An interference single-molecule positioning microscopic imaging device and imaging method based on specular reflection and a single objective

Abstract

The application discloses an interference single-molecule positioning microscopic imaging device and imaging method based on mirror reflection and single objective. The device comprises a light source system, an illumination and modulation system, a fluorescent imaging system and a signal control and processing system. After the excitation light passes through the objective, the light is reflected by the mirror located above the objective, and a standing wave interference field is formed at the sample to excite fluorescent molecules. The axial position of the mirror is accurately adjusted to modulate the illumination phase; based on the change of the fluorescent intensity in the collected single-molecule image with the interference fringes, the phase of the single-molecule image relative to the interference fringes is calculated, and the axial relative position information is combined for analysis, so that the nanoscale axial positioning precision is realized. The application adopts the optical path structure of single objective combined with mirror reflection, avoids the high-precision pairing and complex alignment of the traditional double objective, and realizes the axial resolution close to the double-objective 4Pi single-molecule positioning microscope. Meanwhile, the system complexity, assembly and adjustment difficulty and manufacturing cost are significantly reduced.

Description

Technical Field

[0001] This disclosure relates to the field of super-resolution microscopy imaging technology, and more specifically, to a standing wave interferometry single-molecule localization microscopy imaging device and imaging method based on specular reflection and a single objective lens. Background Technology

[0002] The revolutionary improvement in resolution brought about by fluorescence microscopy has driven significant advances in cell biology, enabling detailed visualization of subcellular structures and precise analysis of protein complexes in situ within the cellular environment. Single-molecule localization microscopy (SMLM) is widely used due to its superior spatial resolution and single-molecule sensitivity. However, traditional three-dimensional SMLM (3D-SMLM) based on point spread function (PSF) engineering suffers from resolution anisotropy: axial resolution is typically about 2–3 times lower than lateral resolution. To improve axial performance, single-molecule localization microscopy using coherent dual-objective detection (4Pi) (4Pi-SMLM) achieves coherent detection through two opposing objectives, improving axial localization accuracy by about 5 times, thus obtaining an isotropic three-dimensional resolution of about 10–15 nm and supporting imaging of multiple targets at the whole-cell scale. Nevertheless, the widespread adoption of 4Pi-SMLM is still limited by its complex instrument structure, stringent alignment requirements, and high system cost and maintenance expenses, making it difficult for most research teams to deploy and operate stably. The aforementioned limitations highlight the urgent need for further simplification of interferometric imaging schemes to improve the accessibility, robustness, and reproducibility of this method in biological research. Summary of the Invention

[0003] In view of the aforementioned problems in the prior art, this invention provides a novel super-resolution interferometric single-molecule localization microscopy imaging device based on specular reflection and a single objective lens (me4Pi-SMLM, i.e., specular enhancement interferometric single-molecule localization imaging microscope) and imaging method. This device replaces the dual-objective geometry of the traditional 4Pi-SMLM by using a single objective lens and a reflector for reflecting the illumination light, completely eliminating the upward optical path in the system and thus eliminating the need for expensive objective lens pairing and complex dual-objective alignment. With the aid of a reflector for reflecting the illumination beam and a piezoelectric actuator that controls the movement of the reflector, rapidly phase-tunable axial illumination interference fringes are generated, achieving imaging resolution comparable to that of a traditional dual-objective 4Pi-SMLM, while significantly reducing system complexity, maintenance costs, and overall expenses.

[0004] The first aspect of this disclosure provides an interferometric single-molecule localization microscopy imaging apparatus based on specular reflection and a single objective lens. This interferometric single-molecule localization microscopy imaging apparatus includes a light source system configured to generate excitation light for illumination. Further, the apparatus includes an illumination and modulation system comprising: an objective lens assembly including a single objective lens; a sample stage arranged translationally on the focal plane of the objective lens for placing a sample; a mirror assembly including a modulation mirror positioned axially on the sample stage opposite to the objective lens, the modulation mirror being parallel to the sample stage; wherein the excitation light from the light source system, after passing through the objective lens and the sample, is reflected back by the modulation mirror to generate an axial standing wave interference pattern illuminating the sample on the focal plane; and a mirror piezoelectric actuation assembly configured to actuate the modulation mirror relative to the sample in the axial direction of the objective lens during a single localization cycle, thereby obtaining the standing wave interference pattern corresponding to at least three phases. Furthermore, the interferometric single-molecule localization microscopy imaging device includes a fluorescence imaging system configured to acquire phase-shift images corresponding to each of the standing-wave interference patterns in a single localization period for each single molecule of the sample, thereby obtaining a fluorescence signal containing at least three phase-shift images. Furthermore, the interferometric single-molecule localization microscopy imaging device includes a signal control and processing system configured to control the light source system to generate the excitation light, control the actuation of the modulation mirror by the piezoelectric actuation component of the mirror, and control the imaging of the fluorescence imaging system, and based on the fluorescence signal obtained by the fluorescence imaging system, locate each single molecule of the sample, thereby reconstructing an image of the sample.

[0005] The second aspect of this disclosure provides a microscopic imaging method using an interferometric single-molecule localization microscopy imaging apparatus according to the first aspect. The method includes: the signal control processing system controlling the light source system to emit modulated excitation light, the excitation light being guided to the objective lens assembly of the illumination and modulation system. Further, the method includes: in a single localization cycle, the signal control processing system controlling the piezoelectric actuation assembly of the mirror to actuate the modulation mirror relative to the sample stage in the axial direction of the objective lens, thereby obtaining the standing wave interference pattern corresponding to at least three phases. Further, the method includes: for each single molecule of the sample, the fluorescence imaging system collecting phase-shifted images corresponding to each of the standing wave interference patterns in the single localization cycle, thereby obtaining a fluorescence signal containing at least three fluorescence images. Further, the method includes: the signal control processing system localizing each single molecule of the sample based on the fluorescence signal obtained by the fluorescence imaging system, thereby reconstructing an image of the sample.

[0006] The super-resolution interferometric single-molecule localization microscopy imaging device (me4Pi-SMLM) disclosed herein employs a single-objective configuration, utilizing the specular reflection of the illumination beam to generate phase-tunable standing-wave interference fringes. This innovative design improves the axial resolution of astigmatic methods by approximately five times, providing performance comparable to conventional 4Pi-SMLMs, while significantly reducing system complexity and maintenance difficulty. In biological samples, me4Pi-SMLM achieves isotropic localization accuracy of 2–3 nm, enabling clear resolution of various ultrastructural features. Furthermore, it supports high-fidelity two-color imaging and nanoscale resolution whole-cell reconstruction. Importantly, me4Pi-SMLM can be seamlessly integrated into existing 3D-SMLM systems, significantly improving performance with minimal cost and effort. These advantages make me4Pi-SMLM an accessible and powerful platform for nanoscale cell imaging. Attached Figure Description

[0007] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this disclosure. Where appropriate, the same reference numerals are used in all drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of this device or method. In the drawings:

[0008] Figure 1 This is a schematic diagram of the structure of an interferometric single-molecule localization microscopy imaging device (me4Pi-SMLM) based on specular reflection and a single objective lens according to an embodiment of the present disclosure.

[0009] Figure 2 This is a schematic diagram illustrating the principle and verification of the me4Pi-SMLM according to an embodiment of this disclosure. a: Simplified schematic diagram of the me4Pi-SMLM; a laser is focused onto the back focal plane of the objective lens to illuminate the sample. After passing through the objective lens and the sample, the laser beam is reflected by a mirror, generating an axial standing wave interference pattern. A piezoelectric actuator rapidly translates the mirror within milliseconds, moving the interference pattern with sub-nanometer precision. The fluorescence signal is collected by the objective lens and imaged onto a camera; L4: fourth lens; M9: modulation mirror; DM2: second dichroic mirror; OBJ: objective lens; TL: tube lens; CL1: first cylindrical lens; CAM1: first camera. b: Top and middle images: simulated PSF under illumination modes at different axial positions and phases; bottom image: intensity distribution curves of the three sub-images at different axial positions. c: Relative 3D position distribution of two 40 nm fluorescent beads reconstructed using me4Pi-SMLM and 3D-SMLM. The results of 3D-SMLM were obtained by astigmatic Gaussian fitting of the summed phase images. The photon counts of the two fluorescent beads are 5527±358 and 5091±246 (mean ± standard deviation), respectively. d: Histograms of the center-to-center distance between the two 40 nm fluorescent beads in each direction in c, reconstructed using me4Pi-SMLM. For comparison, axial results reconstructed using 3D-SMLM are also shown.

[0010] Figures 3-5 This is a schematic flowchart of a me4Pi-SMLM microscopic imaging process according to an embodiment of the present disclosure. Three sub-images corresponding to three phase-shift illumination patterns were acquired. A 2D astigmatic Gaussian fit was performed to determine the lateral position (x, y) of the molecules and the PSF width, and these widths were used to estimate a coarse axial z-position. The intensity of the three sub-images was analyzed to calculate phase information. After phase drift correction, an axial phase unwrapping algorithm was used to convert the phase data into a precise axial z-position. Then, a 3D drift correction algorithm was applied to compensate for sample drift and residual phase drift. In filtering step 1, non-fluorescent molecule signals in the three frames were excluded based on modulation depth. In filtering step 2, molecules were further excluded based on criteria such as photon count and positioning accuracy. For me4Pi-SMLM, a precise z-position was used, while for 3D-SMLM, a coarse z-position was used.

[0011] Figure 6This is a timing diagram showing the control of a piezoelectric actuator, an AOTF, and an sCMOS camera by a signal control processing system according to an embodiment of this disclosure. The positions of the modulation mirror and the piezoelectric actuator are controlled by three voltages, each applied for 15 ms. The sCMOS camera trigger signal is delayed by 3 ms relative to the piezoelectric actuator signal, the camera exposure time is set to 10 ms, and the AOTF remains on throughout the imaging process, with one imaging cycle being 45 ms.

[0012] Figure 7 This is a schematic diagram of axial positioning calibration according to an embodiment of the present disclosure. a: Relationship curve between the PSF width of the 40 nm fluorescent bead and the axial position. b: Relationship curve between the measured z-position and the piezoelectric z-position. c: Intensity curves of three sub-images at different axial positions, with a period of approximately 210 nm.

[0013] Figure 8 This is a schematic diagram of ridge finding in axial phase expansion according to an embodiment of the present disclosure. Dots represent the normalized metric and phase values ​​of a single molecule, and circles highlight the monotonic ridges of the metric versus phase curve.

[0014] Figure 9 This is a schematic diagram illustrating voltage estimation for the piezoelectric actuator of the me4Pi-SMLM according to an embodiment of this disclosure. a: The intensity oscillation of the 40 nm fluorescent bead was recorded when the piezoelectric actuator was driven by a 4-6V AO signal. The intensity distribution was fitted to a sine function to determine the period of the AO signal. b: The average AO signal period was calculated through multiple experiments, with one period being 0.324 V, equivalent to a 2π / 3 phase shift of 0.108 V.

[0015] Figure 10 This is a schematic diagram of modulation depth according to an embodiment of the present disclosure. a: Modulation depth of a 40 nm phosphor bead within the field of view (FOV), scale bar: 5 μm. b: Histogram of modulation depth in a.

[0016] Figure 11Schematic diagram of subcellular structures resolved by me4Pi-SMLM according to embodiments of this disclosure. a: me4Pi-SMLM image of microtubules in fixed COS-7 cells. b: 200 nm thick cross-section at the locations indicated by numbers in a, reconstructed using me4Pi-SMLM and 3D-SMLM. c: 3D localization distribution generated by molecules emitting ≥10 frames. The photon number of these molecules is 5550 ± 1332 (mean ± standard deviation). d: Histogram of the distribution in c, fitted with a Gaussian function, with standard deviation reported. e: me4Pi-SMLM image of nucleoporin Nup96 in fixed U-2 OS cells. f: Enlarged image of the blue box region in e and xz view of the yellow box region in f, reconstructed using me4Pi-SMLM and 3D-SMLM. g: 3D particle averaging result of 500 nucleoporin complexes (NPCs) in the cell nucleus shown in e, with a 3D visualization of the average NPCs after cluster analysis shown in the lower right corner. h: me4Pi-SMLM image of the endoplasmic reticulum (ER) in fixed COS-7 cells. i: 50 nm thick xy slice of the lower yellow box region in h, reconstructed using me4Pi-SMLM and 3D-SMLM, and a 100 nm thick xz cross section along the dashed line. j: Enlarged image of the upper blue box region in h, reconstructed using me4Pi-SMLM and 3D-SMLM, and a 100 nm thick xz cross section along the numbered dashed line. Color bars indicate depth. Scale bar: 5 μm (a, e, h), 500 nm (top of i, top of j), 200 nm (f, bottom of i, bottom of j), 50 nm (b), 20 nm (g).

[0017] Figure 12 The resolution curve of the microtube according to an embodiment of this disclosure is shown. a: Figure 11 Figure a shows a histogram of the distances between location points in adjacent frames of microtube data. Figure b: Figure 11 Figure a shows the FRC curves for various dimensions of the microtubule data.

[0018] Figure 13 Transmission curves of different dyes according to embodiments of this disclosure. a: Transmission curves of a dichroic spectrometer and emission filter for monochromatic imaging, and the emission spectrum of Cy3B. The red area represents the combined transmission curve of Cy3B through the dichroic spectrometer and emission filter. b: Transmission curves of a dichroic spectrometer and emission filter for duochromatic imaging. The red area represents the combined transmission curve of the conventional fluorescence path, while the green area represents the combined transmission curve of the recovered fluorescence path. cd: Emission spectra of Cy3B and AF568 (a) or Cy3B and ATTO Rho11 (b), and transmission curves of conventional and recovered fluorescence.

[0019] Figure 14This is a schematic diagram of me4Pi-SMLM two-color imaging according to an embodiment of the present disclosure. a: Two-color me4Pi-SMLM image of ER and microtubules in fixed COS-7 cells, with a depth color-coded image shown in the lower left corner and a merged image of the two markers shown in the upper right corner. b: Enlarged image of the area within the yellow box on the right in a. c: A 10 nm thick xy slice of the area within the blue box on the left in a. d: A 500 nm thick yz cross section and a 100 nm thick xz cross section along the dashed line in b. e: Two-color me4Pi-SMLM image of ER membrane proteins Sec61β and KDEL in fixed COS-7 cells. f: Enlarged image of the area within the yellow box in e. KDEL and Sec61β markers are shown in the lower left and upper right corners, respectively. g: A 50 nm thick xy slice of the area within the yellow box in e; h: A 200 nm thick xz cross section at the position indicated by the number in g; i: An intensity distribution curve along the white line in g. The color bars represent depth. Scale bar: 5 μm (a, e), 1 μm (b, f, g), 500 nm (c, h), 200 nm (d).

[0020] Figure 15 This is a schematic diagram of me4Pi-SMLM whole-cell imaging according to an embodiment of this disclosure. a: 3D image of synaptic complexes in immobilized mouse spermatocytes imaged using me4Pi-SMLM. b: yz cross-section of the region indicated by the yellow dashed line in a. c: yz view of a. d: Enlarged image of 19 synaptic complexes segmented and extracted from a complete image of the mouse spermatocyte nucleus. e: Representative 3D views of the two synaptic complexes shown in d. f: 3D whole-cell image of mitochondria in HeLa cells imaged using me4Pi-SMLM. g: Cross-section of f with xy projection and numerical representation of location. Color bars indicate depth. All cross-sectional slices are 100 nm thick. Scale bar: 2 μm (b, d, g). Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the described embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0022] Unless otherwise defined, the technical or scientific terms used in this disclosure shall have the ordinary meaning understood by one of ordinary skill in the art to which this disclosure pertains. The terms “first,” “second,” and similar terms used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as “comprising” or “including” mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects. Terms such as “connected” or “linked” are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as “upper,” “lower,” “left,” and “right” are used only to indicate relative positional relationships, and these relative positional relationships may change accordingly when the absolute position of the described objects changes.

[0023] To keep the following description of the embodiments of this disclosure clear and concise, detailed descriptions of known functions and known components are omitted.

[0024] The super-resolution interferometric single-molecule localization microscopy imaging device provided in this disclosure employs a single objective lens configuration to perform lateral and axial localization of molecules in the sample, thereby achieving super-resolution microscopic imaging of the sample. In the following description, a super-resolution interferometric single-molecule localization microscope based on specular reflection and a single objective lens (i.e., specular enhancement interferometric single-molecule localization imaging microscope, me4Pi-SMLM) is used as an example to describe the super-resolution single-molecule localization microscopy imaging device and its imaging method provided in this disclosure. However, it should be understood that the super-resolution single-molecule localization microscopy imaging device provided in this disclosure can also be other devices or systems besides microscope devices, as long as it has the configuration provided in this disclosure and uses the corresponding imaging principle for microscopic imaging.

[0025] The following section will provide a detailed explanation of the configuration of this mirror-enhanced interferometric single-molecule localization imaging microscope and its imaging principle for samples.

[0026] like Figure 1 As shown, the me4Pi-SMLM 100 provided in this disclosure may include: a light source system 110, an illumination and modulation system 120, a fluorescence imaging system 130, and a signal control and processing system (not shown), wherein each system in Figure 1The image is defined by a dashed box. The light source system 110 generates excitation light (such as a laser) for illumination. This excitation light is directed to an illumination and modulation system 120 to provide excitation illumination to the sample 200. The illumination and modulation system 120 simultaneously modulates the excitation light to obtain multiple interference patterns with phase differences to excite the sample 200. The fluorescence imaging system 130 collects and scans phase-shift images (fluorescent sub-images) corresponding to each interference pattern in a single positioning period for each single molecule of the sample 200, thereby obtaining a fluorescence signal containing multiple fluorescence images. The signal control and processing system controls the light source system 110 to generate excitation light, controls the illumination and modulation system 120 to generate multiple interference patterns, and controls the fluorescence imaging system 130 to perform imaging. Based on the fluorescence signal obtained by the fluorescence imaging system 130, it positions each single molecule of the sample 200, thereby reconstructing an image of the sample 200.

[0027] In a preferred embodiment, a laser can be selected as the excitation light to excite and illuminate the sample molecules. In this embodiment, the light source system 110 may include: a first laser 111, a first reflector M1, a second reflector M2, a second laser 112, a third reflector M3, and a first dichroic mirror DM1, a fourth reflector M4, a fifth reflector M5, an acousto-optic tunable filter (AOTF), a sixth reflector M6, a seventh reflector M7, a coupling lens 114, and a single-mode fiber SMF arranged along the optical path.

[0028] The first laser 111 and the second laser 112 can each emit laser light of different wavelengths. For example, the first laser 111 can emit a first laser with a wavelength of 488 nm for wide-field illumination to locate cell sample 200, while the second laser 112 can emit a second laser with a wavelength of 552 nm for imaging sample 200. Of course, these two lasers can also emit laser light of different wavelengths, depending on the type of sample to be imaged and the imaging requirements.

[0029] The excitation light from the first laser 111 is reflected sequentially by the first reflector M1 and the second reflector M2, and then combined with the excitation light from the second laser 112 via the third reflector M3 at the first dichroic mirror DM1. The combined excitation beam is then reflected sequentially by the fourth reflector M4 and the fifth reflector M5 and modulated by the AOTF to control the output of excitation light of different wavelengths. The modulated excitation light is then reflected sequentially by the sixth reflector M6 and the seventh reflector M7 and coupled to the single-mode fiber SMF through the coupling lens 114, thereby obtaining the modulated excitation light through the light source system 110.

[0030] Further as Figure 1As shown, the illumination and modulation system 120 may include: an objective lens assembly 121, which includes a single objective lens OBJ; a sample stage 122, which is translatably positioned on the focal plane of the objective lens OBJ for placing a sample 200; a mirror assembly 123, which includes a modulation mirror M9, the modulation mirror M9 being axially positioned on the side of the sample stage 122 opposite to the objective lens OBJ, and the modulation mirror M9 being parallel to the sample stage 122; wherein, the excitation light from the light source system 110, after passing through the objective lens OBJ and the sample 200, is reflected back by the modulation mirror M9 to generate an axial standing wave interference pattern, which is focused onto the focal plane to illuminate the sample 200; and a mirror piezoelectric actuation assembly 124, which is configured to actuate the modulation mirror M9 relative to the sample 200 in the axial direction of the objective lens OBJ with sub-nanometer precision in a single positioning cycle to adjust the illumination phase, thereby obtaining a standing wave interference pattern corresponding to at least three phases, such as Figure 2 As shown in b in the figure.

[0031] As the core component of this me4Pi-SMLM, the objective assembly 121 presented in this paper differs from traditional 4Pi-SMLMs that rely on complex dual-objective configurations. It employs a single objective OBJ, which directly generates standing-wave interference fringes by reflecting the illumination beam through a modulation mirror M9 located above the sample 200, thus eliminating the need for expensive objective pairing and dual-objective alignment. To achieve precise axial positioning, a mirror piezoelectric actuation assembly 124 is used to rapidly translate the mirror, moving the interference pattern with sub-nanometer precision, and sequentially acquiring at least three phase-shifted images (see [link to documentation]). Figure 2 (b) This self-referenced interferometry makes the me4Pi-SMLM100 highly resistant to mechanical vibrations and eliminates the need for a dedicated real-time focus locking module. This architecture achieves super-resolution comparable to traditional 4Pi-SMLMs while simplifying mechanical design, photoelectric synchronization, optical alignment, and image processing, significantly simplifying overall operation and creating a cost-effective platform.

[0032] In one embodiment, the mirror assembly 123 further includes a mirror support 123A, and the mirror braking assembly 124 includes a piezoelectric actuator 124A connected to the mirror support 123A. The modulation mirror M9 can be securely mounted on the piezoelectric actuator 124A to move synchronously with the movement of the mirror support 123A and the piezoelectric actuator 124A. The signal control processing system can actuate the modulation mirror M9 by controlling the piezoelectric actuator 124A to actuate the axial translation of the mirror support 123A, thereby obtaining a standing wave interference fringe pattern with different phases. It should be understood that the back surface of the modulation mirror M9 (e.g., by adhesive) is attached to the piezoelectric actuator 124A, and its reflective surface faces the sample 200 in a parallel manner to facilitate the original reflection of the excitation light to form a standing wave interference pattern.

[0033] Here, the illumination and modulation system 120 may further include a first lens L1, a second lens L2, an eighth reflector M8, a third lens L3 and a fourth lens L4, and a second dichroic mirror DM2 arranged sequentially along the optical path.

[0034] The excitation light output from the single-mode fiber SMF is collimated by the first lens L1, and then reflected and expanded by the second lens, the eighth mirror and the third lens in sequence. Finally, it is focused by the fourth lens L4 and reflected by the second dichroic mirror DM2, and then guided to the back focal plane of the objective lens OBJ to achieve excitation illumination of the sample 200.

[0035] Further as Figure 1 As shown, the fluorescence imaging system 130 includes the objective lens OBJ, the second dichroic mirror DM2, the first filter F1, the tube lens TL, the tenth reflecting mirror M10, and a fluorescence imaging assembly arranged sequentially along the optical path. Depending on the imaging requirements, the fluorescence imaging assembly may include a first fluorescence imaging assembly 131 for monochromatic imaging and / or a second fluorescence imaging assembly 132 for dual-color imaging. The first fluorescence imaging assembly 131 and the second fluorescence imaging assembly 132 are... Figure 1 All are delimited by dashed lines.

[0036] As can be seen, in the me4Pi-SMLM 100 disclosed herein, both the objective lens OBJ and the second dichroic mirror DM2 are components of both the objective lens assembly 120 and the fluorescence imaging system 130. The second dichroic mirror DM2 can both, as described above, emit the expanded and focused excitation light from the light source system 110 into the objective lens OBJ, and can also separate the generated fluorescence signal from the excitation light signal output from the objective lens OBJ. Figure 2(as best shown in a), so that the fluorescence signal can enter the fluorescence imaging system 130 through it.

[0037] To address the problem of phase periodic repetition, this disclosure incorporates a cylindrical lens (such as...) in the fluorescence imaging assembly. Figure 1 The first cylindrical lens CL1 and the second cylindrical lens CL2 are used to introduce slight astigmatism, thereby achieving coarse molecular axial positioning and reliable phase unfolding, such as... Figure 2 As shown in b, the same molecule exhibits different shapes at different z-axis heights (such as -300 nm, 0 nm, and 300 nm).

[0038] like Figure 1 and Figure 2 As shown, the first fluorescence imaging assembly 131 includes a first cylindrical lens CL1 for introducing astigmatism, a second filter F2, and a first camera CAM1. The separated fluorescence signal is emitted via the tenth reflector M10, then introduced into astigmatism by the first cylindrical lens CL1, and subsequently focused onto the first camera CAM1 by the second filter F2.

[0039] like Figure 1 As shown, the second fluorescence imaging assembly 132 includes a second cylindrical lens CL2 for introducing astigmatism, an aperture SL, a fifth lens L5, a third dichroic mirror DM3, a third filter F3, a sixth lens L6, an eleventh reflector M11, a fourth filter F4, a seventh lens L7, a twelfth reflector M12, a right-angle prism PM, and a second camera CAM2. The separated fluorescence signal is reflected by the tenth mirror M10, then astigmatism is introduced by the second cylindrical lens CL2, followed by the aperture SL and the fifth lens L5, and then spectrally separated into conventional fluorescence and recovered fluorescence by the third dichroic mirror DM3. The conventional fluorescence passes sequentially through the third filter F3 and the sixth lens L6 and is reflected by the eleventh mirror DM11. The recovered fluorescence passes sequentially through the fourth filter F4 and the seventh lens L7 and is reflected by the twelfth mirror M12. The light beams reflected by the eleventh mirror M11 and the twelfth mirror M12 are guided to the second camera CAM2 by the right-angle prism PM.

[0040] It should be understood that the fluorescence imaging components here may include only one of the first fluorescence imaging component 131 and the second fluorescence imaging component 132, or both. In a configuration that includes both, the optical paths of the corresponding fluorescence imaging components can be turned on and off according to imaging requirements. The tenth reflecting mirror M10 can be switchably disposed inside the microscope, allowing the orientation of M10 to be switched according to the needs of monochrome or dual-color imaging. For example, in a configuration for monochrome imaging of sample 200, the orientation of the tenth reflecting mirror M10 can be switched so that the separated fluorescence signal, after being reflected by the tenth reflecting mirror M10, forms a direct optical path to the first fluorescence imaging component 131, allowing the separated fluorescence signal to directly enter the first fluorescence imaging component 131; while in a configuration for dual-color imaging of sample 200, the other orientation of the tenth reflecting mirror M10 can be set so that the separated fluorescence signal, after being reflected by the tenth reflecting mirror M10, enters the second fluorescence imaging component 132. Therefore, in Figure 1 In the middle, the tenth reflecting mirror M10 is shown as a dashed line.

[0041] The imaging principle of the me4Pi-SMLM 100 disclosed herein will be explained next.

[0042] Figure 3 A flowchart of a method 300 for microscopic imaging of sample 200 using the me4Pi-SMLM 100 of this disclosure is shown. Figure 3 As shown, the method 300 includes the following steps:

[0043] S310, the signal control and processing system controls the light source system 110 to emit modulated excitation light, which is guided to the objective lens assembly 121 of the illumination and modulation system 120;

[0044] S320, in a single positioning cycle, the signal control processing system controls the piezoelectric actuation assembly 124 of the mirror to actuate the modulation mirror M9 relative to the sample stage 122 in the axial direction of the objective lens OBJ, thereby obtaining a standing wave interference pattern corresponding to at least three phases.

[0045] S330, for each single molecule of sample 200, the fluorescence imaging system 130 collects phase-shift images corresponding to each standing wave interference pattern in a single positioning period, thereby obtaining a fluorescence signal containing at least three phase-shift images; and

[0046] S340, the signal control and processing system locates each single molecule of sample 200 based on the fluorescence signal obtained by the fluorescence imaging system 130, thereby reconstructing an image of sample 200.

[0047] Before step S310, the prepared sample 200 should be placed on a self-made sample holder. First, rinse the sample 200 with freshly prepared imaging solution to remove excess solution, then transfer the sample 200 to the sample holder. Next, apply 100 μL of imaging solution to the sample 200, cover with a clean coverslip, and remove any excess solution (e.g., ...). Figure 2 (as shown in a). The coverslip is sealed with two-component silicone (Picodent Twinsil, Picodent) and cured for 10-20 minutes before being used for me4Pi-SMLM imaging provided in this disclosure, and then placed on the sample stage 122.

[0048] In step S310, the signal control processing system first controls the AOTF so that a first laser with a wavelength of, for example, 488 nm, from the first laser 111 is emitted from the light source system 110 to roughly locate the sample 200 in the field of view of the first camera CAM-1 or the second camera CAM-2 of the fluorescence imaging assembly. After the target cell is found, the signal control processing system continues to control the AOTF so that a second laser with a wavelength of, for example, 552 nm, from the second laser 112 is emitted from the light source system 110 and guided to the objective lens assembly 121 of the illumination and modulation system 120 to perform fluorescence imaging and localization of the sample 200.

[0049] As described above, the objective lens assembly 121 of the me4Pi-SMLM 100 provided in this disclosure includes only a single objective lens OBJ, rather than a traditional dual objective lens configuration. Above the single objective lens OBJ, a mirror assembly 123 including a modulation mirror M9 is provided. After the excitation light (second laser) passes through the objective lens OBJ and the sample 200, it is reflected back by the modulation mirror M9, thereby generating an axial standing wave interference pattern, which is focused onto the focal plane of the objective lens OBJ to illuminate and excite the sample.

[0050] In step S320, within a single positioning cycle, the signal control processing system further controls the piezoelectric actuator 124A of the reflector piezoelectric actuator assembly 124 to rapidly axially translate the reflector support 123A within milliseconds, thereby synchronously actuating the axial translation of the modulation reflector M9, moving the aforementioned standing wave interference pattern with sub-nanometer precision, thereby sequentially obtaining standing wave interference patterns corresponding to at least three phases (with phase differences), for example... Figure 2 φ1, φ2 and φ3 are shown in b.

[0051] Sample molecules excited by at least three different standing wave interference patterns correspond to different phase shift images, and the fluorescence signal is composed of at least three phase shift images. For example... Figure 2As shown in b, for the same Z-axis height (e.g., -300 nm), different phases correspond to different phase shift images. The fluorescence signal and the excitation signal are separated at the second dichroic mirror DM2. The fluorescence signal passes through the second dichroic mirror DM2, while the excitation signal is discarded.

[0052] In S330, the fluorescence imaging system 130 acquires phase-shift images corresponding to each standing wave interference pattern in a single positioning period, thereby obtaining a fluorescence signal containing at least three phase-shift images corresponding to each single molecule.

[0053] In S340, the signal control processing system locates and images the corresponding single molecules based on the acquired fluorescence signals.

[0054] Specifically, such as Figure 4 and Figure 5 As shown, step S340 may include the following steps:

[0055] S341, summate at least three phase-shifted images;

[0056] S342, for the fluorescence image obtained after superposition, the lateral position coordinates of the single molecule in the XY plane and the PSF width are estimated by Gaussian fitting;

[0057] S343, using PSF width combined with astigmatism to determine the initial axial position of a single molecule along the Z-axis;

[0058] S344, calculate the intensity of at least three phase-shifted images and extract axial phase information along the Z-axis; and

[0059] S345 uses linear regression to correlate axial phase information with preliminary axial position, determining the precise axial position coordinates of a single molecule along the Z-axis.

[0060] Specifically, taking three phases as an example, the sample molecules are illuminated sequentially with three illumination patterns 1-3. Three phase-shifted images (i.e., sub-images 1-3) are acquired sequentially within the camera's field of view, forming a fluorescence signal, thus eliminating the need for channel registration. The three phase-shifted images are superimposed and summed. Single molecules are detected in the resulting fluorescence image, and a two-dimensional (2D) Gaussian fitting based on astigmatism is applied to estimate the lateral position coordinates (x, y) and point spread function (PSF) width (σx, σy) of the single molecule in the XY plane. The PSF width is used to determine a rough preliminary axial position based on the calibration curve generated by the fluorescent beads on the coverslip (see...). Figure 6Subsequently, the intensity of the three phase-shifted images is analyzed to extract the phase and modulation depth along the Z-axis. Finally, a ridge-finding algorithm is used to unfold the phase and determine the precise Z-axis position. Precise Z-axis position coordinates are used for me4Pi-SMLM, while coarse Z-axis position coordinates are used for conventional 3D-SMLM.

[0061] In one specific embodiment, the me4Pi-SMLM 100 is a super-resolution interferometric single-molecule localization microscope built on the Nikon ECLIPSE Ti2-E inverted microscope frame.

[0062] -Hardware Configuration

[0063] A first laser 111 (OBIS 488 nm LS, 150 mW, Coherent) emitting a first laser beam with a wavelength of 488 nm and a second laser 112 (2RU-VFLP-2000-552, MPB Communications) emitting a second laser beam with a wavelength of 552 nm are combined via a first dichroic mirror DM1 (LM01-503-25, Semrock) and modulated by an acousto-optic tunable filter AOTF (AOTFnC-400.650-TN, AA Opto-Electronic). The combined beam is coupled to a single-mode fiber SMF (P1-488PM-FC-2, Thorlabs) via a coupling lens (PAF2P-A10A). The fiber optic output is collimated by the first lens L1 (f=50 mm, AC254-050-A, Thorlabs), expanded by the second lens L2 (f=100 mm, AC254-100-A, Thorlabs) and the third lens L3 (f=250 mm, ACT508-250-A, Thorlabs), and focused onto the back focal plane of objective OBJ by lens L4 (f=400 mm, ACT508-400-A, Thorlabs) for illumination of sample 200. An oil immersion objective (UPLAPO100XOHR, 100× / 1.5 NA, Olympus) is used for imaging near the coverslip, and a silicone oil immersion objective (UPLSAPO100XS, 100× / 1.35 NA, Olympus) is used for whole-cell imaging. The approximate position of sample 200 was controlled by an automated xyz platform (PZ-2000FT, Applied Scientific Instrumentation). The axial positioning of the objective lens was achieved by a PIFOC piezoelectric scanner (P-721.12Q, Physik Instrumente). After the laser beam passed through objective lens OBJ and sample 200, it was reflected back by a silver-coated mirror with a protective film (modulation mirror M9), generating an axial standing wave interference pattern on the sample plane.

[0064] -Fluorescence detection

[0065] The emitted fluorescence is separated from the excitation light using a second dichroic mirror DM2 (ZT543rdc, Chroma) and a first (bandpass emission) filter F1 (ET560lp, Chroma). For monochromatic imaging, the fluorescence signal exits from the right port of the microscope, passes through a first cylindrical lens CL1 (f=1000 mm, LJ1516RM-A, Thorlabs), which produces slight astigmatism for coarse axial positioning, and is then focused onto the first (sCMOS) camera CAM1 (ORCA Fusion, C14440-20UP, Hamamatsu). An additional second (emission) filter F2 (ET560lp, Chroma) is placed directly in front of the camera. For duochromatic imaging, the fluorescence signal exits from the left port of the microscope, passes through a second cylindrical lens CL2 (f=1000 mm, LJ1516RM-A, Thorlabs). The light is then relayed through the fifth lens L5 and the sixth / seventh lenses L6 / L7 (f=200mm, 49364, Edmund), and spectral separation is achieved using the third dichroic mirror DM3 (ZT561rdc, Chroma). The conventional fluorescence component is filtered out by the third (single-band) filter F3 (ET605 / 70m, Chroma), while the recovered fluorescence is passed through a different fourth (emission) filter F4 (FF01-572 / 28-25, Semrock). A right-angle prism PM (RAP120-RA-A, LBTEK) then guides the two separated channels to different regions of the second (sCMOS) camera CAM2 (ORCA-Fusion, C14440-20UP, Hamamatsu).

[0066] -Axial interference illumination

[0067] To ensure a stable axial standing wave interference pattern, the mirror assembly 123 is isolated from the microscope frame using a custom mount on the optical platform. The key component is located above the sample stage 122 for optimal alignment. A 10 mm diameter silver-coated mirror M9 (34-386, Edmund) with a protective film is connected to a piezoelectric actuator 124A (S23.Z10K, CoreMorrow) for precise axial positioning of the mirror. This assembly is mounted on the mirror holder 123A (motion mirror mount MT-AM1, LBTEK) for coarse horizontal tilt adjustments. A piezoelectric linear stage (N-565.260, Physik Instrumente) is used for coarse axial positioning and provides ample space for sample changes.

[0068] During initial alignment, the illumination beam first passes through objective lens OBJ and is then reflected back to objective lens OBJ by modulation mirror M9. A dense monolayer of 40 nm fluorescent beads (F8793, 580 / 605 nm, Invitrogen) is then mounted onto sample stage 122. Using a piezoelectric platform, the mirror module is brought close to sample 200, and the mirror position is adjusted to optimize the reflected spot size and maximize the modulation depth across the entire field of view (FOV). The motion mount is manually adjusted to widen the interference fringes, while simultaneously adjusting the last mirror in front of objective lens OBJ to center the reflected spot in the field of view. These steps are repeated until a concentric interference profile is obtained.

[0069] -Data Acquisition

[0070] Synchronization control was implemented using an NI-DAQ card (USB-6363, BNC, National Instruments) and a LabVIEW 2022 DAQ module. A periodic voltage waveform consisting of three phases, each lasting 15 ms, was generated via the analog output (AO) port of the NI USB-6363 to control a 124A piezoelectric actuator. Simultaneously, the sCMOS camera operated in outer-edge triggered mode, triggered by the rising edge of the digital output (DO) signal. The DO signal was delayed by 3 ms relative to the AO signal to allow sufficient time for the mirror to stabilize. The camera exposure time was set to 10 ms. The AOTF maintained continuous transmission mode throughout the image acquisition process. For speed-optimized DNA-PAINT, the average intensity of the 552 nm illumination was set to 300–500. For Fluorogenic DNA-PAINT, the average intensity of 552 nm illumination was set to 1500–2000. .

[0071] Image Reconstruction

[0072] Sub-images corresponding to the three phase-shifted illumination modes are summed. The xy position and PSF width (σx, σy) of each molecule are estimated using 2D Gaussian fitting. For the 3D-SMLM, only σx and σy are used to calculate a coarse axial position. The intensity of the sub-images is then analyzed to determine the phase and modulation depth along the z-direction. A ridge-finding algorithm is applied to unfold the phase and determine the precise z-position of the me4Pi-SMLM (see [link to relevant documentation]). Figure 8 ).

[0073] -Drift Correction

[0074] me4Pi-SMLM employs interferometric illumination for precise axial positioning. Therefore, both positional and phase drift affect the final positioning accuracy. To correct for phase drift, the positioned molecules within a time window (typically every 1000-3000 frames) are divided into multiple individual segments. A series of molecular phase representations and normalized metrics are generated. m ( Figure 8 The two-dimensional histogram image of the phase data is obtained and then Gaussian blurred. Cross-correlation is then used to estimate the phase drift between the images, followed by correction via spline interpolation. This method significantly reduces phase drift, thus achieving robust phase unfolding and reliable conversion of phase data to axial coordinates. Figure 8 For 3D sample drift correction, the AIM (Adaptive Cross-Maximization) and DME (Minimum Entropy Drift) algorithms are applied in sequence, while also addressing any residual phase drift during this process.

[0075] -System Calibration

[0076] To generate a calibration file for astigmatic localization, a sparse sample of 40 nm fluorescent beads was imaged. Axial scans of sample 200 within a 1 μm range were performed at three different phases using a piezoelectric actuator to obtain a 3D image stack. The PSF width was calculated to estimate the approximate z-position, and the results showed a linear relationship with the actual axial displacement. To calibrate the interference fringe period, the intensity of single molecules in each phase sub-image was measured. The position and single-molecule intensity were then fitted to determine the axial period of the interference fringes. Figure 7 To determine the voltage of the piezoelectric actuator 124A corresponding to a 2π / 3 offset in the interference fringes, a sparse sample of 40 nm fluorescent beads was imaged while sample 200 remained stationary. The piezoelectric actuator 124A was driven using a 4–6 V AO signal. Frame-by-frame intensity measurements were fitted to a sine function to determine the oscillation period (2π), and the voltage corresponding to the 2π / 3 offset was determined. Figure 9 ).

[0077] The calibration results show that, under the second laser excitation at a wavelength of 552 nm, the interference period is 210 nm. Figure 7 c). Scanning the piezoelectric actuator 124A can determine the voltage corresponding to a 2π / 3 offset in the interference fringes ( Figure 9 The modulation contrast ratio is estimated to be approximately 0.9. Figure 10 Under these conditions, the axial positioning accuracy of me4Pi-SMLM is approximately 5 times higher than that of traditional 3D-SMLM, achieving a positioning accuracy of 2-3 nm in all dimensions, with a photon count of approximately 5300. Figure 2 (c and d in the text).

[0078] Performance evaluation

[0079] The following will combine Figure 11-15 This paper describes the performance evaluation of biological samples after imaging using the me4Pi-SMLM 100 and microscopic imaging method 300 provided in this disclosure.

[0080] - me4Pi-SMLM Analysis of Subcellular Structures

[0081] Since me4Pi-SMLM requires at least three phase images to achieve precise axial localization, this disclosure employs DNA-PAINT technology, which possesses photobleach resistance and adjustable reaction kinetics to achieve optimal performance. Furthermore, where feasible, this disclosure uses nanobodies instead of traditional antibody pairs to reduce label size. As a baseline, this disclosure labels microtubules in COS-7 cells by overexpressing ALFA-labeled kinesin (Ensconsin), followed by immunolabeling with an ALFA nanobody (NbALFA)-conjugated DNA docking strand, and imaging using DNA-PAINT technology. Figure 11 (a) Compared to 3D-SMLM, me4Pi-SMLM's enhanced axial resolution can more effectively resolve annular structures of single and bundled microtubules. Figure 11 (b) Localization analysis of molecules emitting more than 10 frames showed that, in cells with approximately 5500 photons, the localization accuracy of me4Pi-SMLM was 2-3 nm. Figure 11 (c and d in the text). Furthermore, the 3D positioning accuracy estimate for DAFL (Difference between adjacent frame positioning points) is 2.60 nm. Figure 12 In the a), the FRC (Fourier ring correlation) resolution is better than 10 nm in every dimension. Figure 12 (b) in the middle.

[0082] Next, the nuclear pore complex (NPC) in immobilized U-2 OS cells was imaged. Nup96 is an octet symmetric NPC protein that forms two loop structures: a nucleoplasmic loop (NR) and a cytoplasmic loop (CR). Here, this disclosure describes the construction of a homozygous knock-in U-2 OS cell line in which Nup96 was endogenously labeled with an ALFA marker and DNA-paint imaging was performed. Figure 13 (e). Compared to 3D-SMLM, me4Pi-SMLM can more clearly distinguish the nucleoplasmic loop and the cytoplasmic loop (e). Figure 13 f in the text. By using advanced algorithms to perform particle averaging on 500 nuclear pore complexes, me4Pi-SMLM visualization revealed nearly 32 Nup96 copies (f in the text). Figure 13The imaging performance of the me4Pi-SMLM is comparable to that of the traditional 4Pi-SMLM and the angstrom-level precision of the MINSTED. In terms of structural averaging, the me4Pi-SMLM resolved two adjacent Nup96 proteins that were 10 nm apart laterally and 3 nm apart axially, indicating that its three-dimensional resolution is better than 10 nm.

[0083] Furthermore, this disclosure also includes imaging of the endoplasmic reticulum (ER) in immobilized COS-7 cells. me4Pi-SMLM reveals that the ER is a connected network composed of hollow tubular structures (60-100 nm in diameter) and sheet-like structures. Figure 13 The h in the figure shows detailed three-dimensional film profiles in both the xy and xz cross sections, which is difficult to distinguish using traditional super-resolution techniques. Figure 13 (i). Notably, the high axial resolution of me4Pi-SMLM allows for the clear visualization of sheet-like structures with a thickness of 30–50 nm, which are finer than endoplasmic reticulum tubes and consistent with the values ​​obtained by 4Pi-SMLM (i). Figure 13 (j) These observations, along with previous live-cell super-resolution imaging results, support the existence of peripheral endoplasmic reticulum sheets, but their function remains to be elucidated.

[0084] - Dual-color synchronous me4Pi-SMLM imaging

[0085] The high resolution of me4Pi-SMLM is invaluable for resolving subcellular structures. However, multicolor imaging is crucial for studying the spatial organization and interactions within organelles and protein complexes. To achieve this, this disclosure combines an established salvaged fluorescence method with me4Pi-SMLM for simultaneous two-color imaging. In two-color imaging, two fluorescent probes are used to image the cell; these probes should be selected to be excited by the same wavelength and to be spectrally proportional after color separation. For example, in DNA-PAINT at 552 nm excitation, Alexa Fluor 568 (AF568) and ATTO Rho11 were identified as spectrally compatible dyes paired with Cy3B. Figure 13 Alternatively, the combination of the two fluorescent probes mentioned above can also be a spectrally compatible dye of AF647 and CF660C. In COS-7 cells, microtubules and endoplasmic reticulum were co-labeled by stable expression of ALFA-tagged kinesin (ALFA-Ensconsin) and transient overexpression of mEmerald-tagged Sec61β, followed by immunolabeling with anti-mEmerald nanobodies and anti-ALFA nanobodies. Figure 14(a and b in the text). me4Pi-SMLM achieves clear spectral separation with minimal crosstalk, enabling visualization of the hollow centers of endoplasmic reticulum tubes and microtubules in both axial and lateral dimensions. Figure 14 (c and d in the text). To further validate this, specific nanobodies were used to label the outer membrane (Sec61β) and lumen (KDEL) of the endoplasmic reticulum. Figure 14 (e). High axial resolution effectively distinguished these closely adjacent compartments and resolved different endoplasmic reticulum substructures (e). Figure 14 fi in the middle.

[0086] - me4Pi-SMLM Whole-cell Imaging

[0087] The me4Pi-SMLM 100 overcomes axial phase repetition by generating astigmatism through cylindrical lenses (i.e., the first cylindrical lens CL1 and the second cylindrical lens CL2), thus enabling high-resolution imaging of thicker samples. The depth of field of the objective lens OBJ used in the me4Pi-SMLM 100 of this disclosure is approximately 1.2 μm. Therefore, for imaging volumes exceeding this thickness, axial sample scanning is required. To ensure sufficient overlap between optical slices for subsequent alignment, the sample is typically translated axially in steps of 500 nm.

[0088] To demonstrate its whole-cell imaging capabilities, this disclosure presents a visualization of synaptic complexes (SCs) in the nucleus of mouse spermatocytes. These important supramolecular structures mediate chromosome pairing, recombination, and precise segregation during meiosis. Through immunolabeling of the SYCP3 scaffold protein, me4Pi-SMLM clearly resolved fully paired chromosomes within the nucleus, clearly revealing the unique double helix structure of the SYCP3 substructure regardless of their spatial orientation and depth. Figure 15 In addition, the outer membrane of mitochondria in HeLa cells was imaged at a depth of 4.5 μm. The image was reconstructed from 7 optical slices, clearly resolving the membrane outline and complex interconnections, and no artifacts were detected. Figure 15 f and g in the text.

[0089] In summary, the super-resolution interferometric single-molecule localization microscopy imaging device and method based on specular reflection and a single objective lens disclosed herein can achieve resolutions below 10 nm in three-dimensional space and is applicable to label sizes in biological samples. The performance of the me4Pi-SMLM is comparable to that of the conventional 4Pi-SMLM, but significantly reduces hardware complexity and calibration difficulty. Notably, almost all existing 3D-SMLM devices can be upgraded to me4Pi-SMLM by simply adding the modulation mirror M9 and the inexpensive piezoelectric actuator 124A as described in this disclosure. This contrasts sharply with the conventional 4Pi-SMLM, which requires extensive expertise and substantial financial investment. With its simplicity, stable performance, and wide accessibility, the me4Pi-SMLM holds the promise of becoming a transformative platform in the field of nanoscale bioimaging. Furthermore, in this disclosure, the me4Pi-SMLM achieves superior axial localization specifically through axial illumination interferometry, but can also be seamlessly integrated with transverse illumination interferometry to improve resolution in all dimensions. This strategy has two key advantages: (1) molecular localization in all dimensions is determined directly by the photon emission of molecules relative to the illumination pattern, rather than by the PSF pattern, and is therefore essentially unaffected by optical aberrations; (2) since localization is defined by the illumination wavelength, chromatic aberration in multicolor imaging is eliminated. Combined with an advanced labeling strategy that minimizes probe size and improves efficiency, me4Pi-SMLM enables multichannel, molecular-scale imaging in a variety of biological scenarios.

[0090] It should be understood that, in the various embodiments of this disclosure, the signal control processing system may include a processor. The processor may be a Central Processing Unit (CPU), or other general-purpose processors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. It should be noted that the processor may also integrate memory units and / or cache units for storage.

[0091] In implementation, each step of the above method can be completed by integrated logic circuits in the processor's hardware or by instructions in software. The steps of the method disclosed in this embodiment can be directly implemented by a hardware processor, or by a combination of hardware and software modules within the processor. The software modules can reside in readily available storage media in the art, such as random access memory, flash memory, read-only memory, programmable read-only memory, electrically erasable programmable memory, or registers. This storage medium is located in memory; the processor reads information from the memory and, in conjunction with its hardware, completes the steps of the above method. To avoid repetition, detailed descriptions are omitted here.

[0092] The above description is merely a preferred embodiment of this disclosure and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of this disclosure is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the above-described concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features disclosed in this disclosure that have similar functions.

[0093] Furthermore, while the operations are described in a specific order, this should not be construed as requiring these operations to be performed in the specific order shown or in sequential order. In certain circumstances, multitasking and parallel processing may be advantageous. Similarly, while several specific implementation details are included in the above discussion, these should not be construed as limiting the scope of the invention. Certain features described in the context of individual embodiments may also be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment may also be implemented individually or in any suitable sub-combination in multiple embodiments.

[0094] Although the subject matter has been described using language specific to structural features and / or methodological logic, it should be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or actions described above. Rather, the specific features and actions described above are merely illustrative examples of implementing the claims.

[0095] The foregoing has provided a detailed description of several embodiments of this disclosure. However, this disclosure is not limited to these specific embodiments. Those skilled in the art can make various variations and modifications based on the concept of this disclosure, and all such variations and modifications should fall within the scope of protection claimed by this disclosure.

Claims

1. An interferometric single-molecule localization microscopic imaging device based on specular reflection and a single objective lens, characterized in that, include: A light source system configured to generate excitation light for illumination; Lighting and modulation systems, including: Objective lens assembly, which includes a single objective lens; A sample stage, which is translatably positioned on the focal plane of the objective lens for placing the sample; A mirror assembly includes a modulation mirror positioned axially on the sample stage opposite to the objective lens, the modulation mirror being arranged parallel to and opposite the sample stage; wherein the excitation light from the light source system, after passing through the objective lens and the sample, is reflected back by the modulation mirror to generate an axial standing wave interference pattern, illuminating the sample on the focal plane; and A piezoelectric actuation assembly for a mirror is configured to actuate the modulation mirror relative to the sample in the axial direction of the objective lens during a single positioning cycle, thereby obtaining the standing wave interference pattern corresponding to at least three phases; and A fluorescence imaging system configured to acquire phase-shift images corresponding to each of the standing-wave interference patterns within a single positioning period for each single molecule of the sample, thereby obtaining a fluorescence signal comprising at least three phase-shift images; and A signal control and processing system is configured to control the light source system to generate the excitation light, control the actuation of the mirror piezoelectric actuator component on the modulation mirror, and control the imaging of the fluorescence imaging system, and to locate each single molecule of the sample based on the fluorescence signal obtained by the fluorescence imaging system, thereby reconstructing an image of the sample.

2. The interferometric single-molecule localization microscopic imaging device according to claim 1, characterized in that, The fluorescence imaging system includes a cylindrical lens for introducing astigmatism into the acquired phase-shifted image.

3. The interferometric single-molecule localization microscopic imaging device according to claim 2, characterized in that, The signal control processing system is configured as follows: The phase-shifted images are superimposed and summed at least three of them; For the fluorescence image obtained after superposition, the lateral position coordinates of the single molecule in the XY plane and the width of the point diffusion function are estimated by Gaussian fitting. The initial axial position of the single molecule along the Z-axis is determined by combining the point diffusion function width with the astigmatism. Calculate the intensity of at least three phase-shifted images and extract axial phase information along the Z-axis; as well as By using linear regression to correlate the axial phase information with the preliminary axial position, the precise axial position coordinates of the single molecule along the Z-axis are determined.

4. The interferometric single-molecule localization microscopic imaging device according to any one of claims 1 to 3, characterized in that, The mirror assembly further includes a mirror support, and the mirror piezoelectric actuator assembly includes a piezoelectric actuator fixed to the mirror support. The modulation mirror is mounted on the piezoelectric actuator. The signal control processing system can actuate the modulation mirror by controlling the piezoelectric actuator to actuate the axial translation of the mirror support.

5. The interferometric single-molecule localization microscopic imaging device according to any one of claims 1 to 3, characterized in that, The light source system includes a first laser, a first reflector, a second reflector, a second laser, a third reflector, and a first dichroic mirror, a fourth reflector, a fifth reflector, an acousto-optic tunable filter, a sixth reflector, a seventh reflector, a coupling lens, and a single-mode optical fiber arranged along the optical path. The excitation light from the first laser, after being reflected sequentially by the first and second reflectors, and the excitation light from the second laser, after being reflected sequentially by the third reflector, are combined by the first dichroic mirror. The combined excitation beam is then reflected sequentially by the fourth and fifth reflectors and modulated by the acousto-optic tunable filter. The modulated excitation light is then reflected sequentially by the sixth and seventh reflectors and coupled into the single-mode optical fiber through the coupling lens.

6. The interferometric single-molecule localization microscopic imaging device according to claim 5, characterized in that, The illumination and modulation system also includes a first lens, a second lens, an eighth reflecting mirror, a third lens, a fourth lens, and a second dichroic mirror arranged sequentially along the optical path; The excitation light output from the single-mode optical fiber is collimated by the first lens, and then reflected and expanded by the second lens, the eighth mirror, and the third lens in sequence. Finally, it is focused by the fourth lens and reflected by the second dichroic mirror, and then guided to the focal plane of the objective lens.

7. The interferometric single-molecule localization microscopic imaging device according to claim 6, characterized in that, The fluorescence imaging system includes the objective lens, the second dichroic mirror, the first filter, the tube lens, the tenth reflecting mirror, and the fluorescence imaging assembly arranged sequentially along the optical path. The fluorescence imaging assembly includes a first fluorescence imaging assembly for monochromatic imaging and / or a second fluorescence imaging assembly for dual-color imaging. The second dichroic mirror is configured to separate the generated fluorescence signal from the excitation light. The first fluorescence imaging component includes a first cylindrical lens, a second filter, and a first camera; the separated fluorescence signal is reflected by the tenth mirror, then astigmatism is introduced by the first cylindrical lens, and subsequently focused onto the first camera by the second filter; The second fluorescence imaging component includes a second cylindrical lens, an aperture, a fifth lens, a third dichroic mirror, a third filter, a sixth lens, an eleventh reflecting mirror, a fourth filter, a seventh lens, a twelfth reflecting mirror, a right-angle prism, and a second camera. The separated fluorescence signal is reflected by the tenth reflecting mirror, then astigmatism is introduced by the second cylindrical lens, followed by the aperture and the fifth lens, and then spectrally separated into conventional fluorescence and recovered fluorescence by the third dichroic mirror. The conventional fluorescence passes sequentially through the third filter and the sixth lens and is reflected by the eleventh reflecting mirror, while the recovered fluorescence passes sequentially through the fourth filter and the seventh lens and is reflected by the twelfth reflecting mirror. The light beams reflected by the eleventh reflecting mirror and the twelfth reflecting mirror are guided to the second camera by the right-angle prism.

8. The interferometric single-molecule localization microscopic imaging device according to claim 7, characterized in that, For the aforementioned dual-color imaging, the two fluorescent dyes are excited by the same wavelength of excitation light and their spectra are proportional after color separation.

9. The interferometric single-molecule localization microscopic imaging device according to claim 8, characterized in that, The fluorescent dyes used are spectrally compatible dyes of Cy3B and AF568, or spectrally compatible dyes of Cy3B and ATTO Rho11, or spectrally compatible dyes of AF647 and CF660C.

10. A method for performing microscopic imaging using the interferometric single-molecule localization microscopic imaging apparatus according to any one of claims 1 to 9, comprising: The signal control and processing system controls the light source system to emit modulated excitation light, which is then guided to the objective lens assembly of the illumination and modulation system. In a single positioning cycle, the signal control processing system controls the piezoelectric actuation component of the mirror to actuate the modulation mirror relative to the sample stage in the axial direction of the objective lens, thereby obtaining the standing wave interference pattern corresponding to at least three phases; For each single molecule of the sample, the fluorescence imaging system collects phase-shift images corresponding to each standing wave interference pattern in the single positioning period, thereby obtaining a fluorescence signal containing at least three fluorescence images; as well as The signal control processing system locates each single molecule of the sample based on the fluorescence signal obtained by the fluorescence imaging system, thereby reconstructing an image of the sample.

11. The method according to claim 10, characterized in that, The signal control processing system further includes: The phase-shifted images are superimposed and summed at least three of them; For the fluorescence image obtained after superposition, the lateral position coordinates and point diffusion width of the single molecule in the XY plane are estimated by Gaussian fitting; The initial axial position of the single molecule along the Z-axis is determined by combining the point diffusion function width with astigmatism. Calculate the intensity of at least three phase-shifted images and extract axial phase information along the Z-axis; as well as By using linear regression to correlate the axial phase information with the preliminary axial position, the precise axial position coordinates of the single molecule along the Z-axis are determined.

Images