction xn 、φ yn The phase parameters of the n frame original image in the x and y directions are respectively.
wherein ,
are respectively based on>
The spatial frequency spectrum of the two-dimensional sinusoidal periodic lattice light spot, device for selecting or keeping>
δ is the dirac impulse function for the frequency vector in the spatial frequency domain. />
Under plane wave incidence, the transmittance function of the diffractive optical element DOE is in fourier transform relation with the fraunhofer diffraction field of the objective focal plane (i.e. the sample plane). At this time, how to design the phase distribution of the DOE so that the light intensity distribution of the diffraction field is a two-dimensional sinusoidal periodic lattice spot with a certain spatial frequency is a core problem. For a given target diffraction field distribution, the phase of the DOE can be designed using an Iterative Fourier Transform Algorithm (IFTA). As shown in fig. 6, the main steps include:
1. the initial equal amplitude phase field is imparted to the DOE plane:
where i is the number of iterations,
for the amplitude distribution of the light field in the DOE plane,. Sup.>
Is the phase distribution of the optical field in the plane of the DOE.
2. Calculating the distribution of the plane diffraction field of the sample by Fourier transform:
wherein τ(i) (x) Is the amplitude distribution of the back focal plane (i.e. sample plane) of the objective lens,. Phi (i) (x) Is a phase distribution.
3. In the sample plane, the amplitude distribution of the target diffraction field (two-dimensional structured light illumination pattern) is used to replace the amplitude distribution of the sample plane, the phase distribution is kept unchanged, and a new light field is obtained:
4. calculating DOE planar optical field distribution through inverse Fourier transform:
5. distribution of optical field amplitude of DOE plane
Reset to a uniform amplitude distribution.
And (5) iteratively repeating the steps 2-5. Optical field amplitude distribution at DOE plane
Will converge to an approximately uniform distribution and the amplitude distribution tau of the sample plane light field (i) (x) The amplitude distribution of the target diffraction field (two-dimensional structured light illumination pattern) is converged. The phase profile of the DOE plane then->
Namely, the required two-dimensional sinusoidal periodic lattice light spot can be generated through diffraction according to the DOE phase required to be designed.
The performance of the structured light illuminated microscopy imaging system provided herein is illustrated below with reference to examples.
In the embodiment of the application, 488nm laser can be adopted to generate collimated light with a wide aperture through the collimation beam expanding assembly, the collimated light is modulated by a diffractive optical element DOE arranged at the entrance pupil of the illumination objective or nearby, the illumination objective is incident, and a designed two-dimensional structured light illumination pattern, namely a two-dimensional sine periodic dot matrix light spot, is generated on the back focal plane of the illumination objective.
The diffractive optical element DOE and the two-dimensional sinusoidal periodic lattice light spot generated by diffraction of the diffractive optical element DOE are shown in fig. 7. In fig. 7, (a) shows a phase distribution of the diffractive optical element, (b) shows a light intensity distribution of a two-dimensional sinusoidal periodic lattice spot, (c) and (b) show horizontal scribe line position light intensity curves, and (d) and (b) show vertical scribe line position light intensity curves.
After the target sample is subjected to two-dimensional sinusoidal periodic lattice spot illumination, a corresponding image is imaged by the imaging objective lens and collected by the image sensor, as shown in fig. 8. And performing phase-shift scanning on the target sample by using a displacement table, and shooting original images of the target sample at different positions. Through such a mode, the structured light illumination microscopic imaging system provided by the embodiment of the application has two main elements that the SIM meets the requirements of super-resolution imaging: and illuminating the two-dimensional sinusoidal periodic dot matrix light spot of the target sample and detecting the phase shift of the two-dimensional sinusoidal periodic dot matrix light spot.
In the embodiment of the present application, according to the difference of the illumination objective lens and the design of the diffractive optical element, the generated two-dimensional sinusoidal periodic lattice light spot is correspondingly changed, and the maximum spatial frequency is about 2 times of the spatial frequency corresponding to the diffraction limit resolution determined by the illumination objective lens 1022. The phase-shifting scanning step pitch of the displacement table is set to be about one third of the period of the illumination pattern, so that relatively uniform sampling can be realized in one period of two or more directions of the two-dimensional illumination pattern by phase-shifting scanning, and the reconstruction result can be ensured to obtain a uniform resolution improvement effect in the whole field of view. Further iterative reconstruction can improve the quality of super-resolution microscopic imaging of the sample.
As shown in fig. 9, it is the result of imaging the fluorescent microsphere by the structured light illumination super-resolution microscopic imaging system based on the diffractive optical element. FIG. 9 (a) shows the results of microsphere imaging under wide field illumination. (b) And the super-resolution result under the illumination of two-dimensional sinusoidal periodic lattice light spots generated by the modulation of the diffractive optical element is shown. (c) Showing the intensity curves of the wide field image and the super-resolution image at the selected positions of the scribe lines. It can be seen from fig. 9 that the super-resolution image has a significant improvement in resolution compared to the wide-field image. Wherein the locally magnified images of (a) and (b) show that adjacent microspheres that were not resolved in the original wide-field image are already resolved in the structured-light illuminated super-resolution microscopy imaging system image. And (c) analyzing the light intensity curve at the scribing position, wherein the center-to-center distance between the two microspheres is 567nm after fitting, and the theoretical diffraction limit resolution is 990nm.
Furthermore, in order to verify the biological imaging capability of the structured light illumination microscopic imaging system provided by the embodiment of the application, an imaging experiment is carried out on a fixed cell sample of the A549 cell, wherein the A549 cell adenocarcinoma human alveolar basal epithelial cell is a sample commonly used for researching a II type lung epithelial cell model in drug metabolism. The results of the experiment in which the cell microtubules were labeled with a dye are shown in FIG. 10. In fig. 10, (a) shows the wide-field results, local magnification results (lower right), and spatial frequency spectra of wide-field imaging of cell microtubules (upper right). (b) The spatial spectrum (upper right) representing the super-resolution, local magnification (lower right) and super-resolution imaging of the cell microtubules under illumination by the two-dimensional sinusoidal periodic lattice generated by the modulation of the diffractive optical element 1021. (c) Showing the intensity curves of the wide field image and the super-resolution image at the selected positions of the scribe lines. The comparison between the cell microtubule wide-field image and the super-resolution image also shows a remarkable resolution improvement effect, and a large amount of evidence of resolution improvement can be found in a field of view, as shown in (a) and (b), in a locally amplified region, cell microtubules observed in the wide-field image are blurred, the microtubule structure cannot be distinguished, and the super-resolution image shows a clear microtubule structure. In addition, the spatial spectrum of the super-resolution image is significantly extended compared to the wide-field image, which can also prove that the imaging resolution of the system has exceeded the diffraction limit. And (c) selecting a light intensity curve of the scribing position to the wide field image and the super-resolution image, wherein in the wide field image, two adjacent cell microtubules cannot be resolved, and in the super-resolution image, the two adjacent cell microtubules can be successfully resolved. Through data fitting, the distance between the centers of the two microtubes is about 419nm, which is smaller than the diffraction limit 625nm corresponding to the fluorescence wavelength, and super-resolution imaging is realized.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent application shall be subject to the appended claims.
Claims (9)
1. A structured light illuminated microscopy imaging system, comprising:
an illumination system for emitting a lightwave signal onto the diffractive optical system;
the diffraction optical system is used for modulating the received light wave signal to generate a two-dimensional sinusoidal periodic lattice light spot and scanning a target sample by using the two-dimensional sinusoidal periodic lattice light spot;
the diffraction optical system comprises a diffraction optical element and an illumination objective lens, wherein the diffraction optical element is used for modulating the light field distribution of the received light wave signal and adjusting the period and the number of the two-dimensional sinusoidal periodic lattice light spots on the back focal plane of the illumination objective lens; the illumination objective lens is used for being matched with the diffraction optical element to generate the two-dimensional sine periodic dot matrix light spot on the back focal plane of the illumination objective lens;
the displacement platform is used for moving the target sample so that the two-dimensional sinusoidal periodic lattice light spot performs phase-shifting scanning on the target sample;
the microscopic imaging system is used for acquiring a scanning image of the target sample after the displacement table moves the target sample every time and performing super-resolution reconstruction on the basis of the acquired multiple scanning images;
the two-dimensional sinusoidal periodic lattice light spots are lattice light spots generated by adding or multiplying two or more one-dimensional sinusoidal distribution patterns in different directions in a plane.
2. The structured light illuminated microscopy imaging system of claim 1, wherein the illumination system comprises:
an illumination assembly for emitting the lightwave signal;
and the collimation and beam expansion assembly is used for collimating and expanding the light wave signals so as to adapt the spot size of the light wave signals to the size of a receiving surface of the diffraction optical system.
3. The structured light illuminated microscopy imaging system of claim 2, wherein the illumination assembly is a laser or an LED illuminator.
4. The structured light illuminated microscopy imaging system as claimed in claim 1, wherein the diffractive optical element is attached to or located at a predetermined position in front of the entrance pupil position of the illumination objective, and the aperture size of the diffractive optical element is adapted to the entrance pupil size of the illumination objective, and the target sample is located on the back focal plane of the illumination objective.
5. The structured light illuminated microscopy imaging system of claim 1, wherein the diffractive optical element is any one or any combination of a binary optical element, a holographic optical element, a micro-nano optical element, a super-structured surface and a spatial light modulator.
6. The structured light illuminated microscopy imaging system of claim 1, wherein the system further comprises a control system,
and the control system is used for controlling the displacement table to move and controlling the microscopic imaging system to acquire the scanning image of the target sample.
7. The structured light illuminated microscopy imaging system of claim 1, wherein the microscopy imaging system comprises:
the imaging objective lens is used for imaging the target sample scanned by the two-dimensional sinusoidal periodic dot matrix light spots to obtain an initial image;
the fluorescence color filter is used for filtering the initial image;
a tube lens for performing aberration correction and magnification matching on the initial image;
and the image sensor is used for acquiring the scanning images of the target sample and performing super-resolution reconstruction according to the plurality of scanning images.
8. The structured light illuminated microscopy imaging system of claim 1, wherein the mathematical model of the two-dimensional sinusoidal periodic lattice spot is:
or ,
wherein ,
for a two-dimensional structured light pattern intensity distribution, n is the number of the structured light illumination pattern, and n is usually [1,9 ] in two different patterns]The integer number of the interval (a) is,
for the position vector in the null field,
the spatial frequency phi corresponding to the period of the two-dimensional structured light pattern in the x and y directions xn 、φ yn The phase parameters of the n frame original image in the x and y directions are respectively.
9. A structured light illuminated microscopy imaging method applied to a structured light illuminated microscopy imaging system according to any one of claims 1 to 8, the method comprising:
generating a two-dimensional sinusoidal periodic lattice light spot by using a diffractive optical element, wherein the two-dimensional sinusoidal periodic lattice light spot is imaged on a target sample of a structured light illumination microscope imaging system;
and performing phase-shift scanning on a target sample by using the two-dimensional sinusoidal periodic dot matrix light spot to obtain a plurality of scanning images, and performing super-resolution reconstruction according to the plurality of scanning images.
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