Equal-wavelength periodic grating longitudinal depth structured illumination generator for microscopic imaging

Abstract

The present invention discloses an equal-wavelength periodic grating longitudinal depth structured light illumination generator for microscopic imaging, which can be used for structured light illumination microscopic imaging. The structured light illumination generator includes a glass substrate and an equal-wavelength periodic metal grating. The metal grating is used to be directly irradiated by incident light, and on both sides of the slit of the grating, structured light propagating perpendicular to the surface of the grating appears respectively. This structured light propagates linearly upward along the direction perpendicular to the surface of the grating, and its intensity gradually decreases during the propagation process. Moreover, the generated structured light is a row of parallel stripes, and the lateral period of the stripes is half of the grating period. The present invention uses an equal-wavelength periodic grating to excite periodic structured light, which can be used for structured light illumination microscopic imaging. By performing image reconstruction processing on the results of multiple detections using structured light illumination, super-resolution imaging beyond the diffraction limit can be achieved. The structured light of the present invention has the characteristics of high spatial frequency and stable propagation over a long distance, improving the resolution and detection depth of structured light illumination microscopic imaging in fields such as biomedicine.

Description

Technical Field

[0001] The present invention belongs to the fields of micro-nano optics and super-resolution microscopy, and in particular, to an isowavelength periodic grating longitudinal depth structured illumination generator for microscopy imaging. Background Art

[0002] Structured illumination microscopy imaging technology uses structured light beams of different modes to illuminate a sample and acquires multiple images. By reconstructing these acquired images, a super-resolution image of the target can be obtained. A grating is a commonly used tool for generating structured light, and various different modes of structured light can be generated by utilizing the diffraction and interference characteristics of the grating. In 2000, Gustafsson first verified the super-resolution result of structured illumination microscopy imaging technology in an experiment, and the sinusoidal fringe structured light used in the experiment was generated based on a grating. In this experiment, a linear transmission phase grating was irradiated by linearly polarized light emitted by a multimode fiber, and the +1 and -1 order diffracted lights generated interfered to form a sinusoidal fringe structured light. In 2008, Gustafsson used a multimode fiber to excite linearly polarized light to irradiate a grating, and utilized the interference of the 0 order and ±1 order diffracted lights generated to form three-dimensional structured light, and for the first time realized three-dimensional structured illumination microscopy imaging technology. In addition, a structured illumination microscopy imaging technology based on the Talbot effect of a grating has also been proposed. In the Talbot effect, the diffracted lights of each order excited by the grating interfere with each other, generating a periodic structured light field in the far field. However, whether or not a grating is used to excite structured light, the resolution of conventional structured illumination microscopy imaging technology can at most reach 2 times the diffraction limit, and higher spatial frequency structured light needs to be generated to further improve the resolution.

[0003] Plasmonic structured illumination microscopy imaging technology is a microscopy imaging technology that uses surface plasmons as structured light illumination. Since surface plasmons have the characteristics of sub-wavelength and can generate a structured light distribution pattern exceeding the diffraction limit, plasmonic structured illumination microscopy imaging technology has a higher resolution ability than conventional SIM. A metal grating is an efficient tool for exciting surface plasmons. In 2012, a plasmonic structured illumination microscopy imaging method using a metal grating to excite surface plasmons as structured light was proposed. This method excites surface plasmons through a grating and makes these surface plasmons interfere with each other to form a standing wave fringe structured light. The period of this standing wave fringe structured light is only half of the wavelength of the surface plasmon, and using it for illumination can improve the imaging resolution to more than 3 times the diffraction limit. Plasmonic structured illumination microscopy imaging technology can obtain a higher resolution than conventional structured illumination microscopy imaging technology, but since the surface plasmon structured light is confined to the metal surface, its detection depth is often only one or two hundred nanometers, and it cannot detect and image samples located at a certain depth (such as cell organelles).

[0004] While maintaining the resolution of plasmonic structured illumination microscopy, improving its detection depth is a problem worthy of study. Utilizing the plasmonic Talbot effect is a potential method to increase the detection depth. Different from the Talbot effect of light beams, which is based on the interference of diffracted light, the plasmonic Talbot effect is formed by the interference of scattered electromagnetic waves with the same wavelength as surface plasmons, and can generate an interference light field on the transmission surface of the metal structure. The plasmonic Talbot effect provides an idea for solving the problem of the detection depth of plasmonic structured illumination, that is, generating surface plasmons through a metal grating and generating an interference structured light field affected by surface plasmons on the transmission surface. Summary of the Invention

[0005] The object of the present invention is to provide a structured illuminator capable of generating high spatial frequency and long longitudinal depth for the problem of limited detection distance in plasmonic structured illumination microscopy.

[0006] The technical solution for achieving the object of the present invention is: an equal-wavelength periodic grating longitudinal depth structured illumination generator for microscopy imaging, the structured illumination generator includes a glass substrate and a metal grating deposited on the glass substrate;

[0007] A beam of incident light irradiates the bottom of the metal grating from bottom to top and generates structured light with high spatial frequency and longitudinal depth. The period Λ of the metal grating satisfies Λ = λ, where λ is the wavelength of the incident light;

[0008] The incident light is a linearly polarized plane wave, perpendicularly incident on the metal grating, and the polarization direction is perpendicular to the slit of the metal grating.

[0009] Further, the lateral fringe distribution period of the structured light is half-wavelength size; the structured light is used for illumination detection imaging to achieve super-resolution detection imaging of a target sample at a certain depth.

[0010] Further, during the super-resolution detection imaging process, the target sample to be detected is placed on a glass slide, the glass slide is placed parallel to the upper surface of the metal grating, and the distance from the glass slide to the metal grating is within the longitudinal propagation range of the structured light.

[0011] Further, the target sample to be detected is treated with a fluorescent dye, and the excitation wavelength of the fluorescent dye corresponds to the incident light wavelength λ.

[0012] Further, during the super-resolution detection imaging process, it is necessary to use structured light for illumination in multiple directions to obtain super-resolution images in each direction.

[0013] Further, in each direction, the structured light needs to be laterally displaced to collect multiple sets of images illuminated and detected by structured light with different phases. Based on these images, the signals detected by different frequency components in the structured light can be solved.

[0014] Further, in the Fourier space, the signals detected by different frequency components are moved to their corresponding positions and an inverse Fourier transform is performed to obtain a reconstructed super-resolution image of the target sample.

[0015] Compared with the prior art, the significant advantages of the present invention are as follows:

[0016] 1) The lateral period of the structured light generated by the structured light illuminator of the present invention is half of the wavelength of the incident light, that is, the structure has a spatial frequency twice that of the conventional sinusoidal structured light, can extract more high-frequency information of the target, and can obtain an image with higher resolution.

[0017] 2) Compared with the existing plasma structured light illuminator, the structured light generated by the structured light illuminator of the present invention has a longer propagation distance and can detect targets with a certain depth.

[0018] 3) During the propagation process, the structured light does not diverge as the propagation distance increases and can maintain a stable structured light illumination mode at a long detection depth.

[0019] The present invention will be further described in detail below with reference to the accompanying drawings. Description of the Drawings

[0020] Figure 1 It is a schematic structural diagram of an equal-wavelength periodic grating longitudinal depth structured light illumination generator of the present invention.

[0021] FIG. 2(a) is an intensity distribution diagram of the longitudinal depth fringe structured light field in the x-z plane generated by an equal-wavelength periodic gold grating in an embodiment, and FIGS. 2(b), 2(c) and 2(d) are the electric field components of the structured light in the x, y and z directions respectively.

[0022] Figure 3 It is a relationship diagram between the intensity of the longitudinal depth fringe structured light generated in an embodiment and the longitudinal propagation distance.

[0023] FIG. 4(a) is an electric field intensity distribution diagram at a distance of 5 micrometers from the surface of the gold grating in an embodiment, FIG. 4(b) is a transverse electric field intensity distribution curve corresponding to the electric field intensity distribution, and FIG. 4(c) is the Fourier spectrum corresponding to the electric field intensity distribution.

[0024] Figure 5 It is a schematic diagram of the change of the optical transfer function after the longitudinal depth structured light generated by an equal-wavelength periodic grating in an embodiment is used for super-resolution imaging.

[0025] Figure 6 The simulated quantum dot detection imaging results in an embodiment, where Figure 6 (a) in is the detection result of a diffraction-limited microscope, Figure 6 (b) in is the result diagram of the reconstruction after the longitudinal depth fringe structured light generated by the equal-wavelength periodic grating of the present invention is used for illumination imaging. Detailed implementation manners

[0026] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0027] It should be noted that if there are directional indications (such as up, down, left, right, front, back...) involved in the embodiments of the present invention, the directional indications are only used to explain the relative positional relationship and movement conditions between components in a specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indications will also change accordingly.

[0028] The plasmonic Talbot effect is a self-imaging phenomenon of surface plasmon polaritons, which can generate a structured light field with a certain propagation distance on the transmission surface. When studying this effect, it is found that when the period Λ of the metal grating satisfies Λ = λ, a structured light with a period of half of the incident light wavelength and a stable propagation distance that can reach the order of ten micrometers can be generated. Based on this phenomenon, the present invention proposes an equal-wavelength periodic grating longitudinal depth structured light illumination generator. The lateral distribution characteristics of the structured light generated by using this illumination generator are similar to those of the structured light used in plasmonic structured light microscopy imaging technology. Although the present invention does not use plasmonic structured light for illumination, the resolution ability close to that of plasmonic structured light microscopy imaging technology can be obtained by using this structured light illumination generator. In addition, the structured light generated by the present invention has a certain longitudinal propagation depth and can be used for detecting and imaging samples at a certain depth.

[0029] Specifically, in an embodiment, the present invention provides an equal-wavelength periodic grating longitudinal depth structured light illumination generator for microscopy imaging. The structured light illumination generator includes a glass substrate and a metal grating deposited on the glass substrate;

[0030] A beam of incident light irradiates the bottom of the metal grating from bottom to top and generates a structured light with high spatial frequency and longitudinal depth. The period Λ of the metal grating satisfies Λ = λ; where λ is the wavelength of the incident light;

[0031] The incident light is a linearly polarized plane wave, and the polarization direction is perpendicular to the slit of the metal grating.

[0032] Further, in one embodiment, the incident light is perpendicularly incident on the metal grating.

[0033] Further, in one embodiment, the lateral stripe distribution period of the structured light is half the wavelength. That is, the lateral stripe distribution of the structured light has a period of half the wavelength of the incident light and can maintain a stable illumination intensity during longitudinal propagation.

[0034] Further, in one embodiment, the structured light is used for illumination detection imaging to achieve super-resolution detection imaging of a target sample at a certain depth.

[0035] Here, during the super-resolution detection imaging process, the target sample to be detected is placed on a glass slide, and the glass slide is placed parallel to the upper surface of the metal grating, and the distance from the glass slide to the metal grating is within the longitudinal propagation range of the structured light.

[0036] The target sample to be detected is treated with a fluorescent dye, and the excitation wavelength of the fluorescent dye corresponds to the incident light wavelength λ.

[0037] During the super-resolution detection imaging process, the structured light needs to be used for illumination in multiple directions to obtain super-resolution images in each direction. For example, illuminating in 3 directions, and the illumination direction needs to be switched by 60° after each illumination; finally, illumination is performed in three directions where the rotation angle θ is 0°, 60°, and 120°.

[0038] In each direction, the structured light needs to be laterally displaced to collect multiple groups of images of illumination detection with structured light of different phases, and the signals detected by different frequency components in the structured light can be solved based on these images. For example, in each direction, 3 groups of images of illumination detection with structured light of different phases are collected, and the striped structured light with longitudinal depth is used for 9 illuminations and 9 sub-images are collected.

[0039] Moving the signals detected by different frequency components to the corresponding positions in the Fourier space and performing an inverse Fourier transform can obtain a reconstructed super-resolution image of the target sample.

[0040] As a specific example, in one embodiment, the present invention is further verified and described.

[0041] The present invention provides an equal-wavelength periodic grating longitudinal depth structured light illumination generator. The structured light illumination generator includes a glass (SiO2) as a substrate and an equal-wavelength periodic gold (Au) grating deposited on the substrate, as Figure 1 shown. The target sample to be detected is placed on a glass slide and stained with a fluorescent dye, and the excitation wavelength of the fluorescent dye is 532 nm.

[0042] The substrate of the structured light illuminator described above is glass with a thickness of 500 nm. The period Λ of the gold grating on the substrate is 532 nm, the slit width is 100 nm, and the thickness of the gold grating is 200 nm. In this embodiment, the Lumerical FDTD Solutionos simulation software is used for simulation.

[0043] In the simulation, periodic boundary conditions are used in the x and y directions, and a perfectly matched layer absorbing boundary condition is used in the z direction. The ranges of the simulation region in the x-axis, y-axis, and z-axis directions are 3.724 μm × 1 μm × 10 μm respectively, and the corresponding simulation grid accuracy is 2 nm × 10 nm × 10 nm. The incident light is a linearly polarized plane wave with a wavelength λ of 532 nm, and the polarization direction is perpendicular to the slits of the grating. At y = 0, a monitor with a wavelength band of 532 nm is placed to analyze the propagation electric field of the transmitted light in the x-z plane.

[0044] Figure 2(a) is a distribution diagram of the longitudinal depth structured light electric field intensity generated by the equal-wavelength periodic grating of the present invention in the x-z plane. It can be seen that on both sides of the slits of the grating, structured light propagating perpendicular to the grating surface appears respectively. This structured light propagates linearly upward along the direction perpendicular to the grating surface, and its intensity will gradually decrease during the propagation process. The generated structured light is a row of parallel stripes, and the transverse period of the stripes is half of the grating period. These stripes do not diverge as the propagation distance increases in the z direction. The stripe beam generated in the present invention has the characteristic of maintaining a stable stripe contrast in the propagation path, which is beneficial for stable super-resolution imaging at a certain depth. Figures 2(b) to 2(d) It is the electric field component of the longitudinal depth structured light generated by the equal-wavelength periodic grating of the present invention in the x-z plane. Among them, the electric field component in the x direction shows the characteristic of periodic change in the propagation direction, the intensity of the component in the y direction can be ignored, and the electric field intensity in the z direction is the highest. The distribution characteristics of its electric field component are consistent with the total field, indicating that the structured light is dominated by the electric field in the z direction.

[0045] Figure 3 It is the relationship between the stripe intensity of the stripe structured light and the longitudinal propagation distance, and its intensity is normalized. There is a rapid attenuation of intensity from the exit surface of the gold grating to about 100 nm above it, which reflects the rapid attenuation of the intensity of the local surface plasmon excited on the metal surface in the longitudinal direction. During the longitudinal transmission process from z = 100 nm to z = 10 μm, the intensity of the stripe structured light shows a gradually decreasing trend. At z = 10 μm, the intensity of the stripe structured light is about 35% of the intensity at the exit surface of the gold grating. This intensity is sufficient to support the stripe structured light to detect a target sample at a depth of 10 μm.

[0046] The fringe structured light at a distance of z = 5 μm from the surface of the gold grating is selected for display and analysis below. This fringe structured light is shown in Fig. 4(a). It can be seen that the structured light still maintains a clear periodic fringe distribution pattern after propagating 5 μm, which means that the present invention can perform structured illumination on targets at different depths. Further, the lateral intensity distribution characteristics of this fringe structured light are analyzed, and the corresponding intensity distribution curve is shown in Fig. 4(b). The lateral period of the structured light generated by the present invention is approximately 266 nm, which is exactly half of the grating period and also half of the wavelength of the incident light. The period of the conventional sine fringe structured light is equal to the wavelength of the incident light, which indicates that the fringe structured light used in the present invention has a spatial frequency twice that of the conventional sine fringe structured light. Further, the lateral intensity distribution of the structured light in Fig. 4(b) is subjected to Fourier transform, and the structured light spectrum shown in Fig. 4(c) is obtained. Among them, in addition to the fundamental frequency of the incident light, there are also two frequency components of the second harmonic. That is, the fringe structured light generated by the present invention can be regarded as a linear superposition of a sine fringe structured light of 532 nm and a fringe structured light of the second harmonic. The high-frequency components in these structured lights can extract the high-frequency information of the target object, so that a target image with higher resolution can be obtained.

[0047] The structured light generated in the present invention is a fringe light field with a lateral periodic distribution. In a single direction, to improve the resolution, it is necessary to use three sets of structured lights with different lateral phase distributions to illuminate respectively, and collect multiple sub-images. According to the phase-shift solving method, the signals detected by each structured light frequency component in Fig. 4(c) can be solved. By moving the signals detected by different frequency components to their corresponding positions in the Fourier space, the resolution improvement in one direction can be obtained. In order to obtain a uniform resolution improvement effect in all directions, it is necessary to use three sets of structured lights with different phases to illuminate respectively in each of the three directions (0°, 60°, and 120°), that is, a total of 9 times of structured light illumination are required, and 1 complete super-resolution image is reconstructed by collecting 9 sub-images.

[0048] Figure 5 It is a schematic diagram of the optical transfer function of the structured light illumination generator of the present invention for super-resolution imaging. The small gray circle in the center represents the range of diffraction-limited information, and its cut-off frequency is k cutoff, that is, signals above this frequency are limited by the diffraction limit and will be lost during transmission. The 6 small circles on the periphery represent high-frequency signals detected in 3 directions respectively, and the large dashed circle represents the frequency range that can be detected approximately by using the structured light illumination generator of the present invention. Compared with the diffraction-limited gray area, the radius of the dashed circle area is 3 times that of it, that is, theoretically, the structured light illumination generator of the present invention can achieve super-resolution imaging 3 times the diffraction limit. There are gaps between the 7 small circles in the figure, that is, signals in some areas will be lost, which will cause a slight decrease in the final image resolution or the appearance of some artifacts. These gaps can be filled by increasing the illumination direction. However, considering that increasing the illumination direction will increase the time cost of imaging and the missing gaps are small and have little impact on the super-resolution image, it is preferably designed to illuminate in 3 directions, so that high-frequency signals can be detected most efficiently.

[0049] Figure 6 The resolution improvement effect of the structured light illumination generator of the present invention was tested using an analog quantum dot pattern. Among them, Figure 6 (a) in is the diffraction-limited image of the corresponding quantum dots. Due to the existence of the diffraction effect, some adjacent quantum dots are difficult to distinguish, and the full width at half maximum size of a single quantum dot pattern reaches 252 nm. Figure 6 (b) in is the result of imaging using the fringe structured light of the present invention. It can be seen that some adjacent quantum dots can be clearly distinguished. The full width at half maximum size of a single quantum dot pattern is reduced to 92 nm, which means that the resolution is improved to nearly 2.74 times the diffraction limit. Considering Figure 5 the frequency domain gaps existing in it, this resolution improvement effect is reasonable.

[0050] The present invention uses an equal-wavelength periodic grating to generate structured light with a longitudinal depth structure, which can be used for super-resolution detection imaging. The structured light generated by the present invention has a high spatial frequency and a long longitudinal detection depth, improving the resolution and detection depth when observing microscopic structures in the fields of biomedicine and the like.

[0051] The above shows and describes the phenomena, features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the phenomena of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. An equal-wavelength periodic grating longitudinal depth structured light illumination generator for microscopic imaging, characterized in that The structured light illumination generator includes a glass substrate and a metal grating deposited on the glass substrate; A beam of incident light irradiates the bottom of the metal grating from bottom to top and generates structured light with a high spatial frequency and a longitudinal depth. The period Λ of the metal grating satisfies Λ = λ, where λ is the wavelength of the incident light; The incident light is a linearly polarized plane wave, perpendicularly incident on the metal grating, and the polarization direction is perpendicular to the slits of the metal grating; The transverse fringe distribution period of the structured light is half-wavelength in size, and the structured light has a longitudinal propagation depth. The structured light is used for illumination detection imaging to achieve super-resolution detection imaging of a target sample at a certain depth.

2. The equal-wavelength periodic grating longitudinal depth structured illumination generator for microscopic imaging according to claim 1, wherein During the super-resolution detection imaging process, the target sample to be detected is placed on a glass slide, and the glass slide is placed parallel to the upper surface of the metal grating. The distance from the glass slide to the metal grating is within the longitudinal propagation range of the structured light.

3. The equal-wavelength periodic grating longitudinal depth structured illumination generator for microscopic imaging according to claim 2, wherein The target sample to be detected is treated with a fluorescent dye, and the excitation wavelength of the fluorescent dye corresponds to the incident light wavelength λ.

4. The equal-wavelength periodic grating longitudinal depth structured light illumination generator for microscopic imaging according to claim 1, characterized in that, During the super-resolution detection imaging process, structured light needs to be used for illumination in multiple directions to obtain super-resolution images in each direction.

5. The equal-wavelength periodic grating longitudinal depth structured illumination generator for microscopic imaging according to claim 4, wherein In each direction, the structured light needs to be laterally displaced to collect multiple groups of images of structured light illumination detection with different phases. Based on these images, the signals detected by different frequency components in the structured light can be solved.

6. The equal-wavelength periodic grating longitudinal depth structured illumination generator for microscopic imaging according to claim 5, characterized in that, In the Fourier space, the signals detected by different frequency components are moved to the corresponding positions and subjected to inverse Fourier transform to obtain a reconstructed super-resolution image of the target sample.

Images