Methods for enhancing imaging resolution on arrayed plasmonic nanostructures

Abstract

Angle-multiplex structured-illumination imaging can be uniquely enabled by arrayed plasmonic nanostructures. The arrayed plasmonic nanostructures can be utilized as an incidence angle-multiplex near-field modulation mask and can achieve a resolution gain over the diffraction limit. Angle-multiplex structured-illumination (AMSI) can be implemented as a general technique with any arrayed plasmonic nanostructures to empower them with enhanced-resolution fluorescence in addition to label-free plasmonic sensing and imaging.

Description

Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087METHODS FOR ENHANCING IMAGING RESOLUTION ON ARRAYED PLASMONIC NANOSTRUCTURES BACKGROUND

[0001] This application claims priority to U. S. Provisional Patent Application Serial No. 63 / 710,152, entitled “Methods for Enhancing Imaging Resolution on Arrayed Plasmonic Nanostructures,” filed October 22, 2024, the entire contents of which are hereby incorporated by reference.

[0002] This invention was made with government support under grant R01 EB-030623 awarded by the National Institutes of Health, The government has certain rights in the invention.

[0003] This disclosure pertains to imaging of nanoscale objects using arrayed plasmonic nanostructures.

[0004] Label-free optical detection techniques for nanoscale biological objects (e.g. plasma proteins, vesicles) using standard microscopes have grown interest in the field of biophotonics, bioengineering and medical research. The scattering cross-section of a nanosphere of diameter d and relative index m is given by, oscwhere A islight wavelength and. is refractive index of surrounding medium. This equation accentuates the severe decrease of light scatering as the target size decreases. Various techniques, e.g,, interferometric scattering (iSCAT) microscopy, digital holographic microscopy, guided-mode resonance sensing, and interferometric reflectance imaging sensor (IRIS) are developed to detect tiny phase objects that contribute very’ litle refractive index variation in close surroundings. On the other hand, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR) based detection techniques have taken advantage of the exquisite index change sensitivity for surface adsorbates due to the underlying plasmonic effect. Plasmonic techniques shift the detection objective from the target to target-induced changes in the plasmonic scattering and absorption.

[0005] In general, plasmonic sensing relies on refractive index perturbation on the nanostructure surface and therefore is most sensitive to surface adsorbates. However, the spatial resolution is limited by the pitch of the underlying nanostructures (-460 nm) and ultimately by the optical diffraction limit, which makes it challenging to resolve individual nanoparticles within a cluster. Fluorescence imaging has also not benefited from the use of - 1 - 47028727Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087plasmonic imaging techniques. What is needed, therefore, are methods that enable label-free imaging with enhanced resolution in fluorescence imaging.SUMMARY

[0006] The present disclosure relates generally to methods for improving image resolution in fluorescence imaging using arrayed plasmonic nanostructures and a structured illumination technique,

[0007] A technique for plasmonic nano-aperture label-free imaging has been demonstrated as a label-free alternative for single nanoparticle analysis, allowing for nanoparticle counting, sizing, and three-dimensional localization. The technique (in preferred embodiments referred to as PANORAMA) utilizes arrayed plasmonic nanostructures engineered to provide refractive index sensitivity, plasmonic peak wavelength / width, and packing density' of individual nanostructure units, which are all critical parameters for good performance in practice. Because the imaging technique can be set up on a standard inverted fluorescence microscope, plasmonic and fluorescence imaging can be acquired on an integrated platform owing to the physical nature of localized surface plasmon resonance (LSPR), the plasmonic nano-aperture label-free imaging technique is particularly sensitive to nanoparticles around 100 nm or less, a size range suitable for analyzing small extracellular vesicles (sEVs) such as exosomes, which are emerging biomarkers for diseases including cancer.

[0008] When the plasmonic nano-aperture label-free imaging technique is used in a dynamic fashion, it is capable of tracking individual exosomes as they approach tire plasmonic nanostructures. A threshold sEV count has been observed as a highly sensitive and specific way to distinguish cancer from healthy in both animal models and human subjects. However, the spatial resolution of the technique is limited by the pitch of the underlying nanostructures (-460 nm) and ultimately by the optical diffraction limit, which makes it challenging to resolve individual nanoparticles within a cluster. Although it is possible to achieve single exosome counting if the cluster formation can be monitored in the time domain, the spatial distribution of each individual nanoparticle within the cluster is still unresolvable if only an end-point image is acquired. As sEVs range from -30-150 nm in size, an obvious ambiguity’ is whether the signal comes from a cluster of small sEVs or a single large sEV, if time resolved imaging is not implemented. sEVs are also known to carry' membrane -bound proteins and cargo molecules that are reflective of their parental cells which secreted them. Probing these various molecular species has attracted intense attention lately for its potential47028727Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087use in disease biomarker detection.

[0009] In particular disclosed herein is an angle-multiplex structured-illumination imaging approach that can be uniquely enabled by arrayed plasmonic nanostructures. Structured-illumination fluorescence imaging can be implemented by utilizing the arrayed plasmonic nanostructures as an incidence angle -multipl ex near-field modulation mask and can achieve an approximately 2.3-fold resolution gain over diffraction limit. Angle-multiplex structured-illumination (AMSI) can be implemented as a general technique with any arrayed plasmonic nanostructures to empower them with enhanced-resolution fluorescence in addition to label-free plasmonic sensing and imaging.

[0010] Exemplary' applications of the methods disclosed herein include single extracellular vesicle (of various sizes including exosome, exomere, and the like) detection, counting, sizing, and molecular profiling (both membrane biomarkers such as protein and internal cargo or intravesicular biomarkers such as DNA, RNA mRNA, microRNA, lipids, proteins and the like). The outcome of such profiling can be used for disease screening, detection, diagnostics, and monitoring. In particular, it can be usefill for early detection of various cancers and monitoring of disease progression / regression, treatment monitoring, and future surveillance. This technology' can also be applied to profile similarly sized biological / pharmaceutical objects such as viruses (for diagnostics) and lipid nanoparticles (LNP) for drug / vaccine delivery'. For virus detection and profiling, it can provide rapid screening and testing. For LNP profiling, it can assist in various pharmaceutical processes such as drug discovery' and manufacturing.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1(a) shows a schematic of an exemplary' arrayed gold nanodisk on invisible substrate (AGNIS).

[0012] FIG. 1(b) show's a schematic of a strategy for detection of small extracellular vesicles (sEVs) detection using a technique for plasmonic nano-aperture label-free imaging (PANORAMA).

[0013] FIG. 1(c) shows a scanning electron microscopy (SEM) image of an exemplary' AGNIS structure.

[0014] FIG. 1(d) shows a schematic of an exemplary optical setup for an anglemultiplex structured-illumination (AMSI) approach that uses an exemplary AGNIS structureAttorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087and a magnification of a portion of the exemplary optical setup.

[0015] FIG. 2(a) shows a schematic of a setup for angle scanning on an exemplary AGNIS in an exemplary angle-multiplex structured-illumination imaging approach.

[0016] FIG. 2(b) shows a series of raw illumination patterns generated from the incident angle scan of the exemplary AGNIS structure.

[0017] FIG. 2(c) shows a plot of cross-correlation values with the sequential frames from the incident angle scan.

[0018] FIG. 3(a) shows a SEM image of an exemplary AGNIS structure from a 2D top view (x-y).

[0019] FIG. 3(b) shows a SEM image of an exemplary AGNIS structure from a 3D perspective view (x-y-z) showing mushroom -like structure.

[0020] FIG. 4(a) shows an image of a raw background data frame recorded experimentally.

[0021] FIG. 4(b) shows a histogram plot of count with respect to the background intensity.

[0022] FIG. 5(a) shows detected sEVs obtained from ratioed image through PANORAMA in experiments using a plasma sample.

[0023] FIG. 5(b) shows a histogram of sEV contrast distribution in experiments using a plasma sample.

[0024] FIG. 6(a) shows a widefield image of fluorescent beads.

[0025] FIG. 6(b) shows a AMSI image of fluorescent beads.

[0026] FIG. 6(c) shows a boxplot of the FWHM of fluorescent beads detected in widefield and AMSI images.

[0027] FIG. 6(d) and 6(e) show magnified views of boxes shown in FIG. 6(a) and 6(b).

[0028] FIG. 6(f) shows intensity line profiles across lines in the boxes shown in FIG, 6(d) and 6(e).

[0029] FIG. 7(a) shows detected sEVs obtained though PANORAMA imaging.

[0030] FIG. 7(b) shows detected sEVs obtained through widefield imaging, - 4 -Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087

[0031] FIG. 7(c) shows detected sEVs obtained through AMSI.

[0032] FIG. 7(d) shows a comparison of FWHM of detected sEVs from FIG. 7(a)-7(c).

[0033] FIG. 8(a) shows detected sEVs obtained though PANORAMA imaging.

[0034] FIG. 8(b) shows detected sEVs obtained through widefield imaging.

[0035] FIG. 8(c) shows detected sEVs obtained through AMSI.

[0036] FIG. 8(d), 8(e), and 8(f) show magnified views of solid line boxes in FIG.8(a)-8(c).

[0037] FIG. 8(g), 8(h), and 8(i) show magnified views of dotted line boxes in FIG.8(a)-8(c).

[0038] FIG. 8(j) shows intensity profiles across lines in the boxes shown in FIG.8(d)-8(f).

[0039] FIG. 8(k) shows intensity profiles across lines in the boxes shown in FIG.8(g)-8(i).DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0040] The present disclosure relates to a method to enhance microscopy imaging resolution on arrayed plasmonic nanostructures and belongs to a general scientific field called superresolution. The enhanced resolution is achieved by taking images at multiple incident angles. Due to the intensity modulation by the arrayed plasmonic nanostructures, it naturally provides a “structured illumination” that is different from traditional imaging where the illumination is mostly uniform. When the incident angle is varied, the arrayed plasmonic nanostructures provide variable illumination patterns on the targets in close proximity with the nanostructures. A higher resolution image can be obtained by an image reconstruction process incorporating several images acquired at varying incident angle. The technique is called anglemultiplex structured illumination (AMSI).

[0041] Preferred embodiments disclosed herein utilize AMSI on arrayed plasmonic nanostructures with strong radiative coupling produced either by nanosphere lithography or electron beam lithography with high density. Tire arrayed plasmonic nanostructures are very-important to the performance of the techniques. Examples of arrayed plasmonic nanostructures that could be used herein include any suitable arrayed plasmonic nanoparticles, such as a- 5 -wmT2Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087nanoporous gold disk array, an alloy disk array, and a structure termed an arrayed gold nanodisks on invisible substrates (AGNIS), The imaging can be done either in transmission or reflection modes, using bright-field (either broadband light or narrowband light) or fluorescence.

[0042] Preferred embodiments may utilize a custom-made nanostructured surface referred to as arrayed gold nanodisks on invisible substrates (AGNIS) that features an array of 360 nm diameter gold nanodisks with -460 nm pitch sitting on glass pillars. The AGNIS structure provides a plasmonically modulated nano-aperture in response to surface refractive index changes.

[0043] FIG. 1(a) shows a schematic of an exemplary unit cell of arrayed gold nanodisks on invisible substrate (AGNIS), according to preferred embodiments disclosed herein. The structure in this preferred embodiment is an array of gold (Au) nanodisks on glass substrate where the individual nanodisks are 360 nm in diameter, 80 nm in thickness and 100 nm in edge-to-edge distance.

[0044] FIG. 1 (b) shows a schematic of the preferred process for detection of sEVs using the techniques described in preferred embodiments herein.

[0045] FIG. 1(c) shows a scanning electron microscopy (SEM) image of an exemplary AGNIS structure, where individual gold disks sit on top of glass pillars. The LSPR extinction peak of AGNIS is at — 630 nm in air and - 710 nm in water. Compared to a single Au disk of the same geometry, the LSPR peak of AGNIS features a significant blueshift attributed to arrayed radiative coupling and the substrate undercut. The blueshifted LSPR permits the use of visible light instead of near-infrared and provides a number of benefits such as resolution, high-sensitivity detectors, and the like.

[0046] FIG. 1 (d) shows a schematic of an exemplary optical setup for use according to preferred embodiments herein, with closer view of the circled area. As shown in FIG. 1(d), the exemplary optical setup includes halogen lamp 1, bandpass filter 2 (e.g. 660±10 nm), condenser 3, sample 4, objective lens 5, tube lens 6, dichroic mirror 7, scanner 8, laser fiber 9, mirror 10, and camera 11.

[0047] FIG. 2 shows a schematic of an angle-multiplex structured-illumination imaging approach, including a schematic of the angle scanning on the AGNIS in FIG. 2(a), a- 6 -wmT2Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087stack of raw illumination patterns generated by the AGN IS corresponding to the incident angle scan in FIG. 2(b), and a plot of cross-correlation values with the sequential frames in FIG. 2(c),

[0048] Preferred embodiments described herein relate to a label-free nanoparticle imaging technique particularly suitable for detecting and counting small extracellular vesicles (sEVs) such as exosomes (and referred to as PANORAMA), in an integrated platform that incorporates angle-multiplex structured-illumination (AMSI). Instead of acquiring standard wide-field fluorescence, AMSI explicitly takes advantage of the plasmonic nanostructures (AGNIS) as a variable amplitude mask to produce the necessary images required for image reconstruction. In this approach, AGNIS plays a dual role: it provides the essential plasmonic index sensitivity in bright-field microscopy as well as the speckle illumination patterns for AMSI. The same sEV population has been imaged first by PANORAMA in a label-free fashion, and then by AMSI for additional fluorescence and resolution improvement. The former step generates single sEV counts and size and the latter molecular information regarding surface and / or cargo biomarkers. PANORAMA-AMSI can be implemented as a general technique with any arrayed plasmonic nanostructures to empower them with enhanced-resolution fluorescence in addition to label-free plasmonic sensing and imaging.

[0049] Preferred embodiments described herein utilize a high-density nanodisk array, a polycrystalline array comprising nanodisks of gold, gold / silver alloy, or silver. In preferred embodiments the nanoparticles or nanodisks may be shaped as circles, ovals, squares, triangles, rods, diamonds, or ellipses. In preferred embodiments the nanoparticles may be nanoporous gold disks. In preferred embodiments, the nanostructures in the array measure between 100 and 1000 nm in unit size or diameter, preferably about 360 nm in unit size or diameter, and 20 to 150 nm in thickness, preferably about 50 nm in thickness. Tire edge-to-edge distance between nanostructures (i.e., the gap size) can vary depending on tire unit size of the nanostructures but the edge-to-edge distance should always be less than the unit size or diameter. In some embodiments the nanodisks in the array are positioned apart from each other by about 35 nm in edge-to-edge distance, for exemplary’ 130 nm diameter disks, or up to 100 nm in edge-to-edge distance between exemplary- 360 nm disks. In some embodiments the substrate beneath the nanodisks in the array is partially removed, so each nanodisk is positioned on what is essentially a post of substrate. Undercut nanodisks have higher sensitivity-, but nonundercut substrates will work at a longer wavelength. When the nanodisks are undercut, the substrate posts preferably have diameters of about 200 nm and heights of about 150 nm.- 7 - 47028727Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087

[0050] In preferred embodiments, the present methods and techniques have been demonstrated on a high-density arrayed nanodisks on an “invisible” substrate. Hie arrayed nanodisks may be made of gold, a gold / silver alloy, or silver. In preferred embodiments the nanodisks may be shaped as circles, ovals, squares, triangles, rods, diamonds, or ellipses. In preferred embodiments, the nanodisks in the array measure between 100 and 1000 nm in diameter, preferably about 360 nm in diameter, and 20 to 150 nm in thickness, preferably about 50 nm in thickness. Tire edge-to-edge distance between nanodisks can vary depending on the disk diameter. In some embodiments the nanodisks in the array are positioned apart from each other by about 35 nm in edge-to-edge distance, for exemplary 130 nm diameter disks, or up to 100 nm in edge-to-edge distance between exemplar} / 360 nm disks. The high-density nanodisk array may be fabricated using nanosphere lithography followed by a self-aligned substrate undercut. In particular, a large portion of the glass substrate under the gold nanodisks is removed, which results in the nanodisks sitting on “posts” where the posts have a diameter of about 200 nm and a height of about 150 nm.

[0051] In preferred embodiments utilizing arrayed gold nanodisks, the transformation of arrayed gold nanodisks (AGN) to arrayed gold nanodisks on invisible substrates (AGNIS) utilizes an undercut process that has been studied using a 460 nm pitch array. Various degrees of undercut are obtained by stopping the etching process at selected times, which produces a series of varying extinction spectra and corresponding varying SEM images. In preferred examples of the undercut process, the radial and vertical etch rate was calculated to be 1.28 nm / s and 2.11 nm / s, respectively based on SEM images. During successive undercuts, tire LSPR peak blue-shifted from 820 nm to a plateau value of 688 nm when the radial undercut distance reached 100 nm. In this example, since the nanodisk diameter was 350 nm, the nanodisk after the undercut process sat on underlying glass posts with a top diameter of 190 nm, which provided sufficient adhesion. Further undercut did not result in additional blue-shifts, suggesting the substrate effect was completely removed with a radial undercut distance of 100 nm. The greatest amount of radial undercut that was accomplished was —130 nm with glass posts having a top diameter of 90 nm, beyond which the nanodisks failed to adhere to the glass posts. Even after undercutting, the far-field, radiative coupling is still present for AGNIS although the substrate effect has been eliminated. For the AGN before undercut, a high energy mode at -551 nm was previously identified as a split mode due to the asymmetric superstate / substrate configuration. This peak gradually diminished during the undercut process and disappeared eventually at 100 nm radial undercut, providing anAttorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087independent proof that the substrate effect had been entirely removed.

[0052] In preferred embodiments the high-density nanodisk array features a significantly blue-shifted LSPR extinction peak of at least 688 nm in air due to both far-field plasmonic coupling and substrate undercut. Tire blue-shift originates from far-field radiative coupling among individual nanodisks, mode split due to symmetry breaking of the superstate and substrate refractive indeces, or the partial removal of underneath substrate materials. The blue-shifted LSPR peak provides better diffraction-limited resolution and utilization of high quantum yield in silicon-based cameras. In alternative embodiments, substrate undercut in the nanodisk array might not be necessary when the nanodisks are made smaller. However, smaller nanodisks exhibit less radiative coupling, so the magnitude of blue-shift might not be sufficient. Therefore, undercut still represents an indispensable means and an additional "‘knob” to finetune and achieve the optimal blue-shifts. The current ultra near-field imaging methods have the ability to image dielectric nanoparticles as small as 25 nm using, in preferred embodiments, a standard transmission bright-field microscope with a tungsten-halogen lamp. In addition to ultrahigh sensitivity to deep sub- 100 nanoparticles, the current techniques can also provide their size information. Furthermore, using the arrival time difference in a dynamic imaging mode, individual nanoparticles in a cluster with interparticle distance well below the diffraction limit of the current optical system (330 nm) can be counted. Moreover, the longitudinal distance between a nanoparticle and high-density nanodisk array can be monitored using the dynamic imaging mode.

[0053] Preferred embodiments described herein relate to a method for visualization and detection of structures of interest, which may be any particles or molecules or other chemical or biological structures, by first illuminating an array of nanostructures with bright field illumination passed through one or more of a condenser and a bandpass filter to produce transmitted light. The nanostructures preferably comprise gold, a gold / silver alloy, nanoporous gold, or silver, and are about 100 nm to about 1000 nm in unit size or diameter and about 20 nm to about 150 nm in thickness. In additional preferred embodiments, the nanostructures are any preferred nanoparticles, such as nanodisks. Tire edge-to-edge distance between nanostructures is preferably less than the unit size or diameter of the nanostructures, and the nanostructures have a blue-shifted plasmon resonance peak of at least 688 nm in air. In additional preferred embodiments, the nanostructures in the array are gold nanodisks with a diameter of about 360 nm and a pitch of about 460 nm, and the nanodisks are positioned on_ 9 _47028727Attomey DocketNo.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087undercut glass pillars. The transmitted light is preferably captured with an objective lens and a camera to produce a background image of the array of nanostructures.

[0054] In further preferred steps in the method, the array of nanostructures is exposed to a sample that contains the structures of interest, to produce a targeted array of nanostructures. The targeted array of nanostructures is preferably illuminated with bright field illumination passed through one or more of the condenser and the bandpass filter to produce sample transmitted light, lire sample transmitted light is then preferably captured with the obj ective lens and the camera to produce a sample image of the targeted array of nanostructures. The sample image of the targeted array of nanostructures shows the structures of interest located in proximity to the targeted array of nanostructures. Here, “in proximity to"’ means close enough to be viewed in the sample image but not necessarily in contact with or bound to the array of nanostructures. In further preferred steps, the sample image is divided by the background image to produce a ratioed plasmonic sample image.

[0055] In further subsequent steps in the preferred embodiments of the method, the targeted array of nanostructures is then exposed to detection elements. In some preferred embodiments, the sample is exposed to detection elements before it is exposed to the array of nanostructures. Preferred embodiments of the detection elements may target either target membrane biomarkers or intravesicular biomarkers. In preferred embodiments, the detection elements may be any fluorophore labeled antibodies, aptamers, oligonucleotides, or peptides. In certain preferred embodiments, the detection elements may be molecular beacon probes, Tire detection elements hybridize or chemically bind to the structures of interest and produce fluorescence. An excitation laser is then preferably projected through the objective lens to the targeted array of nanostructures at an incidence angle. A series of fluorescence emission patterns is then preferably captured from the targeted array of nanostructures incremen tally at various angles along the incidence angle using the objective lens. The series of fluorescence emission patterns can be processed using an image reconstruction process to produce an anglemultiplex structured-illumination sample image. Finally, according to preferred embodiments described herein, the ratioed plasmonic sample image is analyzed along with the multiplex structured-illumination sample image to visualize and detect the structures of interest present in the sample.

[0056] In additional preferred embodiments of the method described above, the structures of interest are biological particles such as small extracellular vesicles (sEVs) orAttorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087exosomes. The structures of interest may also be pathogens, such as bacteria or viruses. The structures may be any biological molecules of interest. In other preferred embodiments, the structures of interest may be lipid nanoparticles (LNP) for drug or vaccine delivery.. In additional preferred embodiments, the structures of interest can be tissues, cells, proteins or nucleic acids such as DNA, RNA, or microRNA.

[0057] In additional preferred embodiments, the method further comprises a step of functionalizing the array of nanostructures by attaching recognition elements to the nanostructures, prior to the step of exposing the array of nanostructures to the sample, to produce a functionalized array of nanostructures. The recognition elements are capable of capturing or binding to the structures of interest and may preferably comprise any suitable elements, such as aptamers, proteins, antibodies, or oligonucleotides, in addition to fluorescent labels. In subsequent steps in additional preferred embodiments, the functionalized array of nanostructures may be washed to remove any unbound material, after the step of exposing the array of nanostructures to a sample, to produce a further targeted array of nanostructures for visualization.

[0058] Additional preferred embodiments relate to a method for visualization and detection of structures of interest, comprising exposing an array of nanostructures to a sample, wherein the nanostructures comprise gold, a gold / silver alloy, nanoporous gold, or silver, wherein the nanostructures are about 100 nm to about 1000 nm in unit size and about 20 nm to about 150 nm in thickness, wherein the edge to edge distance between nanostructures is less than the unit size of the nanostructures, and wherein the nanostructures have a blue-shifted plasmon resonance peak of at least 688 nm in air, and wherein the sample comprises the structures of interest, to produce a targeted array of nanostructures. This preferred embodiment also includes a step of exposing the sample to detection elements before or after exposing the array of nanostructures to the sample, wherein the detection elements hybridize or chemically bind to the structures of interest and produce fluorescence. Further steps in this preferred embodiment include projecting an excitation laser through the objective lens to the targeted array of nanostructures at an incidence angle, capturing a series of fluorescence emission patterns from the targeted array of nanostructures incrementally at various angles along the incidence angle using the objective lens, processing the series of fluorescence emission patterns using an image reconstruction process to produce an angle-multiplex structured-illumination sample image, and analyzing the angle-multiplex structured-illumination sample image to- II - 47028727Attomey Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087visualize and detect the structures of interest present in the sample. Preferred embodiments of tiiis method may also include steps of functionalizing the array of nanostructures by attaching recognition elements to the nanostructures prior to the step of exposing the array of nanostructures to the sample to produce a functionalized array of nanostructures, wherein the recognition elements are capable of capturing or binding to the structures of interest and producing fluorescence. Preferred embodiments of this method may also include steps of obtaining a plasmonic sample image by illuminating the targeted array of nanostructures with bright field illumination passed through one or more of a condenser and a bandpass filter to produce sample transmitted light, capturing the sample transmitted light with an objective lens and a camera to produce a plasmonic sample image of the targeted array of nanostructures, wherein the plasmonic sample image of the targeted array of nanostructures shows the structures of interest located in proximity to the targeted array of nanostructures, and analyzing the plasmonic sample image with the angle-multiplex structured-illumination sample image to visualize and detect the structures of in terest present in the sample.EXAMPLE 1FABRICATION AND CHARACTERIZATION OF EXEMPLARY ARRAYED PLASMONIC NANOSTRUCTURES

[0059] An arrayed gold nanodisks on invisible substrate (AGNIS) structure is fabricated using nanosphere lithography with 460 nm diameter polystyrene beads (PSBs, Sigma). At first, polystyrene beads (460 nm, Sigma Aldrich, USA) were subjected to a cleaning process for surfactant removal and hydrophilic modification. The procedure involved immersing the beads in an ethanol-water mixture, followed by iterative centrifugation (5-6 cycles). Simultaneously, glass coverslips (No 1; VWR Crop, USA) measuring 60mm × 25mm underwent cleaning in Piranha solution for 1 hour, followed by acetone and isopropanol baths, with copious DI water rinsing and dried in a stream of clean nitrogen. A deposition of 2 nm of Cr and 80 nm of Au was executed on the clean coverslips via sputtering. A monolayer of polystyrene beads was deposited to the Au thin film using the Langmuir-Blodgett method. Oxygen plasma etching (O2 flow rate 50 seem, chamber temperature 21° C, chamber pressure 30 mTorr and RF power 25 W) was employed to reduce the diameter of the polystyrene beads to 360 nm, thus inducing localized surface resonance modes within the desired wavelength range. Subsequently, argon milling (Ar+ with normal angle of incidence, sample holder distance 30 cm and etch rate 0.62 nm / s) selectively removed the unmasked portion of the gold- 12 -wmT2Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087thin film, and polystyrene beads situated on top of the Au nanodisks were removed by flipping the substrate on the silicon wafer and subjecting it to a 20-minute sonication process in water. The silicon wafer was utilized for substrate friction during this removal step.

[0060] After removal of polystyrene beads, the gold nanodisks were immersed in a buffer hydro-fluoric acid to undercut the glass substrate beneath the disks. The duration of this step, precisely 85 seconds, was meticulously controlled to achieve the desired undercut, culminating in the fabrication of the nano structured AGNIS. The top view (SEM image) of the fabricated AGNIS is shown in FIG. 3(a). The nano-structured chip looks like an array of mushroom with the gold disks on the cover-glass and perspective SEM view is shown in FIG.3(b).EXAMPLE 2SETUP AND IMAGE PROCESSING

[0061] A schematic of an exemplary optical setup on a commercial inverted fluorescence microscope (Olympus 1X83) is shown in FIG. 1(d) where the white light from a tungsten -halogen lamp-house (U-LH100L3, Olympus) is filtered by a bandpass filter (FB660-10, Thorlabs) and then passed through a condenser (IX2-LWUCD, Olympus) for sample illumination. In this example, the transmitted light through the sample is collected with an infinity-corrected dry objective lens (UPlanSApo 40X / 0.95, Olympus) followed by a matched tube lens. The bright-field optical images are recorded using a sCMOS camera (C14440-20UP ORCA-Fusion, Hamamatsu) with an exposure time of 30 ms per frame and 100 frames are averaged to produce one image. If there is no sEV on to the AGNIS surface, the detected image (intensity ratioed) becomes equivalent to the contrast of gray background. When the sEVs come in close proximity, they are detected by the enhanced contrast above the gray background. Since the contrast is size dependent, a smaller sEV manifests lower contrast, while a larger sEV higher contrast. This contrast-based sizing scheme generates consistent size distribution compared to other commercially available nanoparticle analyzers, such as Nanosight (Malvern).

[0062] In operation, a background image is first recorded without sEV. Sample images are acquired at prescribed time after an experiment starts and targets gradually bind to the AGNIS surface, which typically takes an hour. The AGNIS is then washed, and another image is acquired to represent the after-wash sample image. To obtain the image, the sample- 13 - 47028727Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087image is divided by the background image, producing a ratioed image with a mean ~ 1. System noise is estimated by ratioing two background images which typically results in a mean ~1 and standard deviation of σ%. A detection threshold is selected as (3o+0.5) %.

[0063] Speckle-like illumination patterns have been employed for structured illumination fluorescence microscopy. In the general scheme, multiple spatially modulated intensity patterns I (x, y) are required for this purpose so that the average of the patterns becomes uniform ∑ᵢ Iᵢ(x,y) ~ constant, where N is the total number of patterns. Due to inherent disorderliness in the AGNIS arrangement, it can be considered as a variable intensity mask for speckle pattern illumination, and multiple patterns can be generated by scanning the excitation laser incidence angle.

[0064] As shown in FIG. 2(a), angle-scan can be achieved by lateral shift of a laser beam (Oxxius- Simply Light, Model: L6CC-CSB-1511, Power: 200m W) within the back aperture of a high-NA objective lens originally set up for total internal reflection fluorescence (TIRF) imaging. Instead of operating in the TIRF mode, the same setup (Olympus Cell-TIRF) is employed to scan the incidence angle without exceeding the critical angle, and fluorescence imaging is performed in reflection mode (Epi) by an objective lens (UPlanSApo 40X / 0.95, Olympus). Since the arrangement of the AGNIS is not perfectly ordered, the transmitted light mutually interferes and generates speckle-like patterns in close vicinity of the surface. When the incidence angle is scanned, the speckle-like illumination patterns Z;-(x, y) also vary’, The scanning is performed from 2 to 62 degrees with a 2.8-degree step and the corresponding illumination patterns are shown in FIG. 2(b) in a series. The cross-correlation value of the speckle patterns with the number of frames is plotted in FIG. 2(c) that quantifies how the patterns are spatially varying, and such variation leads to the desired phase-shifting and intensity modulation required for structured-illumination fluorescent imaging.EXAMPLE 3IMAGING OF SEVS FROM HUMAN PLASMA

[0065] A control experiment was first conducted to assess background fluctuations prior to recording the actual data. Firstly, two background images (frames without any sample) of the AGNIS substrate were recorded at ~1 min time interval. The experimentally recorded raw background data is shown in FIG. 4(a) which was eventually the optical image of tire AGNIS. Then, a ratioed image was obtained by dividing two background images to determine - 14 - 47028727Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087the background fluctuations of the specific camera being used. It yielded a mean of 1 and a standard deviation of 0.1, The histogram plot of count with respect to background intensity’ is shown in Fig. 4(b), where the pixel value 1 corresponds to the nominal background value (0% contrast). Based on the camera performance, a threshold contrast value was selected as 3 × 0.1×100% + 0.5% = 3.5%. Assuming Gaussian statistics, a contrast value larger than 2% indicates sEV detection.

[0066] Donor plasma sample (20 ul) was dispensed into a poly-dimethyl-siloxane (PDMS) well with AGNIS at the well bottom. Tire background / background histogram gave a mean of 1 with a standard deviation of 0.01, as discussed. So, the threshold was selected to be 1.035 (mean + 3 × standard deviation + 0.005) which is equivalent to 3.5% (Threshold value-1) x 100%) contrast.

[0067] As shown in FIG. 5(a), 430 sEVs were detected from the plasma sample after 60 minutes. The contrast distribution of all detected sEVs is displayed as a histogram in FIG. 5(b) with mean of 8.1% and standard deviation of 2.9 %. It has been demonstrated that sEV contrast highly correlates to its size and a histogram of size distribution can be obtained after calibration.EXAMPLE 4ANGLE-MULTIPLEX STRUCTURED-ILLUMINATION IMAGING

[0068] The angle-multiplex structured-illumination (AMSI) scheme was first demonstrated using fluorescent polystyrene beads (PSBs) of known size in order to characterize the resolution limit. Hence, polystyrene beads of 100 nm in diameter (Thermo Fisher Scientific) were used in 1 / 10000 dilution from stock solution in the imaging system described in the examples above. The PSBs were excited using a 561 nm laser and N = 22 frames in total were recorded by varying the incidence angle from 2° to 62° with 2.8° step. The 40X / 0.95 objective lens was employed both for excitation and fluorescent signal collection, Tire recorded raw moire frames were processed with a suitable reconstruction algorithm, namely, an ImageJ plugin, for the structured illumination imaging, The widefield image of the 100 nm PSBs is shown in FIG. 6(a) and the corresponding AMSI image is shown in FIG. 6(b).

[0069] Using a program for super-resolution microscopy image analysis, namely, ThunderSTORM, an ImageJ plugin typically used, full width at half maximum (FWHM) was- 15 -wmT2Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087extracted for every particle detected in the widefield and AMSI images. FIG. 6(c) shows a boxplot of the FWHM of fluorescent PSBs detected in widefield and AMSI images. As shown in FIG. 6(c), the average FWHM for the AMSI image was -136 nm, while for the widefield image, it was - 351 nm. The theoretical diffraction limit for the imaging configuration was 313 nm. FIGs. 6(d, e) show the magnified views of the white box region from FIGs. 6(a, b), respectively. The intensity linescans across single PSBs identified in FIGs.6(d, e) are shown in FIG. 6(f) where the outer curve represents the average from the widefield image and the inner curve the AMSI image.

[0070] AMSI was next demonstrated with plasma samples where the sEVs within the plasma were targeted by a panel of four biomarkers. The surface modification of the AGNIS substrate was conducted in three sequential steps. Initially, the AGNIS substrate was immersed in a water solution comprising a 1:3 mixture of long (MW. 1 kDa) biotin PEG thiol and short (MW, 0.2 kDa) methyl-PEG-thiol [MT(PEG)4] polymers. This incubated AGNIS substrate was subsequently stored at 4°C for approximately 16 hours to facilitate the binding of the thiol groups with the AGNIS disks. In the second step, the modified substrate was incubated by a water solution of 3.3 pM neutravidin and stored at 4°C for two hours. Finally, the substrate was immersed in a mixture water solution containing 0.5 mg / mL of CD9, CD63, CD81 antibodies, and 2,5% BSA and stored at 4°C for an additional 2 hours to allow for antibody modification. Tire resulting chip was utilized for tire subsequent experiments.

[0071] Molecular beacon probes (MBPs) with sequences complementary to 4 miR, namely miR-126-3p, miR-222-3p, miR-31 and miR-21, shown in Table 1 below were prepared.Table 1- 16 - 47028727Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087

[0072] The MBPs were labeled with a cy3 fluorophore on the 5’ end and BH quencher on the 3' end. The plasma sample was mixed with the MBPs (250 nM of each probe) and incubated for 2 hours at 40°C temperature. A volume of 20 μl from the breast cancer donor plasma sample was utilized to incubate. Four distinct MBPs were mixed with the plasma sample in volumes of 2 μl each. This procedure was conducted in a light-restricted environment to prevent potential photobleaching of the molecular beacons. The resulting mixture was then subjected to incubation in a beaker filled with water at a temperature of 40°C. The heat during incubation facilitated the penetration of molecular beacons into sEVs and induced the unwinding of their hairpin structures to enable hybridization with the target sequences present within the vesicles. Probes that successfully hybridized with the target sequences remained in an elongated conformation, positioning the Cy3 fluorophore and black hole (BH) quencher far apart from each other. This structural change enabled the emission of a fluorescent signal to be detected, indicating successful hybridization.

[0073] The emission band-pass filter was chosen according to the peak emission wavelength of the fluorophore Cy3 ( — 570 nm). The same objective lens (40X / 0.95) and system combination as shown in FIG. 1(d) were used for recording the raw data with an exposure time of 400 ms. The AMSI images were acquired immediately after the image acquisition using the plasmonic nano-aperture label-free imaging technique (PANORAMA) described herein. FIGs.7(a) and 7(b) show the PANORAMA and widefield images. FIG. 7(c) shows a reconstructed angle multiplexed structured illumination (AMSI) image. To quantitatively assess the resolution of each image, the FWHM of all the detected particles in each image was calculated (using ThunderSTORM). As shown in FIG. 7(d), the PANORAMA image showed a FWHM of ~ 478 nm, which is close to the pitch of AGNIS. The widefield and AMSI showed a FWHM of ~306 nm and -138 nm, respectively, AMSI showed significant reduction in the FWTIM compared to both the PANORAMA and widefield fluorescence imaging.

[0074] To demonstrate the enhanced resolution provided by AMSI compared to PANORAMA and widefield fluorescence, two examples in boxes shown in FIGs. 8 (a, b, c) -taken from a cropped-out region in FIG. 5(a) ~ were selected for comparison. FIG. 8 shows experimental results for imaging sEVs using (a) PANORAMA, (b) widefield, and (c) reconstructed angle multiplexed structured illumination (AMSI) imaging. The boxed regions were selected as R1 for two nearby sEVs, shown in zoom in FIGs. 8 (d, e, f) and R2 for two indistinguishable sEVS in PANORAMA and widefield images that are clearly resolved inAttorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087AMSI as shown in zoom in FIGs. 8 (g, h, i). The intensity profiles across the dotted line of R1 and R2 region are plotted in FIGs. 8 (j, k), respectively. In both cases, significant reduction in the FWHM was observed from AMSI compared to both PANORAMA and widefield images.- 18 - 47028727

Claims

Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087WHAT IS CLAIMED IS:

1. A method for visualization and detection of structures of in terest, comprising:(a) illuminating an array of nanostructures with bright field illumination passed through one or more of a condenser and a bandpass filter to produce transmitted light, wherein the nanostructures comprise gold, a gold / silver alloy, nanoporous gold, or silver, wherein the nanostructures are about 100 nm to about 1000 nm in unit size and about 20 nm to about 150 nm in thickness, wherein the edge to edge distance between nanostructures is less than the unit size of the nanostructures, and wherein the nanostructures have a blue-shifted plasmon resonance peak of at least 688 nm in air;(b) capturing the transmitted light with an objective lens and a camera to produce a background image of the array of nanostructures;(c) exposing the array of nanostructures to a sample, wherein the sample comprises the structures of interest, to produce a targeted array of nanostructures;(d) illuminating the targeted array of nanostructures with bright field illumination passed through one or more of the condenser and the bandpass filter to produce sample transmitted light;(e) capturing the sample transmitted light with the objective lens and the camera to produce a sample image of the targeted array of nanostructures, wherein the sample image of tire targeted array of nanostructures shows the structures of interest located in proximity to the targeted array of nanostructures;(f) dividing the sample image by the background image to produce a ratioed plasmonic sample image;(g) exposing the targeted array of nanostructures to detection elements, wherein the detection elements hybridize or chemically bind to the structures of interest and produce fluorescence;(h) projecting an excitation laser through the objective lens to the targeted array of nanostructures at an incidence angle;Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087(i) capturing a series of fluorescence emission patterns from the targeted array of nanostructures incrementally at various angles along the incidence angle using tire objective lens;(j) processing the series of fluorescence emission patterns using an image reconstruction process to produce an angle-multiplex structured- illumination sample image; and(k) analyzing the ratioed plasmonic sample image with the angle-multiplex structured-illumination sample image to visualize and detect tire structures of interest present in the sample.

2. The method of claim 1, wherein the structures of interest are biological particles or biological molecules.

3. The method of claim 2, wherein the biological particles or biological molecules of interest are small extracellular vesicles.

4. The method of claim 2, wherein the biological particles or biological molecules of interest are exosomes,5. The method of claim 1, wherein the structures of interest are tissues, cells, pathogens, lipid nanoparticles, proteins, or nucleic acids.

6. The method of claim 1, wherein the nanostructures are nanodisks.

7. The method of claim 6, wherein the nanodisks are gold nanodisks with a diameter of about 360 nm and a pitch of about 460 nm, and wherein the nanodisks are positioned on undercut glass pillars.

8. The method of claim 1, wherein the detection elements comprise molecular beacon probes.

9. The method of claim 1, further comprising a step of functionalizing the array of nanostructures by attaching recognition elements to the nanostructures prior to the step of exposing the array of nanostructures to the sample to produce a functionalized array of nanostructures, wherein the recognition elements are capable of capturing or binding to the structures of in terest.Attorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-08710. The method of claim 9, further comprising a step of washing the functionalized array of nanostructures to remove any unbound material after the step of exposing the array of nanostructures to a sample, to produce a further targeted array of nanostructures for visualization.

11. The method of claim 9, wherein the recognition elements comprise fluorescent labels.

12. A method for visualization and detection of structures of interest, comprising:(a) exposing an array of nanostructures to a sample, wherein the sample comprises the structures of interest, to produce a targeted array of nanostructures;(b) exposing the sample to detection elements before or after exposing the array of nanostructures to the sample, wherein the detection elements hybridize or chemically bind to the structures of interest and produce fluorescence;(c) projecting an excitation laser through the objective lens to the targeted array of nanostructures at an incidence angle;(d) capturing a series of fluorescence emission patterns from the targeted array of nanostructures incrementally at various angles along the incidence angle using the objective lens;(e) processing the series of fluorescence emission patterns using an image reconstruction process to produce an angle-multiplex structured- illumination sample image; and(k) analyzing the angle -multiplex structured-illumination sample image to visualize and detect the structures of interest present in the sample.

13. The method of claim 12, wherein the structures of interest are biological particles or biological molecules.

14. The method of claim 12, wherein the nanostructures compose gold, a gold / silver alloy, nanoporous gold, or silver, wherein the nanostructures are about 100 nm toAttorney Docket No.: UNIH-0317WO (109293.00327) PATENT APPLICATION UHID 2024-087about 1000 nm in unit size and about 20 nm to about 150 nm in thickness, wherein the edge to edge distance between nanostructures is less than the unit size of the nanostructures, and wherein the nanostructures have a blue-shifted plasmon resonance peak of at least 688 nm in air.

15. The method of claim 14, wherein the nanostructures are gold nanodisks with a diameter of about 360 nm and a pitch of about 460 nm, and wherein the nanodisks are positioned on undercut glass pillars.

16. The method of claim 12, further comprising a step of functionalizing the array of nanostructures by attaching recognition elements to the nanostructures prior to the step of exposing the array of nanostructures to the sample to produce a functionalized array of nanostructures, wherein the recognition elements are capable of capturing or binding to the structures of interest and producing fluorescence.

17. The method of claim 12, further comprising the steps of obtaining a plasmonic sample image by illuminating the targeted array of nanostructures with bright field illumination passed through one or more of a condenser and a bandpass filter to produce sample transmitted light, capturing the sample transmitted light with an objective lens and a camera to produce a plasmonic sample image of the targeted array of nanostructures, wherein the plasmonic sample image of the targeted array of nanostructures shows the structures of interest located in proximity to the targeted array of nanostructures, and analyzing the plasmonic sample image with the angle-multiplex structured-illumination sample image to visualize and detect the structures of in terest present in the sample.- 22 -

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