Four-Dimensional Multiangle Illumination-Based Light-Sheet Super-Resolution Imaging System

Abstract

The present invention relates to a beam sheet ultra-high resolution imaging system based on four-dimensional multi-angle illumination. According to the present invention, a beam sheet ultra-high resolution imaging system comprises an illumination objective lens positioned in an optical path and focused on a sample stage, a sensing objective lens positioned above the sample stage, a differential interference microscope positioned below the sensing objective lens, and a prism unit positioned between the sensing objective lens and the differential interference microscope.

Description

Technology Field

[0001] The present invention relates to a beam sheet ultra-high resolution imaging system based on four-dimensional multi-angle illumination, and more specifically, to a beam sheet ultra-high resolution imaging system that minimizes optical illusions by changing the direction of light coming from a light irradiation lens using a rotatable polygonal prism. Background Technology

[0002] One of the powerful detection techniques that bypasses the limitations of physical barriers within the diffraction limit, which observes the finest structures in nature, is super-resolution microscopy (SRM). To enhance detection capabilities at the single-molecule level, 2D detection has been improved to 3D super-resolution microscopy (SRM), thereby enhancing axial resolution across the x, y, and z coordinate ranges. As the excitent emitter travels along the plane's x and y coordinates while adjusting the cylindrical lens in the imaging system's optical path, photon localization acts on the point spread function (PSF) based on the depth of the sampled image in the z direction. Furthermore, super-resolution microscopy (SRM) has been developed alongside various detection approaches, including light-sheet microscopy (LSM).

[0003] Light sheet microscopy (LSM) was originally developed to overcome the diffraction limit of specimen detection. LSM is based on a thin plane of light passing through the specimen to form a focal region for optically cutting the entire specimen. Generally, LSM offers the advantage of minimizing photodamage and phototoxicity with faster and more convenient detection capabilities compatible with imaging living organisms compared to other microscopes. Therefore, since both LSM and SRM effectively overcome the diffraction limit of light, light sheet-based SRM is one of the most accurate observation methods.

[0004] In addition, dividing the sample into beam sheets in the lateral direction can improve the analysis volume, position, and resolution in multiple dimensions. Therefore, beam sheet-based 3D imaging research has been actively conducted in recent years. The problem to be solved

[0005] As such, according to the present invention, a beamsheet ultra-high resolution imaging system is provided that minimizes optical illusions by changing the direction of light coming from a light irradiation lens using a rotatable polygonal prism. means of solving the problem

[0006] According to an embodiment of the present invention for achieving such technical challenges, a beam sheet ultra-high resolution imaging system comprises an illumination objective lens positioned in an optical path and focused on a sample stage, a sensing objective lens positioned above the sample stage, a differential interference microscope positioned below the sensing objective lens, and a prism unit positioned between the sensing objective lens and the differential interference microscope.

[0007] The above prism unit may include a prism that performs the role of a sample stage and concentrates light from a sample that has passed through a sensing objective lens, a rotary holder that fixes the prism, and a base holder on which the rotary holder is mounted.

[0008] The above prism is formed as a polyhedron and tapers inward from the upper side to the lower side, and a cut surface is formed on the lower side parallel to the upper side so that the area of ​​the cut surface on the lower side can be formed smaller than the area of ​​the upper side.

[0009] The above-described rotary holder can be rotated by a preset angle according to the lighting direction while the prism is seated on the upper side.

[0010] The above-described rotary holder has multiple grooves formed on each side for total reflection fluorescence illumination, and a hole may be formed on the bottom for detection by a differential interference microscope. Effects of the invention

[0011] As such, according to the present invention, optical illusions can be minimized by using reconstructed images obtained by multiple illuminations from various angles to uniformly identify the location of target substances (e.g., nanoparticles and molecules) within a single cell. Brief explanation of the drawing

[0012] FIG. 1 is a drawing for explaining a beamsheet ultra-high resolution imaging system according to an embodiment of the present invention. Figure 2 is a drawing for explaining the prism unit illustrated in Figure 1. FIG. 3 is an illustrative diagram for explaining the position of particles according to the rotation of a rotary holder in an embodiment of the present invention. Figure 4 is an example diagram illustrating plasmonic aggregates and scattering images when FeMP (black ball) is used as a lighting obstacle on a PLL coated cover slip. Figure 5 is an example diagram illustrating 3D immobilized NP and FeMP aggregates in a PVA solution and scattering images. Figure 6 is an example diagram illustrating NP and FeMP aggregates and scattering images when double-sided tape is used as a multi-angle illumination obstacle instead of FeMP. Figure 7 is an example of a 3D cultured single macrophage fixed on PCL nanofibers. Figure 8 is an example diagram showing a series of scattering images of aggregated NPs at multiple illumination angles (0°, 90°, 180°, and 270°). Specific details for implementing the invention

[0013] Preferred embodiments according to the present invention will be described in detail below with reference to the attached drawings. In this process, the thickness of lines or the size of components shown in the drawings may be exaggerated for clarity and convenience of explanation.

[0014] Furthermore, the terms described below are defined in consideration of their functions within the present invention, and these may vary depending on the intent or practice of the user or operator. Therefore, the definitions of these terms should be based on the content throughout this specification.

[0015] Hereinafter, a beamsheet ultra-high resolution imaging system based on 4-dimensional multi-angle illumination according to an embodiment of the present invention will be described in more detail using FIGS. 1 to 8.

[0016] FIG. 1 is a drawing for explaining a beam sheet ultra-high resolution imaging system according to an embodiment of the present invention, and FIG. 2 is a drawing for explaining a prism unit illustrated in FIG. 1.

[0017] As illustrated in FIG. 1, a beam sheet ultra-high resolution imaging system (100) according to an embodiment of the present invention includes an illumination objective lens (IOL) (110) located in the optical path and focused on a sample stage, a detecting objective lens (DOL) (120) located above the sample stage, and a differential interference contrast (DIC) (130) located below the detecting objective lens (DOL) (120).

[0018] The above-mentioned illumination objective lens (IOL) (110), detection objective lens (DOL) (120), and differential interference sensor (DIC) (130) may be commercially available and, as they are not core technologies of the present invention, a detailed description of the illumination objective lens (IOL) (110), detection objective lens (DOL) (120), and differential interference sensor (DIC) (130) is omitted.

[0019] Meanwhile, since multi-angle illumination for image reconstruction is essential to increase the accuracy and precision of the ultra-high-resolution image of the cell environment, the present invention further includes a prism unit (140) between the sensing objective lens (DOL) (120) and the differential interference sensor (DIC) (130).

[0020] The prism unit (140) performs the role of a sample holder and includes a prism (141) that concentrates light from a sample passing through a sensing objective lens (DOL) (120), a rotary holder (142) that fixes the prism (141), and a base holder (143) on which the rotary holder (142) is mounted.

[0021] Specifically, the prism (141) is formed as a polyhedron, tapering inward from the upper side to the lower side, and a cut surface is formed on the lower side parallel to the upper side. Accordingly, the area of ​​the cut surface on the lower side is formed to be smaller than the area of ​​the upper side.

[0022] The rotary holder (142) rotates the prism (141) by a preset angle according to the lighting direction while the prism is seated on the upper side.

[0023] For example, assuming the prism (141) is a rectangular prism, the rotary holder (142) rotates it to any one of 90°, 180°, and 270°. Also, assuming the prism (352) is a hexagonal prism, the rotary holder (142) rotates it to any one of 60°, 120°, 180°, 240°, and 300°.

[0024] Additionally, the rotary holder (142) has multiple grooves formed on each side for Total Internal Reflection Fluorescence (TIRF) illumination and a hole formed on the bottom for detecting Differential Interference Detector (DIC) (130).

[0025] FIG. 3 is an illustrative diagram for explaining the position of particles according to the rotation of a rotary holder in an embodiment of the present invention.

[0026] As shown in FIG. 3, when light emitted from the illumination objective lens (IOL) (110) illuminates the sample, the obstacle (represented by a large black ball, FeMP) in front of the target particles (AgNP, AuNP and AuNR) absorbs the incident light.

[0027] The sample is rotated at a different angle (e.g., 90°) to refract and scatter the incident light and maximize the scattering intensity of the target particles. Individual particles of the aggregate exhibit different scattering intensities at various illumination angles. Higher intensities aid in better image acquisition, and using the maximum scattering intensity, individual particles of the aggregate can be reconstructed in 3D using an astigmatism-based minimum cubic algorithm. Thus, the beamsheet ultra-high resolution imaging system according to an embodiment of the present invention can reconstruct plasmonic aggregates in 3D cultured cells.

[0028] Specifically, to visualize 3D images, three intracellular NP aggregates (80nm AgNPs, 100nm AuNPs, and 40nm AuNRs) are selected as imaging probes. The selected aggregates have high scattering and absorption efficiencies and possess high photostability and biocompatibility.

[0029] The illumination laser and bandpass filter are selected based on the ultraviolet-visible (UV-Vis) absorption spectrum as a 473 nm laser and a 488 / 10 nm filter for 80 nm AgNPs, a 594 nm laser and a 610 / 10 nm filter for 100 nm AuNPs, and a 671 nm laser and a 680 / 10 nm filter for 40 nm AuNRs.

[0030] At this time, 2-3 μm FeM is used as a model obstacle to induce optical illusion, and when examining the scattering signals of three NPs measured after modulation with a specific laser and bandpass filter, it can be seen that each is observed and distinguished at a unique wavelength. That is, AgNPs, AuNPs, and AuNRs are rarely dispersed on the PLL-coated coverslip to form individual NPs.

[0031] At this time, multi-angle illumination and imaging were performed every 90° for individual NPs, and FeMPs and double-sided tape were used as model obstacles to artificially induce a stronger illusion. In addition, the coverslip used was deformed into a positive charge through PLL-coating.

[0032] Figure 4 is an example diagram illustrating plasmonic aggregates and scattering images when FeMP (black ball) is used as an illumination obstacle on a PLL-coated coverslip. As shown in Figure 4, a mixed solution of three NPs (AgNP, AuNP, and AuNR) and FeMP are adsorbed onto a PLL-coated coverslip. The adsorbed solution of AgNP, AuNP, and AuNR can be spread across multiple pixels and appear blurred, as shown in Figures 4 (b) to (d), depending on the optical diffraction limit.

[0033] And as shown in Fig. 4(e), when the prism is in the shape of a rectangular parallelepiped and the position is not changed, that is, when a large FeMP (i.e., an obstacle) is located in the illumination path, AgNP, AuNP, and AuNR exhibit the weakest scattering intensity, resulting in low accuracy in position identification.

[0034] On the other hand, when the prism is rotated 90° so that the position of FeMP (obstacle) changes to the side of the NP aggregate due to sample rotation, the scattering intensity of AgNP, AuNP, and AuNR increases; therefore, at 90°, aggregated AuNP is investigated in direct comparison with AgNP and AuNR. Consequently, aggregated AuNP exhibited the highest scattering intensity at a 90° sample rotation.

[0035] In other words, absorption, refraction, and scattering of AuNPs can reduce the excitation efficiency of AgNPs and AuNRs, and sample rotation allows AgNPs and AuNRs to be directly illuminated at 180° and 270°, respectively, to generate an optimal signal-to-noise ratio. Additionally, the optimal scattering image of each NP in the aggregate was used to calculate a superlocalization precision similar to that of individual Ns.

[0036] Afterwards, as shown in Fig. 4(e), the SR image of the NP aggregate can be reconstructed in 4D using the optimal scattering image and localization precision.

[0037] Therefore, the present invention can minimize optical illusions in spatial super-resolution imaging of plasmonic aggregates.

[0038] Meanwhile, as shown in (g) of Fig. 4, since three NP aggregates are adsorbed onto a flat cover slip, the z-coordinates of the aggregates are the same, and plasmonic coupling cannot be avoided when the NPs get closer at a distance smaller than their size.

[0039] However, at the optimal illumination angle, the localization precision (sx, sy, sz) of each NP in the aggregated region appears similar to that of a single N. This is because the plasmonic coupling effect can be ignored by wavelength modulation using a bandpass filter based on the maximum scattering characteristics of each NP.

[0040] Figure 5 is an example diagram illustrating 3D immobilized NP and FeMP aggregates in a PVA solution and scattering images.

[0041] As shown in Fig. 5, plasmonic aggregates of 80 nm AgNPs (blue spheres), 100 nm AuNPs (yellow spheres), 40 nm AuNRs (red bars), and FeMPs (black spheres) exist within the solidified PVA. The sample coverslip is prepared by adding the solidified PVA solution to the 3D immobilized NPs and FeMPs on the PLL-coated coverslip, and multi-angle illumination scattering images were acquired using the same image acquisition process as in Fig. 4. In this case, the scattering intensity of each NP in the aggregate varies at various illumination angles, and the optimal image is determined by comparing the scattering intensity with the superlocalization precision. Consequently, an optimal 4D CMLS SR image of the NP aggregate can be obtained. However, as shown in Fig. 5(g), the NPs in the solidified PVA have different z-heights.

[0042] Figure 6 is an example diagram illustrating NP and FeMP aggregates and scattering images when double-sided tape is used as a multi-angle illumination obstacle instead of FeMP.

[0043] As shown in Fig. 6, the double-sided tape has a rough surface and low light transmittance, which blocks light intensity, distorts the direction of light, and reduces light intensity. In addition, NP scattering intensity is significantly reduced, and sample rotation and direct NP illumination can minimize optical illusions, thereby generating high-quality 4D CMLS SR images of aggregates.

[0044] As previously explained, compared to cells cultured in coverslips or petri dishes (cells cultured in a 2D form), cells cultured in a 3D form exhibit characteristics closer to cells under complex in vivo conditions.

[0045] Figure 7 is an example of a 3D cultured single macrophage fixed on PCL nanofibers.

[0046] As shown in Figures 7 (a) and (b), RAW264.7 macrophage cells are cultured on a PCL nanofiber membrane to form a 3D cell morphology.

[0047] PCL nanofibers serve as a support for a 3D model of cell growth, and the scattering signal of the PCL nanofiber membrane can be completely eliminated through wavelength-modulation. This means that the PCL nanofiber membrane did not increase the background signal of NPs in the cell.

[0048] Figure 7(b) shows the mitochondria (green) and nuclei (blue) of RAW264.7 macrophages stained with fluorescent dye, and optical sections were taken at 10 nm intervals in the z-direction to confirm the 3D cell morphology. The z-height of the mitochondria and nuclei was greater than that of cells grown in a flat matrix. This indicates that the cells were cultured as a 3D model.

[0049] Features of beamsheet ultra-high resolution technology are helpful for 3D culture cell imaging, such as reduced photodamage, reduced photobleaching, and whole-cell imaging by z-slicing.

[0050] Accordingly, in an embodiment of the present invention, cells were observed using DIC and LS detection modes, and after culturing 3D cultured cells with 3 NPs and FeMP for 4 hours, NP aggregates were detected using a beamsheet ultra-high resolution imaging system composed of 3 NPs (see Fig. 7c and 7d).

[0051] Figure 8 is an example diagram showing a series of scattering images of aggregated NPs at multiple illumination angles (0°, 90°, 180°, and 270°).

[0052] As shown in Fig. 8, aggregated AgNPs exhibit the highest scattering intensity and optimal positional accuracy at an illumination angle of 0°, whereas at other illumination angles (0°, 90°, 180°, and 270°). At an illumination angle of 0°, the positional accuracy of aggregated AgNPs was close to that of individual AgNPs. Therefore, the scattering image of aggregated AgNs at an illumination angle of 0° is used to construct a 4D CMLS image to minimize optical illusions. Based on the same principle, 3D images of aggregated AuNPs and AuNRs with scattering images at illumination angles of 90° and 180°, respectively, are constructed. These three optimal 3D construction images are merged to display the spatial distribution of intracellular plasmonic aggregates. Thus, the developed 4D CMLS imaging system can resolve the spatial distribution of NPs in aggregated regions by minimizing optical illusions using multi-angle illumination imaging.

[0054] The present invention has been described with reference to the embodiments illustrated in the drawings, but this is merely illustrative, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible therefrom. Accordingly, the true technical scope of protection of the present invention should be determined by the technical spirit of the following claims. Explanation of the symbols

[0055] 100: Beam Sheet Ultra-High Resolution Imaging System 110 : Illuminating Objective (IOL) 120 : Detection Objective (DOL) 130 : Differential Interference Microscope (DIC) 140 : Prism Unit 141 : Prism 142 : Rotating holder 143 : Base holder

Claims

Claim 1 The apparatus comprises an illumination objective lens positioned in the optical path and focused on a sample stage, a sensing objective lens positioned above the sample stage, a differential interference microscope positioned below the sensing objective lens, and a prism unit positioned between the sensing objective lens and the differential interference microscope. The prism unit includes a prism that functions as a sample stage and collects light from a sample passing through the sensing objective lens, a rotary holder that fixes the prism, and a base holder on which the rotary holder is mounted. The rotary holder is configured to rotate by a preset angle according to the illumination direction while the prism is seated on its upper side. Multiple grooves are formed on each side for total reflection fluorescence illumination, and a hole is formed on the bottom for detection by the differential interference microscope. become is a beamsheet ultra-high resolution imaging system. Claim 2 delete Claim 3 A beam sheet ultra-high resolution imaging system according to claim 1, wherein the prism is formed as a polyhedron, tapers inward from the upper side to the lower side, and has a cutting surface formed on the lower side parallel to the upper side so that the area of ​​the cutting surface on the lower side is smaller than the area of ​​the upper side. Claim 4 delete Claim 5 delete

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