Apparatuses and methods for the three-dimensional imaging of an object

Abstract

A 3D single-molecule super-resolution imaging technique is disclosed herein. In particular, the techniques described herein combine an optical platform including a tilted single-objective dithered light sheet illumination, a microfluidic system, and point spread function engineering to achieve precise 3D single-molecule super-resolution imaging of subcellular structures (e.g., a sample (110)) with easy control of the extracellular environment. In certain embodiments, the single-objective dithered light sheet (102) of the optical setup (100) includes a plurality of galvanometric mirrors (116, 118, 124) and a tunable lens (122) that may steer a light sheet (102) for optimized optical sectioning of the sample (110). In some embodiments, the sample (110) is positioned in the microfluidic device (160) during imaging. In this way, traditional challenges (e.g., high out-of-focus background fluorescence and lack of environmental control) generally facing single-molecule localization microscopy may be overcome.

Description

APPARATUSES AND METHODS FOR THE THREE-DIMENSIONAL IMAGINGOF AN OBJECTSTATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under Grant No. R00GM134187 and R35GM155365 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0002] The subject matter disclosed herein relates to systems, methods, and techniques of three-dimensional imaging of an object within various imaging chambers, such as a microfluidic chip.

[0003] Single-molecule localization microscopy (SMLM) may provide information of subcellular structures at the nanoscale. For instance, innovations in single-molecule super-resolution (SR) imaging allows for localization of individual fluorescent molecules, surpassing the resolution set by the diffraction limit of light. Further, sequential DNA Points Accumulation for Imaging in Nanoscale Topography (DNA- PAINT) may provide localization-based SR imaging. However, current SMLM techniques may suffer from high fluorescence background, slow acquisition speeds, and limited three-dimensional (3D) imaging capabilities. Further, current imaging of objects in complex environments (e.g.. biological environments) may be limited due to inflexible and complex microfluidic incorporation with standard SMLM techniques and / or difficulty achieving precise extracellular control. As such, there is a need for 3D single-molecule SR imaging of complex biological environments in tunable microfluidic systems.

[0004] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present techniques, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of the various aspects of thepresent disclosure. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0005] This invention was funded in part by the Robert A. Welch Foundation under Welch Grant No. C-2064-20210327.BRIEF DESCRIPTION

[0006] A summary of certain embodiments disclosed herein is set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of these certain embodiments and that these aspects are not intended to limit the scope of this disclosure. Indeed, this disclosure may encompass a variety of aspects that may not be set forth below.

[0007] The present disclosure is directed to a 3D single-molecule super-resolution (SR) imaging technique. In particular, the techniques described herein combine an optical platform including a tilted single-objective dithered light sheet illumination, a microfluidic system, and point spread function engineering to achieve precise 3D single-molecule SR imaging of a sample (e.g.. subcellular structures) with easy control of the extracellular environment. In certain embodiments, the single-objective dithered light sheet of the optical setup includes a plurality of galvanometric mirrors and a tunable lens that may steer a light sheet for optimized optical sectioning of the sample. In some embodiments, the sample is positioned in the microfluidic device during imaging. The microfluidic system may be fabricated according to methods described herein. In this way, traditional challenges (e.g., high out-of-focus background fluorescence) generally facing SMLM may be overcome.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

[0009] FIG. 1 is a schematic illustration of an optical setup including an epiillumination path and a light sheet (LS) illumination path separated by a flip mirror;

[0010] FIG. 2 is a scanning electron micrograph of the microfluidic chip insert of FIG. 2A that may be used for LS reflection, with a scale bar of 50 pm, in accordance with aspects of the present approach;

[0011] FIG. 3 is an image of a thin end of the LS imaged in a fluorescent solution with a scale bar 10 pm, in accordance with aspects of the present approach;

[0012] FIG. 4 is a graph of including line scans of a diffraction-limited signal of lamin Bl in U2OS cells in epi -illumination and LS illumination illustrating a distance (pm) versus a normalized signal, in accordance with aspects of the present approach;

[0013] FIG. 5 is a graph of including line scans of a single-molecule signal of lamin Bl in U2OS cells in epi-illumination and LS illumination illustrating a distance (pm) versus a normalized signal, in accordance with aspects of the present approach;

[0014] FIG. 6 is a graph of localization precision in a lateral dimension for epi- illumination and LS illumination, with aspects of the present approach;

[0015] FIG. 7 is a graph of localization precision in an axial dimension for epi- illumination and LS illumination., in accordance with aspects of the present approach;

[0016] FIG. 8 is a graph of a number of localizations per length of microtubule over time localized using Easy-DHPSF and DECODE, in accordance with aspects of the present approach;

[0017] FIG. 9 is a graph illustrating Gaussian fits to an example line scan across a nuclear rim of an entire U2OS cell nucleus labeled for lamin bl, LAP2, and lamin A / C, in accordance with aspects of the present approach;

[0018] FIG. 10 is a schematic illustration of a protocol used to fabricate channels out of PDMS using soft lithography with molds made from SU8-100 photoresist on Si wafers including schematic illustration of a 3D nanoprinted metalized insert beingpositioned inside of a PDMS channel, in accordance with aspects of the present approach; and

[0019] FIG. 11 is a schematic illustration of a protocol used to fabricate a PDMS insert including schematic illustration of a 3D nanoprinted metalized insert being positioned inside of the PDMS insert, in accordance with aspects of the present approach.DETAILED DESCRIPTION

[0020] When introducing elements of various embodiments of the present subject matter, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. One or more specific implementations will be described below. In an effort to provide a concise description of these implementations, not all features of an actual implementation are described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0021] As mentioned above, single-molecule localization microscopy (SMLM) provides tools for subcellular investigation at the nanoscale. In single-molecule superresolution (SR) imaging, individual fluorescent molecules are temporally separated and localized, thereby surpassing a resolution limit set by the diffraction limit of light. Exchange-PAINT (e.g., sequential DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT)) is a localization-based SR approach that involves labeling multiple targets of interest with antibodies conjugated with oligonucleotide strands (e.g., docking strands) and imaging the targets sequentially byintroducing complementary oligonucleotide-dye conjugated sequences (e.g., imager strands). As multiple structures can be labeled using different oligonucleotide pairs and imaged sequentially using the same fluorophore. offsets caused by chromatic aberrations typical in multi-color approaches can be avoided. One challenge with Exchange-PAINT is that it requires perfusion of the various imager strand solutions and buffers without disturbing the image acquisition process. Another challenge with this method is the increased fluorescence background from freely diffusing imager strands, which increases the achievable single-molecule localization precision. The requirement of low imager strand concentrations for reduced fluorescence background leads to long acquisition times in order to acquire sufficient localization density to achieve good resolution, especially when imaging multiple targets. Recently developed self-quenching fluorogenic DNA probes reduce background fluorescence in DNA- PAINT imaging, but suffer from an increased localization precision compared to conventional DNA-PAINT due to their short binding times which restrict photon collection.

[0022] Further, it is presently known, LS illumination may be defined as illumination of a sample with a thin sheet of light at an image plane. LS illumination may provide a reduced fluorescence background, phototoxicity, and / or photobleaching of fluorophores within the image plane. Additionally, certain LS approaches may require separate objectives (e g., more than one objective, multi-objective systems) for illumination and detection during single-molecule imaging of cells. However, it should be noted, certain LS approaches may suffer from optical complexity, low numerical aperture (NA) objectives, steric hindrance, inability to optically section adherent cells, incompatibility with microfluidic systems, and / or drift between separate objectives. Additionally, it is known that various single-objective LS designs have reduced complexity and limitations of multi-objective systems, however a portion of the various single-objective LS designs have been limited by beam thickness, a need for beam scanning, and / or may have a limited effective NA in a detection path. Further, the portion of the various single-objective LS designs may require post-processing due to an illumination beam that is not aligned to a detection axis.

[0023] Accordingly, the present disclosure provides a platform including steerable, dithered, single-objective tilted light sheet (LS) and a nanoprinted microfluidic chip combined with point spread function (PSF) engineering, deep learning, and active stabilization drift correction to achieve fast, accurate, and precise 3D single-molecule multi-target SR whole-cell imaging using Exchange-PAINT. In some embodiments, a single high-NA objective lens is used to focus a LS. In this manner, high photon collection efficiency without steric hindrance and relative drift is achieved. Further, optical sectioning of adherent cells cultured within microfluidic chips may be used while avoiding LS aberrations.

[0024] In certain embodiments, a LS of an optical excitation path of the platform may be formed by one or more cylindrical lens. As such, the light sheet may be steered (e.g., laterally and / or axially) by one or more galvanometric mirrors conjugated to the back focal plane of an objective. Further, in some embodiments, an additional galvanometric mirror conjugated to a sample plane may be used for dithering. Additionally and / or alternatively, a tunable lens may be conjugated to the back focal plane of the objective for focal steering. One or more optical lenses (of precalculated focal lengths) may be placed between one or more fundamental optical elements (e.g., cylindrical lens, galvanometric mirror, objective and the like) to tune one or more dimensions of the LS.

[0025] In some embodiments, one or more conventional optical lenses of precalculated focal lengths may be placed between fundamental optical elements to tune the dimensions of the light sheet in accordance with Equations 1-3. on 1It should be noted, that w is the width of the light sheet, is the thickness, and b is the confocal parameter. / j_6, as shown in Equation 1, refers to the focal lengths of one or more lenses placed in the optical path, fcyirefers to the focal length of the cylindrical lens, fobj refers to the focal length of the objective, d0refers to the size of the beam before entering the cylindrical lens. DobJ- refers to the back aperture of the objectivelens, and A refers to the wavelength of light being used. It should be noted, excitation lasers of tunable wavelengths may be used to allow the optical setup to adapt to multiple different applications.

[0026] In some embodiments, use of LS illumination for Exchange-PAINT imaging, allows freely diffusing imager strands (e.g., outside of the focal plane) to not be excited by the LS. As such, an intensity of a fluorescence background may be reduced, improving single-molecule localization precision. In this manner, tilting the LS may enable cellular imaging all the way down to a coverslip without special sample mounts. In addition, a single-objective LS is dithered to achieve homogeneous illumination and reduce shadowing artifacts. In certain embodiments, one or more microfluidic chips are formed. The microfluidic chips may consist of a polydimethylsiloxane (PDMS) channel with a 3D nanoprinted metalized insert. The 3D nanoprinted metalized insert may reflect the LS into the sample and enable convenient perfusion of solutions during multi-target Exchange-PAINT imaging. In some instances, it may be advantageous to use the microfluidic cell with the 3D nanoprinted metalized insert to allow customization of the microfluidic cell. For example, use of a customized microfluidic cell may reduce a need for complex reflection mechanisms (e.g., a micromirror, an AFM cantilever mirror, pyramidal microcavities, a glass prism, and a microfluidic chamber with metalized side walls) to redirect the LS into the sample. Further, the customized microfluidic device may provide active solution exchange, compatibility with epi -illumination, and / or compatibility' with various transmission microscopy techniques. It should be noted, that in some instances, the customizable 3D nanoprinted microfluidic insert in a PDMS channel may allow a user to specify a tilt angle of the LS and chip dimensions of the microfluidic cell. Further, the customizable 3D nanoprinted insert may provide a transparent top for flexible combination with other imaging modalities. For example, macroscopic 3D printing (or other additive manufacturing approach) may be used to create molds for PDMS inserts of customizable dimensions. A 3D nanoprinted metalized insert may be incorporated into the PDMS inserts to provide integration into any user-defined imaging chamber for flexible combination with various imaging modalities.

[0027] In some embodiments, SR imaging is extended to 3D by employing soTILT3D. SoTILT3D employs PSF engineering, where a shape of a PSF is modulated to encode axial information about a molecule of interest over several microns of axial range in a single slice. As such, several different PSFs have been identified for use in soTILT3D. In the present disclosure, a double-helix PSF (DH-PSF) is used. The DH-PSF is robust to aberrations, offers good 3D single-molecule localization precision, and requires relatively low computational costs.

[0028] With the foregoing in mind, soTILT3D combines PSF engineering with a deep learning based single-molecule localization software (DECODE) to enable localization of overlapping emitters in 3D. Due to enlarged footprints of engineered PSFs, conventional 3D imaging with engineered PSFs requires low emitter density to ensure that no overlap occurs between PSFs. It should be noted, combination of low imager strand concentrations (e.g., used to reduce fluorescence background) during Exchange- PAINT imaging and whole-cell 3D single-molecule SR imaging with engineered PSFs can be a time-consuming process, often taking multiple days to complete. In this manner, incorporation of a neural network for localization of overlapping PSFs may enable use of higher imager strand concentrations, leading to denser localizations and drastically increased imaging speeds. While previous work has used PAINT approaches and deep learning to localize 2D data, the disclosed approach implements a deep learning algorithm with PAINT and the DH-PSF to localize 3D data.

[0029] With the foregoing in mind, soTILT3D utilizes an active stabilization feedback loop to correct for drift during image acquisition and enable long-term imaging. In this manner, soTILT3D may combine a steerable, dithered, single-objective tilted LS with microfluidics, PSF engineering, deep learning, and active stabilization drift correction to create a flexible imaging platform for whole-cell multi-target 3D single-molecule SR imaging through Exchange-PAINT with improved localization precision and imaging speeds.

[0030] In some embodiments, a background reduction and 3D localization precision improvement achieved by performing 3D single-molecule SR imaging of a nuclear lamina protein lamin Bl with soTILT3D is quantified and compared to conventionalepi-illumination. In this manner, a speed improvement is quantified by imaging a- tubulin a number of localizations per length of microtubule over time (e.g., a period of time). The number of localizations are then analyzed through comparison of the number of localizations analyzed by DECODE compared with a non-neural networkbased localization software. Further, demonstration of a performance of soTILT3D is disclosed herein for multi-target 3D single-molecule SR imaging of nuclear lamina proteins and mitochondria by quantification of a relative distances between nuclear lamina proteins lamin Bl, lamin A / C, and lamina associated protein 2 (LAP2). In some embodiments, soTILT3D achieves up to ten-fold faster whole-cell multi-target 3D SR imaging with Fourier ring correlation (FRC) resolution values below 30 nm laterally and 40 nm axially, enabling a range of applications for correlating proteins on a nanoscale.

[0031] With the foregoing in mind, FIG. 1 is a schematic illustration of an optical setup 100 (e.g., optical platform) of a 3D single-molecule SR imaging technique used to form a LS 102.Optical Setup

[0032] In the illustrated embodiment the optical setup 100 may be controlled to establish multiple illumination geometries. For example, an epi-illumination path 104, a light sheet (LS) illumination path 106, and / or a transmission illumination path may be established. The epi-illumination path 104 and the LS illumination path 106 may be separated by a flip mirror 108. In this manner, the optical setup 100 may be controlled to allow the epi-illumination path 104 and / or the LS illumination path 106 to illuminate a sample 110. In some embodiments, the LS illumination path 104 may include a laser beam 112 directed to a first cylindrical lens 114 (e.g.. Lcyl). In this manner, the laser beam 112 may be focused in at least one dimension to form a LS. In this manner, the laser beam may be reflected by a first galvanometric mirror 116 (e.g., GalvoY). The first galvanometric mirror 116 may be conjugated to the back focal plane of the objective 118. The first galvanometric mirror 116 may be controlled to steer the LS in the y direction. In some embodiments, a second galvanometric minor 120 (e.g., GalvoX) may be conjugated to the back focal plane of the objective 118. As such, thesecond galvanometric mirror 120 may steer the LS in an x direction. In some embodiments, a focus of the laser beam 112 is tuned by one or more tunable lens. In this manner, a tunable lens 122 may be conjugated to the back focal plane of the objective 118. In some instances, the LS may be dithered with a third galvanometric mirror 124 (e.g., GalvoD) conjugated to a sample plane 126. With this in mind, in some embodiments, emitted light may be collected through a tw o-channel 4f system 128. In some instances, a transmissive dielectric phase mask 130 is used to modulate the emitted light. Further, the emitted light may be imaged on an EMCCD camera 132 after transmission through the transmissive dielectric phase mask 130. Further, the transmitted light may be used to image a plurality of polystyrene beads on a CMOS camera 134. In some instances, imaging of the polystyrene beads may be used to conduct active stabilization. It should be noted, the schematic illustrated in FIG. 1, is not drawn to scale.

[0033] The optical setup 100 is built around a conventional inverted microscope 136 (1X83, Olympus). An excitation pathway includes one or more illumination lasers 138 (e.g., 560 nm, 1000 mW, MPB Communications; 642 nm, 1000 mW, MPB Communications) which may be spectrally fdtered (FF01-554 / 23-25, Semrock; FF01- 631 / 36-25, Semrock) and / or circularly polarized (560 nm: Z-10-A-.250-B-556 quarterwave plate. Tower Optical; 642 nm: LPVISC050-MP2 polarizer. Thorlabs, and Z-10- A-.250-B-647 quarter-wave plate, Tower Optical) before being expanded and collimated (LA1951-A, f = 25.4 mm, Thorlabs; LA1417-A, f = 150 mm, Thorlabs). In some embodiments, switching between lasers may be controlled with a plurality shutters (VS14S2Z1. Vincent Associates Uniblitz) connected to a shutter control box (VDM-D3, Vincent Associates Uniblitz). In this manner, one or more laser pathways 140 may be merged into a single optical path (e.g., laser path 112) with a dichroic mirror 142 (T5901pxr-UF2, Chroma). As such, laser light may be sent to one or more pathways that may include the epi-illumination pathway 104 and / or the single-obj ective LS illumination pathway 106. In some instances, laser light may be switched between the pathways by manually flipping one or more flip mirrors, such as the flip mirror 108. In certain embodiments, in the epi-illumination pathway 104, a laser beam may be further expanded and collimated (AC508-075-A, f = 75 mm, Thorlabs; AC508-300-A,f = 300 mm, Thorlabs) to achieve a laser spot size that may illuminate an entire field of view (FOV) of ~50 pm x 50 pm. Further, in some embodiments, a beam may be reflected off a flip mirror (e.g., the flip mirror 108) and directed through a Kohler lens 146 (AC508-300-A, f = 300 mm, Thorlabs) that may focus the beam at the back aperture of a high-NA oil immersion objective (UPLXAPOlOOx, lOOx, NA 1.45, Olympus) (e.g., the objective 118). In some instances, the high-NA oil immersion objective may be used for both illumination and detection to generate collimated widefield epi-illumination in the sample plane 126. With this in mind, along the LS illumination pathway, the beam may be reduced and collimated (AC254-15-A-ML, f = 150 mm, Thorlabs; AC254-075-A-ML, f = 75 mm, Thorlabs) before passing through a cylindrical lens (ACY254-150-A, f = 150 mm, Thorlabs) which may focus the light in at least one dimension onto 106 the first galvanometric mirror 116 oriented horizontally and conjugated to the back focal plane of the objective lens for beam steering in the sample plane in the y direction (GVS211, Thorlabs; GPS011-US Galvo Power Supply, Thorlabs). The beam may then be reduced and collimated once again (AC254-150-A- ML, f = 150 mm, Thorlabs; AC254-075-A-ML. f = 75 mm, Thorlabs) and may be focused onto the second galvanometric mirror oriented 120 vertically and conjugated to the back focal plane of the objective lens for beam steering in a sample plane in a x direction (GVS211, Thorlabs; GPS011-US Galvo Power Supply. Thorlabs). In some instances, the beam may be reduced and collimated once again (AC254-150-A-ML. f = 150 mm, Thorlabs; AC254-075-A-ML, f = 75 mm, Thorlabs) and may be focused in one dimension onto the tunable lens 122 (EL-3-10-VIS-26D-FPS, Optotune; EL-E-4 Electrical Lens Driver, Optotune) conjugated to the back focal plane of the objective lens for focal steering in the sample plane. Further, the beam may be directed through a lens 148 (AC508-300-A, f = 300 mm, Thorlabs) focusing the beam onto the third galvanometric mirror 124 (SP30Y-AG, Thorlabs; GPWR15 Galvo Power Supply, Thorlabs; CBLS3F Galvo System Cable Set, Thorlabs) oriented horizontally and conjugated to the sample plane 126 for rapid dithering of the LS with a function generator (15 MHz DDS Signal Generator / Counter, Koolertron). In this manner, the beam may be directed through the Kohler lens 146. It should be noted that the Kohler lens 146 may be a common element between both the epi- and the LS illumination paths. It should be noted, without wishing to be bound by theory, that lenses in theillumination path may be selected to generate a LS with dimensions suitable for mammalian cell imaging and / or one or more additional types of samples.

[0034] In some embodiments, the high-NA objective lens may focus the LS 102 into a sample imaging chamber 150 bonded to a coverslip (vide infra) and the LS 102 may be reflected off of a metalized side wall 152 of the sample imaging chamber 150 at a 12° angle for 3D imaging or at a 0° angle for 2D imaging. In this manner, the LS 102 may be directed into the sample 110. In some embodiments, for whole-cell SR imaging, one or more mirrors on one or more removable magnetic mounts (not shown) in a red epi illumination path may be used for simultaneous epi-illumination for fiducial bead-based drift correction. Further, in some embodiments, a dichroic mirror (not shown) (69-216, 600 nm Dichroic Shortpass Filter, Edmund Optics) may be placed on a sidewaysmounted magnetic mount in place of the flip mirror 108 (just before the Kohler lens 146) to allow for simultaneous LS illumination with the one or more illumination lasers 138 such as a 560 nm laser and epi-illumination with a 642 nm laser. In this manner, fiducial bead-based drift correction may be achieved using one or more 0.20 pm 660 / 680 fiducial beads (F8807, Invitrogen) in both lateral and axial dimensions wftile acquiring single-molecule data with the LS 102. Further, real-time active stabilization was achieved using an infrared (IR) LED (BLS-LCS-0850-03-22. Mightex; BLS- SA02-US LED Control Box. Mightex) mounted in place of a microscope's white light halogen lamp for IR brightfield illumination.

[0035] In certain embodiments, a dichroic mirror 154 (e.g., an 800 nm dichroic short-pass mirror 14-015, Edmund) may be mounted between the objective 118 and the Kohler lens 146 to direct transmitted IR light 156 to a separate path where it may be focused using a tube lens 158 (ACT508-200-B, f = 200 nm, Thorlabs) onto a sensor of the CMOS camera 134 (CS235MU, Thorlabs) aligned in an optical cage system. Optical cleanup filters (not shown) (FFO 1-842 / 56-25, Semrock) may be placed between the LED and sample and in front of the CMOS camera 134 to filter stray IR wavelengths in the transmission path. Furthermore, a short-pass cleanup filter (not shown) (BSP01- 785R-25, Semrock) may be placed in a fluorescence detection path to remove residual IR wavelengths from the fluorescence emission light. In this way, active stabilization of the optical system 100 using the standard PSF of a plurality of nonfluorescent 3 pmpolystyrene beads (C37484, Invitrogen) in both lateral and axial dimensions may be achieved using an open-source active stabilization Image J / Micromanager Plugin while acquiring single-molecule data.

[0036] The sample imaging chamber 150 may be mounted in a stage top incubation chamber (P-736-ZR1S / ZR2S, OkoLab) compatible with an xy translation stage (OPH- XYS-O, Physik Instrumente) and a fine adjustment xyz piezoelectric translation stage (OPH-PINANO-XYZ, Physik Instrumente). In some embodiments, a sample imaging chamber may be mounted to the stage top using one or more imaging modalities. The sample imaging chamber may include an embedded metalized insert such as a chip. For example, the sample imaging chamber may be mounted in a macroscopic imaging well, live cell culture chambers, perfusion chambers, one or more custom imaging chambers, and the like. In some embodiments, microfluidic chambers 160 may be used in combination with the sample imaging chamber 150. In this manner, solutions maybe perfused using a pressure-based flow control pump 162 (LU-FEZ-0345, Fluigent, Inc.) connected to compressed air. In some instances, switching between solutions may be achieved with an l l-port / 10-position bidirectional valve connected to multiple solutions (ESSMSW003, Fluigent, Inc.) and locally controlled using a microfluidic flow controller (ELUSEZ. Fluigent, Inc.). Tubing 164 (FEP 1 / 16’' OD, 1 / 100” ID, Cole-Parmer) may be connected from the pump 162 to the sample imaging chamber 150 for solution perfusion. In some embodiments, the cell may be imaged independently of solution perfusion, such as in embodiments in which the sample imaging chamber may be coupled with macroscopic imaging wells.

[0037] In some embodiments, emission from a plurality of fluorophores may be collected by the high-NA objective lens (UPLXAPO 1 OOx, lOOx, NA 1.45, Olympus) used for epi- and LS illumination, spectrally filtered by a dichroic mirror 166 (ZT405 / 488 / 561 / 640rpcV3 3 mm thick dichroic filter set mounted in Chroma BX3 cube. Chroma; ZET642NF, ZET561NF, both Chroma), and may be focused by the microscope tube lens to form an image at the intermediate image plane (IIP) 168. In this manner, light may be directed to a first lens 170 of the 4f system 128 (AC508-080- AB, f = 80 mm, Thorlabs) positioned one focal length away from the IIP 168. In some instances, an iris attached to a lens tube on the emission port of the microscope mayallow for control of the emission spot size on the camera sensor. A dichroic mirror 172 (T6601pxr-UF3, Chroma) may be used to split emission into two color channels: a “green path” 174 (e.g., wavelengths shorter than 660 nm) and a “red path” 176 (e.g.. wavelengths longer than 660 nm). In some instances, a second 4f lens 178 (AC508- 080- AB, f = 80 mm, Thorlabs) may be placed in each path two focal lengths away from the first 4f lens 170. In this manner, one focal length after the first 4f lens 170, the Fourier plane of the microscope may be accessible, and the phase of emitted light may be modulated to engineer a PSFs to encode axial positions of emitters in each path. As such, encoding of axial position of the PSFs may be achieved using a first phase mask 180 such as a 2-pm axial range dielectric DH phase mask (DH-1 phase mask, 590 nm with a diameter of 2.484 mm, Double-Helix Optics, LLC) in the green channel 174 for single-molecule data collection and a 12-pm axial range DH phase mask (DH-12 phase mask, 670 nm with diameter of 2.484 mm, Double-Helix Optics, LLC) and / or a second phase mask 182 in the red channel 176 for bead localizations and drift correction. In some embodiments, after phase modulation with one or more phase masks 180, 182, emitted light in each color channel (e.g. red channel and / or green channel) may be focused by the second 4f lens 178 in each channel and directed onto opposite comers of the sensor of the EMCCD camera 132 (iXon Ultra 897, Andor, Oxford Instruments).

[0038] In certain embodiments, to achieve fluorescence imaging, a set EM gain of 200 may be used, corresponding to a calibrated EM gain of 182 for the EMCCD camera. Further, the EMCCD camera 132 conversion gain may be experimentally determined to be 4.41 photoelectrons per A / D count. As such, the EMCCD camera 132 may be operated at a shift speed of 1.7 pm / s and normal vertical clock voltage amplitude. Further, a read-out rate may be selected as 10 MHz at 16 bits using a preamplifier gain of 3. In some instances, a calibrated pixel size of the camera was 159 nm / pixel vertically and 157 nm / pixel horizontally. Further, to enable transmission imaging, the CMOS camera 134 was set to a gain of 20 photoelectrons per A / D count, and the calibrated pixel size was 42.7 nm / pixel.SoTILT3D design and performance

[0039] FIG. 2 is a scanning electron micrograph of the microfluidic chip insert used for LS reflection. Scale bar 50 gm. SoTILT3D enables 3D single-molecule imaging by reflecting a LS off of a metalized insert with an angled side wall inside of a microfluidic PDMS chip, as shown in FIG. 2. The LS is formed using a cylindrical lens. The LS is steered with a plurality of galvanometric mirrors in the x and y directions and defocused with a tunable lens. An additional galvanometric mirror is used to dither the LS at a half-angle of 20° in a plane of the LS at a frequency of 100 Hz to reduce striping and shadowing effects. FIG. 3 is an image of a thin end of the LS imaged in a fluorescent solution with a scale bar of 10 pm. In some embodiments, the LS has a thickness of approximately 1.1 pm (e.g., l / e2 beam waist radius) of as shown in FIG. 3. Further, the LS has a width of approximately 74.7 pm (l / e2 diameter). In some instances, the LS has a confocal parameter of approximately, 18.0 pm (l / e2). It should be noted, that the width, the thickness, the confocal parameter and one or more additional parameters may be tuned based on a choice of one or more lenses. In some embodiments, a nanoprinted insert of the microfluidic PDMS chip shown in FIG. 2 may be formed with one or more angles and / or one or more dimensions. In some instances, the dimensions of the PDMS chip may be tuned. In certain embodiments, a LS beam angle of 12° is used to allow7imaging of one or more whole-cell. As such, sectioning of one or more adherent cells may be achieved to a level of a coverslip (support of the one or more whole cells). In some instances, a LS beam angle of 0° is used to allow for 2D imaging at a certain image plane away from the coverslip. In some embodiment, a transparent top of the microfluidic chip allow s for incorporation and / or combination with one or more additional epi- and transmission illumination modalities.

[0040] With the foregoing in mind, FIG. 1 shows a 4f-system that may be implemented in an emission path. In this manner, a Fourier plane may be accessed to enable PSF engineering. As show n, a DH phase mask may enable 3D localization of a plurality of single molecules. Further, PSF engineering using the DH phase mask and the 4f system allows for acquisition of information in one or more dimensions (e.g., three dimensions).SoTILT3D improves the localization precision of 3D single-molecule imaging

[0041] In some embodiments, the LS may improve the signal-to-background ratio (SBR). FIG. 4 is a graph of including line scans of a diffraction limited signal of laminBl in U2OS cells in epi-illumination and LS illumination illustrating a distance (pm) versus a normalized signal. In the illustrated embodiment, the SBR may be improved by approximately four times for diffraction-limited imaging. FIG. 5 is a graph of including line scans of a single-molecule signal of laminBl in U2OS cells in epi-illumination and LS illumination illustrating a distance (pm) versus a normalized signal. In certain embodiments, as shown in FIG. 5, the SBR may be improved by at least six times for single-molecule imaging. For example, imaging of lamin Bl in a plurality of U2OS cells shows an improved SBR.

[0042] FIGS. 6 and 7 illustrate localization precision in xy and z for epi-illumination and LS illumination. In some embodiments, to assess a performance (e.g., localization precision) of soTILT3D single-molecule DNA-PAINT data of lamin Bl in U2OS cells is acquired in 3D using the DH-PSF with epi- illumination. In some embodiments, single-molecule DNA-PAINT data of lamin Bl in U2OS cells is acquired in 3D using the DH-PSF with LS illumination. In some instances, a fluorescence background may be reduced (median background values of 125 photons / pixel for epi- compared to 64 photons / pixel for LS illumination) in LS configuration while maintaining signal intensities of individual molecules (median values of 8,400 photons / localization for epi- and 11,400 photons / localization for LS illumination). As such, one or more improvements in lateral localization precisions may be made. As shown in FIG. 6, median values for epi-illumination may be 11.2 nm in xy and / or median values for LS illumination may be 7.8 nm in xy. FIG. 7 shows the axial localization precisions. In the illustrated embodiment, the axial localization precision (e.g., z) in the epi- illumination is 16.9 nm and in the LS illumination is 11.8 nm.SoTILT3D improves the speed of 3D single-molecule imaging

[0043] In some embodiments, a speed improvement of soTILT3D may be quantified. In some instances, a deep learning analysis system may be quantified by 3D singlemolecule SR imaging of microtubules in the plurality of U2OS cells. In certain embodiments, an approach was benchmarked by analysis of corresponding single-molecule data and fluorescent bead data with Easy-DHPSF, an open-source leastsquares fitting-based analysis software, and DECODE. Analysis of the approach generated comparable localization precisions. Further, high density data may be acquired with a 0.2 nM concentration of imager strands. It should be noted, that standard concentration data for non-overlapping emitters may be acquired at 0.025 nM.

[0044] FIG. 8 is a graph of a number of localizations per length of microtubule over time localized using Easy-DHPSF and DECODE. In some embodiments, quantitative comparison of the average localizations per pm per second may demonstrate a ten-fold increase in speed when using DECODE. For example. Easy-DHPSF may localize 0.055 localizations per pm per second. Further, DECODE may localize 0.567 localizations per pm per second. It should be noted, each data point on the graph represents the average localizations per length of microtubule for three different microtubule sections and error bars are ± standard deviations of illustrated data sets. In some embodiments, non-overlapping single molecule data may be localized with Easy- DHPSF. In this manner, high-density data may be localized with a DECODE model. In some instances, the DECODE model may be trained on a 0.025 nM microtubule data set and may converge to a Jaccard Index of 0.69 after 1000 epochs. As such, in some embodiments, a more than ten-fold increase in imaging speed when using DECODE may be achieved. In certain embodiments, resolutions after 50,000 frames may be estimated using FRC calculations in a xy, ayz, and / or a xz plane. As such, resolutions of 35.4 / 38.6 / 36.9 nm and 28.4 / 32.1 / 33.0 nm for Easy-DHPSF data set and DECODE data set may be reported, respectively. In this manner, in some embodiments, the resolutions may demonstrate overall improved resolution when using DECODE for a same number of frames.Whole-cell multi-target 3D single-molecule SR imaging with soTILT3D

[0045] FIG. 9 is a graph illustrating Gaussian fits to an example line scan across a nuclear rim of an entire U2OS cell nucleus labeled for lamin bl, LAP2, and lamin A / C. In this manner, FIG. 9 demonstrates separation distances between lamin Bl (LB1), LAP2, and lamin A / C (LA / C) in nm. In some embodiments, whole-cell singlemolecule data may be acquired using soTILT3D and DECODE for analysis. In thismanner, a performance of soTILT3D for accurate and precise multi-target 3D SR imaging may be demonstrated as shown in FIG. 9. In this manner, the focal plane and / or the LS may be moved in 1 pm steps to image one or more target in overlapping slices. In some instances, a pressure-driven pump may be used to introduce imager strand sequences sequentially. For example, 3D two-target imaging may be demonstrated by imaging of mitochondria and lamin A / C. In this manner, hollow structures of the mitochondrial outer membrane (TOMM20) may be visualized. As such, FRC resolutions may be 39.3 / 41. 1 / 44.2 nm for lamin A / C and / or 34.1 / 37.6 / 42.1 nm for mitochondria.

[0046] With the foregoing in mind, whole-cell multi-target imaging may be demonstrated on a plurality of nuclear proteins lamin Bl, LAP2, and / or lamin A / C in a plurality of U2OS cells. In this manner, a nuclear protein distribution may be resolved with approximately 10,000 frames per slice. As such, high-density data may be analyzed with DECODE allowing a greater number of localizations to be acquired in a shorter time. Further, nanoscale separation (e.g., separation between these nuclear targets) may be quantified from 3D SR reconstructions using Gaussian fits of line scans across a nuclear rim. Nanoscale separation was found to be 41 ± 8 nm for lamin Bl and lamin A / C, 29 ± 9 nm for lamin Bl and LAP2. and 12 ± 10 nm for LAP2 and lamin A / C (reported as mean ± standard error of the mean for 25 line scans). FIG. 9 shows representative fits to a line scan in which nuclear protein distribution distances are found to be 41 nm between lamin Bl and lamin A / C, 27 nm between lamin Bl and LAP2, and 14 nm between LAP2 and lamin A / C. In this manner, one or more average nuclear target distributions across all line scans may be shown pairwise with Gaussian fits to the average distributions. As such, soTILT3D maintained high resolution throughout a cell during whole-cell, multi-target, 3D SR imaging. As shown, in Table 1, high resolution may be demonstrated by Fourier ring correlation (FRC) analysis. Without wishing to be bound by theory, FRC analysis may report on resolutions in the xy / yz / xz plane.

[0047] In certain embodiments, a combination of a steerable, dithered, single-objective tilted LS with microfluidics, PSF engineering, deep learning, active drift stabilization, and / or Exchange-PAINT may result in improved localization precision and imaging speeds for whole-cell multi-target 3D single-molecule SR imaging. With the foregoing in mind, the optical system, as shown in FIG. 1, may be implemented on conventional microscopes. Further, the disclosed procedure for fabricating one or more microfluidic devices may be compatible with single-objective LS illumination, widefield epiillumination, and / or transmission microscopy. Flexibility of the 3D nanoprinted insert geometry along with one or more design options for fabrication of PDMS channels may allow for adaptability and implementation for various applications. In this manner, in some non-limiting embodiment, a soTILT3D platform may be extended to live-cell imaging and / or single-particle tracking (SPT). As such, biocompatibility and / or a gas permeability of the 3D nanoprinted insert and PDMS may ensure that one or more microfluidic channels may be suitable for live-cell studies. In this manner, one or more cells may be cultured inside a chip for imaging. While not wishing to be bound by theory', solution perfusion through the microfluidic channel may allow precise control of an extracellular environment and / or removal of heat or waste products coupled with a constantly replenished fluid reservoir. Further, the single-objective light sheet design may be compatible with standard microscope stage top incubators. Furthermore, LS illumination may minimize photodamage to a specimen. In some instances, autostabilization drift correction scheme allows for easy decoupling of cell movement from sample drift over long acquisition periods.

[0048] In certain embodiments, design flexibility of a soTILT3D setup may allow for implementation with a plurality of additional live-cells, one or more compatible single-molecule imaging modalities, such as Peptide-PAINT and SR imaging using SiR dyes. With this in mind, one or more fluorophore developments, including self-quenched DNA-PAINT probes may be used to improve a performance of soT!LT3D imaging. As such, soTILT3D may offer a simple and / or flexible approach to improve 3D SR imaging and / or SPT that may be adapted and / or utilized for various single-molecule imaging applications for fast, efficient and / or precise nanoscale investigation of cellular structures and / or molecular dynamics.Fabrication process for microfluidic chips with reflective angled side walls

[0049] FIG. 10 is a schematic illustration of a protocol 200 used to fabricate channels out of PDMS using soft lithography with molds made from SU8-100 photoresist 202 on Si wafers 204 including schematic illustration of a 3D nanoprinted metalized insert 206 being positioned inside of a PDMS channel 208. As shown, FIG. 10 illustrates an overview of a fabrication process to form one or more microfluidic chips 210. The microfluidic chip 210 consists of two main parts: the 3D nanoprinted metalized insert 206 and the PDMS channel 208. In some embodiments, a process of fabricating the 3D nanoprinted metalized insert 206 is demonstrated. In this manner, the 3D nanoprinted metalized insert 206 of one or more desired dimensions may be designed using AutoCAD (Autodesk). Further, the 3D nanoprinted metalized insert 206 may be sliced and hatched for 3D nanoprinting in Describe (Nanoscribe GmbH & Co. KG) with a lOx Silicon Shell recipe using a slicing distance of 0.3 pm and a hatching distance of 0.3 pm with a hatching angle of -6° and a hatching angle offset of 0°. In some instances, the block size (X: 840 pm, Y:800 pm, Z: 4000 pm), block offset (X:400 pm. Y:425 pm, Z:0 pm), and block shear angle (0°) may be adjusted to avoid formation of stitching lines throughout the printed structure. In some embodiment, characterization of the LS was performed in fluorescent solution. In this manner, the 3D nanoprinted metalized inserts 206 may be printed to be 105 pm tall, 300 pm wide, and 2.8 mm long. As such, a side wall 212 of the 3D nanoprinted metalized insert 206 may have an angle of 45° and a height of 70 pm. In embodiments, in which the 3D nanoprinted metalized insert 206 were used for imaging of mammalian cells, the 3D nanoprinted metalized insert 206 may be printed to be 105 pm tall, 300 pm wide, and 4 mm long. As such, the side wall 212 may have an angle of 39° and a height of 15 pm to allow7for a reflected LSwith a 12° tilt. With this in mind, a design of the 3D nanoprinted metalized insert 206 may be printed using two-photon polymerization direct laser writing on a fused silica substrate with a Nanoscribe Photonic Professional GT2 (Nanoscribe GmbH & Co. KG). Further, the 3D nanoprinted metalized insert 206 was printed using a lOx objective with IP-Visio resin (Nanoscribe GmbH & Co. KG), a non-cytotoxic, optically transparent, nonfluorescent methacrylate-based resin, with a laser power set to 100% and a scan speed of 30 mm / s.

[0050] In some embodiments, the 3D nanoprinted metalized insert 206 may be briefly (e.g., seconds, minutes) immersed in SU8 developer (mr-Dev 600, Kayaku Advanced Materials, Inc.). Immersion of the 3D nanoprinted metalized insert 206 into SU8 developer may include use of a pipette to gently dissolve excess unpolymerized resin from a surface of the 3D nanoprinted metalized insert. In some instances, the 3D nanoprinted metalized insert 206 may be briefly immersed in isopropyl alcohol (MPX18304, Fisher Scientific) and dried with nitrogen gas. The 3D nanoprinted metalized insert 206 may then be treated for approximately 15 minutes with an 18 W UV light source (140010, Vacuum UV-Exposure Box, Gie-Tec GmbH). In some embodiments, the 3D nanoprinted metalized insert 206 may be mounted on a side (e.g., a side of the 3D nanoprinted metalized insert) to allow for vapor deposition. In this manner, mounting on the side of the 3D nanoprinted metalized insert 206 may ensure an edge of the 3D nanoprinted metalized insert 206 containing a side wall 212 may be metalized to reduce scattering from reflectivity of other portions of the 3D nanoprinted metalized insert. In some embodiments, the 3D nanoprinted metalized insert 206 may be mounted in an e-beam vapor deposition chamber. In this manner, approximately 200 nm of silica may be deposited (EVMSIO21-5D, Kurt J. Lesker), followed by approximately 350 nm of aluminum (EVMAL50EXEB, Kurt J. Lesker), and followed by approximately 5 nm of silica (EVMSIO21-5D, Kurt J. Lesker). While not wishing to be bound by theory, in some instances, a first layer of silica may provide an insulating layer to the 3D nanoprinted metalized insert 206 (e.g., plastic insert) that may protect the 3D nanoprinted metalized insert 206 from one or more laser intensities used during imaging. Further, a layer of aluminum may provide a reflective layer for singleobjective LS reflection. A final layer of silica provides a dielectric coating for a mirrorsurface (e.g., the reflective layer). It should be noted, that one or more layers may be optimized to provide heat protection for a thermoplastic base and may provide good reflectivity. It should be noted, scans profiling a surface of the insert side wall 212 may be performed using atomic force microscopy (AFM). In this manner, a 10 pm x 10 pm region of the side wall may be scanned (NCHR-10, NanoWorld; NX20, Park Systems) and analyzed (XEI 4.3.4, Park Systems), to determine a root mean square (RMS) roughness of 25.9 nm for the entire 100 pm2scanned region.

[0051] In some embodiments, a PDMS base and / or one or more PDMS (SYLGARD 184 Silicone Elastomer Kit, Dow Inc.) channels 208 may be formed from one or more SU8 molds 214 made on silicon wafers 204 (444, University Wafer, Inc.) with SUS- 100 photoresist 202 (Y131273, Kayaku Advanced Materials, Inc.). The one or more SU8 molds 214 may be prepared by spin-coating (WS-650Mz-23NPPB, Laurell Technologies) approximately 2 mL of photoresist onto a silicon wafer 204. The protocol 200 includes spin coating the silicon wafer 204 with SU8-100 photoresist 202 to form a treated silicon wafer 216. In some embodiments, spin-coating may be performed at a first setting of 500 revolutions per minute (rpm) at 100 rpm / second acceleration. In some instances, after the first setting a second setting may be performed at 500 rpm for 10 seconds. Further, a third setting may be performed to ramp spincoating to 3000 rpm at an acceleration rate of 300 rpm / second. The third setting may be held at 3000 rpm for approximately 30 seconds. In some embodiments, the one or more SU8 molds may be heated on a hot plate at 65°C for 10 minutes followed by 95°C for 30 minutes. In some embodiments, a custom film photomask 218 (Micro Lithography Services Ltd) designed in AutoCAD (Autodesk) with desired channel dimensions may be applied to one or more photoresist-coated silicon wafers (e.g., the treated silicon wafer 216). The photoresist-coated silicon wafers 216 may be exposed to UV light under vacuum (140010, Vacuum UV -Exposure Box, Gie-Tec GmbH) with an 18 W light source for approximately 27 seconds. The SU8 molds 214 may be heated again on a hot plate at 65°C for 1 minute followed by 95°C for 10 minutes. The SU8 molds 214 may be placed in a beaker with approximately 20 mL of SU8 developer 220 (mr-Dev 600, Kayaku Advanced Materials, Inc.) and gently swirled for 10 minutes to dissolve residual untreated photoresist. It should be noted, that the outlined steps maybe performed in accordance with a procedure outlined on the technical data sheet for SU8-100 negative epoxy photoresist. The SU8 molds 214 may be designed to have channels with dimensions of 700 pm wide, 100 pm tall, and 1 cm long. In some embodiments, approximately 25 rnL of PDMS 222 may be prepared from a two-part system of a 10: 1 ratio of elastomer to curing agent and poured over SU8 molds 214 placed in 60 mm petri dishes 224 (FB0875713A, Fisher Scientific). Further, pins may be placed on both ends of the channel 208 prior to pouring the PDMS 222 and may be left in place as the PDMS 222 cured generating holes for tubing. In this manner, the PDMS 222 may be left to cure at room temperature overnight followed by an additional 2 hours at 65°C in an oven. Once the PDMS 222 is cured, the PDMS slab may be peeled off the SU8 mold, and the 3D nanoprinted metalized insert 206 may be placed into an insert 224 within the PDMS channel 208. It should be noted, that care must be taken to ensure the 3D nanoprinted metalized insert 206 is seated in the correct orientation within the PDMS channel 208 for LS reflection into a sample.

[0052] In some embodiments, bonding of the microfluidic chip 210 to coverslips (#1.5, 22 mm x 22 mm, Electron Microscopy Sciences) may be performed. Coverslips 226 may be cleaned via sonication (Branson Ultrasonics Cleaning Bath, 15-336-120, Fisher Scientific) in acetone (BP2403-4. Fischer Scientific) for approximately 25 minutes followed by plasma treatment (PDC-32G, Harrick Plasma Inc) with argon gas for approximately 25 minutes. In this manner, a clean coverslip 226 may be placed with the PDMS insert 208 into a plasma cleaner (PDC-32G, Harrick Plasma Inc) and may be treated with air plasma for approximately 30 seconds. In some instances, immediately after removal of the clean coverslip 226 from the plasma cleaner, the clean coverslip 226 and the PDMS insert 208 (e.g., the 214 may be pressed together, allowing covalent bonds to form between the clean coverslip and PDMS. In this manner, the clean coverslip 226 and the PDMS insert 208 may be bound to the microfluidic chip 210 to form a sealed channel 228.

[0053] FIG. 11 is a schematic illustration of a protocol 300 used to fabricate a PDMS insert 302 including schematic illustration of a 3D nanoprinted metalized insert 304 being positioned inside of the PDMS insert 302, in accordance with aspects of the present approach. As shown, FIG. 11 illustrates an overview of a fabrication processto form one or more customizable imaging inserts 306 for use in various imaging modalities in combination with SoTILT3D. The imaging insert 306 consists of two main parts: a 3D nanoprinted metahzed insert 304 and a PDMS insert 302.

[0054] As described in reference to FIG. 10, the 3D nanoprinted metalized insert 304 fabrication process may be used to design a sample imaging chamber (e.g. such as microfluidic chips) of customizable dimensions. In some embodiments, one or more structures of desired dimensions of the 3D nanoprinted metalized inserts 304 may be designed using AutoCAD (Autodesk). For example, the structures may be prepared for 3D nanoprinting in Describe (Nanoscribe GmbH & Co. KG) using a lOx Silicon Shell recipe. Dimensions of the 3D nanoprinted metalized inserts 304 may be tailored to match one or more SU8 molds 308 designed to be compatible with various imaging systems such as imaging wells. In this manner, a length and a width of the 3D nanoprinted metahzed inserts 304 may be determined based on dimensions of a photomask 310, and a height of the 3D nanoprinted metahzed inserts 304 may be based on profiling the SU8 molds 308 using a NPFlex profiler (Bruker). Control of the dimensions of the 3D nanoprinted metalized inserts 304 may ensure a seamless fit of the 3D nanoprinted metalized insert 304 within the PDMS insert 302. In some embodiments, the 3D nanoprinted metalized inserts 304 may be designed with a sidewall angle of 45° and a height of 70 pm, to enable a reflected LS with no tilt. In some embodiments, dimensions of the 3D nanoprinted metalized inserts 304 may be changed based on a type of imaging. For example, in mammalian cell imaging, the 3D nanoprinted metalized inserts 304 may be adjusted to a 39° angle and 15 pm height, producing a reflected LS with a 12° tilt. The 3D nanoprinted metahzed inserts 304 may be fabricated via two-photon polymerization direct laser writing on a fused silica substrate, using the Nanoscribe Photonic Professional GT2 system and IP-Visio resin (Nanoscribe GmbH & Co. KG). Printing may be performed with a lOx objective at 100% laser power and a scan speed of 40 mm / s. As described in reference to FIG. 10 the 3D nanoprinted metahzed inserts 304 may be prepared using SU8 developer 312. photolithography 314, e-beam vapor deposition, or a combination thereof. For example, as shown in a first step 316 a silicon wafer 318 may be treated via spin coating with a photoresist to form a treated silicon wafer 320. In a second step 322 a photomask310 may be placed on the treated silicon wafer 320 and treated with UV light and the like. In a third step 324, the treated silicon wafer 320 may be washed with developer.

[0055] In some embodiments, as shown, the PDMS well insert 302 may be fabricated by pouring PDMS and a curing agent into a 3D-printed cavity 326 positioned over a mold 328 in a fourth step 330. For example, the PDMS well insert 302 may be fabricated by pouring PDMS (SYLGARD 184 Silicone Elastomer Kit. Dow Inc.) in a 10: 1 ratio of elastomer to curing agent into the 3D-printed cavity 326 positioned over an SU8 mold (e.g., the mold 328). As such, the PDMS well insert 302 may be fitted into an imaging chamber while providing a groove 332 for embedding the 3D nanoprinted metalized insert 304 for LS reflection. That is. the PDMS well insert 302 may be designed and fabricated to enable coupling of the 3D nanoprinted metalized inserts into various imaging geometries.

[0056] In one embodiment, an SU8 mold 308 with approximately 330 pm height was prepared by spin-coating (WS-650Mz-23NPPB, Laurell Technologies) approximately 3 mL of the SU8-100 photoresist onto a silicon wafer. For example, the silicon wafer 318 may be prepared in the first step 316 by spin casting photoresist at 500 revolutions per minute (rpm) at 100 rpm / second acceleration, then held at 500 rpm for 10 seconds. The silicon wafer 318 was then ramped to 1000 rpm at an acceleration of 300 rpm / second and held at this speed for 30 seconds. The SU8 molds 308 may be heated on a hot plate at approximately 65°C for approximately four hours followed by heating to 95°C overnight. Photomasks 310 may be designed using suitable dimensions to account for imaging geometry and / or to generate a molded structure (e.g., groove, channel, depression, etc.) of the PDMS insert 302 to enabling coupling of the 3D printed metallized inserts 304 to the PDMS insert 302. For example, a custom film photomask may be designed in AutoCAD. The custom film photomask may be applied to one or more photoresist-coated silicon wafers 320. The photoresist-coated silicon wafers 320, in the second step 322, may be exposed to UV light (18 W light source) under vacuum (140010, Vacuum UV-Exposure Box, Gie-Tec GmbH) for approximately 45 seconds. Photomasks 320 may be designed independently for each imaging chamber within a multi well imaging chamber. An additional region 334 for insertion of the 3D nanoprinted metallized insert 304 may be designed within the photomasks 310. TheSU8 molds 308 may be heated on a hot plate at approximately 65°C for approximately one minute followed by approximately 95°C for approximately 20 minutes. In the third step 324. the SU8 molds 308 may be placed in a beaker with approximately 20 mL of SU8 developer (mr-Dev 600, Kayaku Advanced Materials, Inc.) and gently swirled for 25 minutes to dissolve residual untreated photoresist. In other embodiments, one or more additional and / or alternative steps may be used to treat the SU8 molds 308 based on developer protocols.

[0057] In some embodiments, a macroscopic 3D printed plastic cavity 326 may be placed over the SU8 mold 308 to form a boundary to enclose PDMS and form an chamber 328 as a mold to form the PDMS insert 302 that matches one or more imaging chamber dimensions. The 3D printed plastic cavity 326 may be designed with one or more indents to match the profiled height of the SU8 mold to enable the 3D printed plastic cavity 326 to fit (e.g., click) into place on top of the photoresist. Fitting the 3D printed plastic cavity 326 on top of the SU8 mold 308 forms the chamber 328 for pouring PDMS. The chamber 328 may be used to form an imaging insert 306 that may be inserted into one or more imaging modalities with various dimensions while providing a groove 332 (e.g., a location) to insert the 3D nanoprinted metallized insert 304. In this manner, the chamber 328 formed by the 3D printed plastic cavity 326 may mimic dimensions of an imaging chamber while providing for placement of the 3D nanoprinted metallized insert 304. In some embodiments, the 3D printed plastic cavity 326 and / or the chamber 328 formed by the 3D printed plastic cavity7326 are cured. For example, the 3D printed plastic cavity' 326 may be cured at approximately 65°C for approximately 24 hours to prevent material of the 3D printed plastic cavity 326 from inhibiting PDMS curing. In certain embodiments, the PDMS in the 3D printed plastic cavity 326 may be allowed to cure at approximately 65°C for approximately 4 hours. After curing, the PDMS may be removed from the chamber 328 in a fifth step 336. It should be noted, in some embodiments the 3D printed plastic cavity' 326 may be made of any suitable material. That is. plastic is a non-limiting example and one or more additional materials may be used in place of or in addition to such plastic. Additionally and / or alternatively, one or more additional macroscopic forms may be used to generate a chamber to enable formation of the PDMS insert 302 of customizable dimensionsincluding to enable customizable fitting in various imaging modalities while incorporating the 3D nanoprinted metallized insert 304.

[0058] As shown in FIG. 11, the 3D nanoprinted metallized insert 304 may be positioned into the PDMS insert as shown in a sixth step 338 of the protocol 300. Such assembly and insertion of the 3D nanoprinted metallized insert 304 may enable compatibility of sample into imaging chamber of various geometries. In some embodiments, to assemble the 3D nanoprinted metallized insert 304, the 3D nanoprinted metallized insert 304 is placed into the groove in the PDMS insert 302. It should be noted, care must be taken to ensure the 3D nanoprinted metallized insert 304 is seated in the groove within the PDMS for LS reflection into the imaging chamber. Confirmation of proper assembly of the 3D nanoprinted metallized insert 304 into the PDMS insert 302 forming an assembled insert 342, shown in a seventh step 340 of the protocol 300, may be accomplished with a standard optical microscope.

[0059] In certain embodiments, the assembled insert 342 may be secured in the imaging chamber using one or more bonding techniques such as plasma bonding, forming a pressure fit, and the like. For example, plasma bonding may be achieved by treating the PDMS insert 302 and the imaging chamber with air plasma (PDC-32G, Harrick Plasma Inc.) for a suitable duration of time (e.g., approximately one minute). The 3D nanoprinted metallized insert 304 may be aligned and pressed against a bottom end of the imaging chamber to form covalent bonds. Alternatively, a pressure fit may be formed using the flexibility of cured PDMS to enable a pressure fit to secure the assembled insert 342. The assembled insert 342 may be used as an imaging insert to perform LS imaging and cell culture within imaging chambers of various geometries. In this manner, sample preparation including the 3D nanoprinted metallized insert 304 may be customized to support various imaging modalities during LS imaging.Cell culture

[0060] In some embodiments, the microfluidic channel may be cleaned with 70% ethanol (BP82031GAL, Fisher Scientific), nanopure water, and coated with a 1 : 100 solution of fibronectin (F0895, Sigma-Aldrich) in phosphate-buffered saline (PBS)(SH3025601, Fisher Scientific). As such, a plurality' of human osteosarcoma cells (U- 2 OS - HTB-96. ATCC) may be seeded into the sample imaging chamber 24 hours before labeling and incubated at 37°C and 5% carbon dioxide (Thermo Scientific Heracell 150i CO2 Incubator, 51-032-871, Fisher Scientific) in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 25 mM HEPES and supplemented with 10% (v / v / ) fetal bovine serum (FBS, Gibco) and 100 mM sodium pyruvate (Gibco).Sample Preparation

[0061] In some embodiments, a plurality of U2OS cells is cultured within the sample imaging chamber for approximately 24 hours. As such, the U2OS cells may be fixed and immunolabeled inside the sample imaging chamber. In this manner, for fixation, the U2OS cells may be first washed three times in PBS (SH3025601, Fisher Scientific), fixed for 20 min in chilled 4% formaldehyde solution made by dissolving paraformaldehyde (PF A, Electron Microscopy Sciences) in PBS, washed once more in PBS, and incubated with 10 rnM ammonium chloride (Sigma- Aldrich) in PBS for approximately 10 minutes. In some instance, the U2OS cells may be permeabilized with three washing steps with 0.2% (v / v) Triton X-100 (Sigma- Aldrich) in PBS with an approximately five-minute incubation period between each wash, and blocked with 3% (w / v) bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1 hour.

[0062] With this in mind, the U2OS cells may be labeled with rabbit anti-lamin Bl (ab!6048, Abeam) primary antibodies using a 1 : 1.000 dilution in 1% (w / v) BSA in PBS for two hours. In this manner, the U2OS cells may be prepared for diffraction-limited imaging. The U2OS cells may be washed three times with 0.1% (v / v) Triton X-100 in PBS with a three-minute incubation period during each wash before being labeled with donkey anti-rabbit secondary’ antibodies conjugated with dye CF568 (20098-1, Biotium) at a 1 : 100 dilution in 1% (w / v) BSA in PBS for one hour. Further, the U2OS cells may be washed five times in 0.1% (v / v) Triton X-100 in PBS. In some instances, 0.1 gm 580 / 605 nm fiducial beads (F8801, Invitrogen) at a dilution of 1 : 100,000 in PBS may be introduced to the U2OS cells via flow just before (e.g., seconds, minutes) imaging. In some embodiments, an oxygen scavenging buffer containing 100 mMTris-HCl (J22638-K2, Thermo Scientific), 10% (w / v) glucose (215530, BD Difco), 2 (il / ml catalase (C100, Sigma-Aldrich), and 560 pg / ml glucose oxidase (G2133, Sigma- Aldrich) may be used during imaging to reduce photobleaching.

[0063] In certain embodiments, single-molecule imaging may be conducted in one or more illumination geometries. In this way, comparison of epi-illumination and LS illumination may be conducted. With this in mind, the U2OS cells may be labeled with rabbit anti-lamin Bl (ab 16048, Abeam) primary antibodies using a 1 : 1,000 dilution in 1% (w / v) BSA in PBS for two hours. The U2OS cells may then be washed three times with 0.1% (v / v) Triton X-100 in PBS with a three-minute incubation period during each wash before being labeled with donkey anti-rabbit oligonucleotide-conjugated secondary antibodies (Massive Photonics) in antibody incubation buffer (Massive Photonics) for one hour. The U2OS cells may be further washed three times with IX washing buffer (Massive Photonics) in nanopure water, three times with 0.2% (v / v) Triton X-100 in PBS, and once in imaging buffer (500 mM NaCl in PBS, pH 8). A solution of 0.1 pm 580 / 605 nm fiducial beads (F8801, Invitrogen) at a dilution of 1 : 100,000 in nanopure water may be flowed in just (e.g., seconds, minutes) before single-molecule imaging. In this manner, single-molecule data may be obtained by flowing in imager strand solutions containing complementary oligonucleotide-Cy3B dye conjugates (Massive Photonics) diluted in imaging buffer to a concentration of 0.01 nM. It should be noted, that while the U2OS cells are included in the sample preparation procedure additional and / or alternative cells may be used for singlemolecule imaging.

[0064] In certain embodiments, single-molecule imaging of microtubules may be performed to compare acquisition speed and / or conduct analysis method control. As such, a plurality of cells may be labeled with mouse anti-a-tubulin (T5168, Sigma- Aldrich) primary antibodies using a 1:500 dilution in 1% (w / v) BSA in PBS for two hours. The cells may be washed three times with 0.1% (v / v) Triton X-100 in PBS with a three-minute incubation period during each wash before being labeled with donkey anti-mouse oligonucleotide-conjugated secondary' antibodies (Massive Photonics) at a dilution of 1 : 100 in antibody incubation buffer (Massive Photonics) for approximately one hour. The cells may be further washed three times with IX washing buffer(Massive Photonics) in nanopure water, three times with 0.2% (v / v) Triton X-100 in PBS, and once in imaging buffer (500 mM NaCl in PBS, pH 8). In some instances, before imaging, 0.1 pm 580 / 605 nm fiducial beads (F8801. Invitrogen) at a dilution of 1 : 100,000 in nanopure water may be flowed in. Further, complimentary oligonucleotide-Cy3B dye conjugates (Massive Photonics) diluted in imaging buffer may be flowed in at a concentration of 0.025 nM (standard concentration) or 0.2 nM (high concentration) just before imaging.In certain embodiments, two-target imaging of Tomm20 and lamin A / C may be conducted. In this manner, the sample imaging chambers may be first flowed through with 3 pm carboxylated polystyrene fiducial beads (C37484, Invitrogen) and 0.2 pm 660 / 680 nm fiducial beads (F8807, Invitrogen) and placed on a hotplate at 200°C for 1 minute and then at 100°C for 5 minutes to facilitate active stabilization and drift correction in post-processing. In this manner, after fixation, permeabilization, and / or blocking, cells may be labeled with rabbit anti-Tomm20 (abl 86735, Abeam) and mouse anti-lamin A / C (sc-376248, Santa Cruz Biotechnology) primary antibodies at a dilution of 1:200 and 1: 100, respectively, in 1% (w / v) BSA, 5% (v / v) salmon sperm ssDNA (ab229278, Abeam), and 10% donkey serum (ab7475, Abeam) in PBS for two hours. Cells may be washed three times with 0.1% (v / v) Triton X-100 in PBS with a three- minute incubation period during each wash before being labeled with donkey antirabbit and donkey anti -mouse oligonucleotide-conjugated secondary antibodies (Massive Photonics) at a dilution of 1: 100 in antibody incubation buffer (Massive Photonics) for one hour. Further, cells may be washed three times with IX washing buffer (Massive Photonics) in nanopure water, three times with 0.2% (v / v) Triton X- 100 in PBS, and once in imaging buffer (500 mM NaCl in PBS, pH 8) before imaging. Further, complimentary oligonucleotide-Cy3B dye conjugates (Massive Photonics) diluted in imaging buffer may be flowed in at concentrations of 0.04 nM for Tomm20 and 0.08 nM for lamin A / C sequentially for the two targets during imaging.

[0065] In some embodiments, multi-target imaging may be conducted. As such, imaging of a variety7of cells that may include lamin Bl cells, lamin A / C cells, and / or LAP2 cells may be conducted. In some embodiments, the variety of cells may be labeled with rabbit anti-lamin Bl (abl6048, Abeam), mouse anti-lamin A / C (sc-376248, Santa Cruz Biotechnology), and goat anti-thymopoietin (AF843, R&D Systems) primary’ antibodies at a dilution of 1: 1000, 1 : 100, and 1:50, respectively, in 1% (w / v) BSA, 5% (v / v) salmon sperm ssDNA (ab229278. Abeam), and 10% donkey serum (ab7475. Abeam) in PBS for two hours. In this manner, the variety' of cells may be washed three times with 0.1% (v / v) Triton X-100 in PBS with a three-minute incubation period during each wash before being labeled with donkey anti-rabbit, donkey anti-mouse, and donkey anti-goat oligonucleotide-conjugated secondary antibodies (Massive Photonics) at a dilution of 1: 100 in antibody incubation buffer (Massive Photonics) for one hour. The variety7of cells may be further washed three times with IX washing buffer (Massive Photonics) in nanopure water, three times with 0.2% (v / v) Triton X-100 in PBS, and once in imaging buffer (500 mM NaCl in PBS, pH 8). Before imaging, 0.2 pm 660 / 680 nm fiducial beads (F8807, Invitrogen) at a dilution of 1 : 100,000 may be flowed in. Further, complimentary7oligonucleotide-Cy3B dye conjugates (Massive Photonics) diluted in imaging buffer may be flowed into the sample imaging chambers at concentrations of 0.04 nM for lamin Bl, 0.08 nM for lamin A / C, and 0.08 nM for LAP2 sequentially. In this way. three targets may be analyzed during imaging. It should be noted, multi-target sequential labeling controls may be considered and executed.Imaging procedure and setings

[0066] In some embodiments, diffraction-limited imaging of cells may be conducted. In this manner, cells may be imaged with a 560 nm laser at ~90 W / cm2 and an exposure time of 50 ms.

[0067] In certain embodiments, single-molecule imaging comparison of the illumination geometries may be performed. For example, in some instances, comparing epi-illumination and LS illumination may be executed. Further, an analysis speed may be compared between using Easy-DHPSF or DECODE. In this manner, the 560 nm laser may be used at ~580 W / cm2 for epi-illumination and at ~800 W / cm2 for LS illumination using an exposure time of 100 ms to match the on / off binding rate of imager-docking strands. As such, 50,000 frames were acquired for the epi-illumination. Further, 100,000 frames were acquired in the LS illumination. In this manner, afterimaging, dark frames may be acquired with lasers turned off and camera shutters closed. Further, z-scan calibration stacks of the 2- pm axial range DH-PSF in a green channel were acquired with fiducial beads (T7280. TetraSpeck, 0.2 pm, Invitrogen) spin coated in 1% (w / w) PVA (Mowiol 4-88, #17951, Polysciences Inc.) in nanopure water on a coverslip.

[0068] In some embodiments, two-target single-molecule imaging of mitochondria and lamin A / C may be performed. In this manner, 50,000 frames may be acquired for each target (e.g., mitochondria and lamin A / C) using the 560 nm laser at -550 W / cm2 and an exposure time of 100 ms. In some embodiments, fiducial beads were included to account for drift correction. In this manner, fiducial beads of 0.2 pm 660 / 680 nm may be excited using a 642 nm epi-illumination at -3 W / cm2. It should be noted, to combat fiducial bead saturation, a 0.6 ND filter may be placed in a red channel of the emission path. Further, for active stabilization, imaging of 3 pm carboxylated polystyrene fiducial beads may be performed with an 850 nm LED at - 1.3 W / cm2The CMOS camera may be operated at an exposure time of 10 ms with a correction speed of 400 ms for active stabilization. With this in mind, imager strands for each target (e.g., mitochondria and lamin A / C) and / or a solution of washing buffer that may be introduced between targets may be flowed through the sample imaging chambers using a pressure-based flow control pump set to a pressure of 345 mbar. It should be noted that the pressure may correspond to a calibrated flow rate of 58 pL / min. In some embodiments, after imaging, dark frames may be acquired with lasers turned off and camera shutters closed. Further, z-scan calibration stacks of the 12-pm axial range DH- PSF in the red channel and the 2-pm axial range DH-PSF in the green channel may be acquired with fiducial beads (T7280, TetraSpeck, 0.2 pm, Invitrogen) spin coated in 1% (w / w) PVA in nanopure water on a coverslip. Registration images of both channels may be acquired using the standard PSF with fiducial beads (T7280, TetraSpeck, 0.2 pm, Invitrogen) spin coated in 1% (w / w) PVA in nanopure water on a coverslip.

[0069] In certain embodiments, multi-target single-molecule imaging of the three nuclear targets may be performed. In this manner, 10,000 frames may be acquired for each target at each slice using the 560 nm laser at -190 W / cm2 and an exposure time of 100 ms. Fiducial bead drift correction may be performed, using 0.2 pm 660 / 680 nmfiducial beads that may be excited using 642 nm epi-illumination at ~3 W / cm2. To combat fiducial bead saturation, a 1.0 ND filter may be placed in the red channel of the emission path. Imager strands for each target and washing buffer between targets may be flowed through the microfluidic channel with the pressure-based flow control pump set to a pressure of 345 mbar, corresponding to a calibrated flow' rate of 58 pL / min. In this manner, after imaging, dark frames may be acquired with lasers turned off and camera shutters closed. Further, z-scan calibration stacks of the 12-pm axial range DH- PSF in the red channel and the 2-pm axial range DH-PSF in the green channel may be acquired with fiducial beads (T7280, TetraSpeck, 0.2 pm, Invitrogen) spin coated in 1% (w / w) PVA in nanopure water on a coverslip. Registration images of both channels may be acquired using the standard PSF with fiducial beads (T7280, TetraSpeck, 0.2 pm, Invitrogen) spin coated in 1% (w / w) PVA in nanopure water on a coverslip.Data analysis

[0070] In some embodiments, 3D single-molecule images may be analyzed. In some instances, one or more analysis methods may be compared. For example, 3D single-molecule images acquired using a DH phase mask in the green channel may be imported into Easy-DHPSF (e.g., MATLAB-based open-source localization software for non-overlapping emitters), and / or a deep learning based single-molecule detection and localization tool DECODE optimized for analysis of overlapping emitters. As such, 2D single-molecule data for Exchange-PAINT labeling and imaging controls may be analyzed in ThunderSTORM, an open-source ImageJ plugin. In some embodiments, drift correction may be applied to data. For example, one channel imaging (e.g., where the fiducial bead was detected in the same channel as the single-molecule data) data localized by Easy-DHPSF may be drift corrected with a built in Easy-DHPSF drift correction. Further, DECODE-localized data may be drift corrected separately with a custom MATLAB script using fiducial bead data localized in Easy-DHPSF. Further, for two channel whole-cell imaging (e.g., fiducial bead data was acquired in the red channel with the 12-pm range DH-PSF and single-molecule data was acquired in the green channel with the 2-pm range DH-PSF) the 12-pm fiducial bead data may be localized with a custom-edited version of Easy-DHPSF compatible with the 12-pm range DH-PSF. The 12-pm range fiducial bead data may be transformed into the greenchannel with a custom 2D registration code which utilizes an affine transformation to map beads from one channel to another. Tracked motion of the fiducial bead may be smoothed with a cubic spline fitting function and subtracted from the high-density green channel single-molecule data localized by DECODE using a custom MATLAB script. In some embodiments, whole-cell imaging data analysis was conducted. For example, slices may be stitched together with custom scripts which first shift data sets based on a position of fiducial beads that were detectable across multiple slices owing to the very long axial range of the 12-pm DH-PSF. Further, correction of any residual offsets in z (e.g., z-axis) may be executed using cross-correlation between adjacent slices. Further, localizations may be filtered along a gradient from the top of each slice to avoid appearance of harsh lines between stitched slices.

[0071] In some embodiments, once 3D single-molecule data is localized, drift corrected, and stitched together, the 3D single-molecule data may be rendered using Vutara SRX (Bruker) (e.g., software) for visualization. As such, localizations may be visualized by a 3D Gaussian with a 16 nm diameter using point splat rendering. In some instances, epi-illumination may be compared to LS illumination. For example, as shown in FIG. 9, comparison data of lamin Bl may be filtered to remove any localizations with localization precisions in x, y or z greater than 100 nm, photon counts greater than 50,000, and a DH lobe separation below 4.5 or greater than 8.5 pixels. In some embodiments, analysis protocols may be compared. For example, Easy-DHPSF versus DECODE comparison data of microtubules, as shown in FIG. 8, may illustrate localizations with a localization precision greater than 20 nm in xy or greater than 30 nm in z were filtered out. Further, data sets may be filtered for 20-nearest neighbors with a denoise range of 0.01 to 10 to remove spurious localizations. In this manner, the lamin A / C and mitochondria data sets may be filtered to remove localizations with localization precision greater than 30 nm in xy or greater than 50 nm in z. In some embodiments, whole-cell lamin Bl, lamin A / C, and LAP2 data sets as shown in FIG.9 may be filtered to remove localizations with localization precision greater than 30 nm in xy and greater than 150 in z. As such, each slice is filtered to remove localizations with z values greater than 1 pm (the upper limit of our 2-pm DH-PSF).

[0072] In some embodiments, as shown in Table 2, resolution analysis may be executed. For example, Fourier ring correlation (FRC) may be calculated in Vutara SRX and may be used to analyze data in xy. yz, and / or xz planes. In this manner, a SR pixel size of 8 nm may be used with a threshold of 0.143 ( 1 / 7) to extract the resolution.

[0073] In certain embodiments, DECODE training may be executed. In this manner, DECODE receives experimental single-molecule data and simulates realistic singlemolecule data for training. In some embodiments, a DECODE model was trained to detect high density data acquired with the 2-pm range DH-PSF. Further, training may be performed by feeding a DECODE environment a calibration file based on one or more experimental parameters of the optical setup (e.g., imaging system). In some instances the experimental parameters may include signal photons ranging from 0 to 18,000 photons per localization, background levels of 0 to 200 photons per pixel, a dark level of 477 A / D counts, an EM gain of 182, a conversion gain of 4.41 photoelectrons per A / D count, and / or the axial range of our 2-pm PSF. Further, in some instances, the experimental parameters may include sparse 3D single-molecule data acquired with the single-objective light sheet. Further, one or more high density (2.17 pm’2) data sets may be simulated based on the one or more experimental parameters and / or additional features of the optical setup. In this manner, a model of DECODE may be trained on 0.025 nM microtubule data set (e.g., collected with the optical setup) and converged to a Jaccard index of 0.69 after 1000 epochs. In this manner, a performance of the model may be benchmarked. Benchmarking may be executed in terms of precision through comparison of a localization precision of single-molecule data from 200,000 frames toa localization precision of three different fiducial beads localized in over 25,000 frames using both DECODE and Easy-DHPSF). In some embodiments, the localization precisions in x, y, and z for the three beads were extracted from Gaussian fits of the localization distributions and the localization precisions for the single-molecule data were extracted from each software (e.g., DECODE and Easy-DHPSF). It should be noted that results of the comparison showed comparable values between the two approaches (e.g.. determination of localization precisions using DECODE and Easy- DHPSF) both for the fiduciary beads and single-molecule data.Control system and laser system

[0074] The system (e g., optical setup, deep neural learning software, and the like) may include a control system and a laser system. The control system may include communication circuitry, one or more processors, a memory, instructions, an input / output (I / O) port, a power supply (e.g., wired power, a battery), a display, a user interface, and the like. The control system may enable communication between various components of the optical setup. It should be noted, that one or more of the various components of the optical data setup may be omitted.

[0075] The communication circuitry may facilitate wired or wireless communication between various components of the control system as well as with external devices (e.g., mobile device, tablets, personal devices, etc.). The one or more processor may be any suitable type of computer processor or microprocessor capable of executing computer-executable code. Moreover, the processor may include multiple microprocessors, one or more "general-purpose" microprocessors, one or more specialpurpose microprocessors, and / or one or more application specific integrated circuits (ASICs), or some combination thereof. For example, the processor may include one or more than one reduced instruction set (RISC) or complex instruction set (CISC) processors. In some embodiments, the processor may receive inputs transmitted from the user interface and communicate with the optical setup. For example, an input with an instruction to close a shutter of one or more cameras (e.g., EMCCD, CMOS, and the like) may be executed.

[0076] The memory of the control system may also be used to store the data, various other software applications, and the like that are executed by the processors. The memory may represent non-transitory computer-readable media (e.g., any suitable form of memory' or storage) that may store the processor-executable code used by the processors to perform various techniques described herein. The processors may be configured to execute instructions. The instructions, when executed by the processors, may enable the control system to control the user interface to selectively query a user to enable generation of the light sheet, dithering of the light sheet, and the like. The user interface may include a display that is configured to display text or images transferred to it from the one or more processors. In addition to and / or alternative to the display, the user interface may include other devices for interfacing with the user, such as lights (e.g., LEDs), the laser system, speakers, and the like. The I / O ports may be interfaces that may7couple to other peripheral components such as input devices (e.g., keyboard, mouse), sensors, input / output (I / O) modules, and the like. The power supply may provide power to one or more components of the control system.

[0077] The laser system of the optical setup may include one or more emitters and / or one or more detectors. The emitters may include one or more lasers, one or more LEDs, arc lamps, tungsten halogen sources. IR emitters, deuterium light sources, and the like. The lasers may generate light through stimulated emission and may include a continuous wave laser, a pulsed laser, a quasi -continuous wave laser, or a combination thereof. The emitters may emit light across the electromagnetic spectrum through a diffuse and / or a continuous source. For example, the emitters may emit light ranging from 200 nm to 400 nm, 400 nm to 600 nm. and / or 480 nm to 880 nm. The lasers may emit light at a single frequency (e.g., continuous, pulsed) that may include 405 nm, 454 nm, 488 nm, 530 nm, 560 nm, 632 nm, 642 nm, 647 nm, 676 nm, 780 nm, 1064 nm, and the like. The lasers may include gas lasers, solid-state lasers, fiber lasers, liquid lasers, semiconductor lasers, laser diodes, or a combination thereof. The detectors may include a thermal imager, a complementary metal-oxide-semiconductor (CMOS) camera, a charge-coupled device (CCD), electron-multiplier charge-coupled device (EMCCD), one or more photodiodes, pyroelectric sensors, one or more photodetectors, a photomultiplier tube (PMT), and / or other suitable detectors.Technical Effects

[0078] The present disclosure is directed to a single-objective tilted light sheet fluorescence microscopy setup for SR microscopy that: (i) implements a sample imaging chamber with reflective sidewalls for control of the extracellular environment, (ii) enables multitarget imaging using only one channel with sequential Exchange- PAINT, (iii) allows for 3D imaging using point spread function engineering, and (iv) utilizes deep learning to localize overlapping emitter concentrations and reduce acquisition times. In certain embodiments, the single-objective tilted light sheet fluorescence microscopy setup offers improved localization precision, extracellular environment control, multi-target information, and / or three-dimensional imaging that may be implemented for fast, accurate SR whole cell imaging with a simple optical setup and without the need for complex microfabrication processes. In some embodiments, active stabilization drift correction may enable collection of SR images of subcellular structures.

[0079] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims

CLAIMS:

1. A method of forming a sample imaging chamber, comprising: forming a three dimensional nanoprinted metalized insert, via a method comprising: forming, via two-photon polymerization direct laser writing, a three dimensional nanoprinted insert comprising a material on a surface of a fused silica substrate, wherein the material is a nonfluorescent methacry late-based resin; and depositing, via electron beam vapor deposition, one or more layers of one or more materials on at least a side of the three dimensional nanoprinted insert, forming the three dimensional nanoprinted metalized insert; forming, via photolithography, a polydimethylsiloxane base, wherein the poly dimethylsiloxane base comprises one or more channels; and inserting the three dimensional nanoprinted metalized insert into the poly dimethylsiloxane base..

2. The method of claim 1, further comprising binding the three dimensional nanoprinted metalized insert disposed within the polydimethylsiloxane base forming the sample imaging chamber.

3. The method of claim 1, comprising: depositing a first layer of the one or more layers, wherein the first layer is silica; depositing a second layer of the one or more layers, wherein the second layer is aluminum; and depositing a third layer of the one or more layers, wherein the third layer is silica.

4. The method of claim 3. wherein the first layer is thinner than the second layer.

5. The method of claim 3, wherein the third layer is thinner than the second layer and the first layer.

6. The method of claim 1, wherein binding the three dimensional nanoprinted metalized insert disposed within the polydimethylsiloxane base to the coverslip comprises: treating, via air plasma, the coverslip and the polydimethylsiloxane base, for approximately 30 seconds; and binding the coverslip and the polydimethylsiloxane base, wherein binding forms covalent bonds.

7. The method of claim 1, wherein the one or more channels are configured to specify a tilt angle of a light sheet generated by an optical setup within the sample imaging chamber.

8. The method of claim 1, wherein forming, via photolithography, a polydimethylsiloxane base, comprises forming a mold comprising: coating, via spin casting, a silicon wafer with one or more layers of photoresist, wherein the one or more layers of photoresist are drop cast on the silicon wafer; heating the silicon wafer with one or more layers of photoresist for a first period of time; designing, via lithography, a custom film photomask, wherein the custom film photomask comprises one or more channel masks; disposing the custom film photomask on a surface of the silicon wafer coated with one or more layers of photoresist; exposing, via ultraviolet light irradiation, the surface of the silicon wafer for a second period of time; heating the exposed silicon wafer for a third period of time; developing, via a developer, the heated silicon wafer; and outputting, the mold.

9. The method of claim 8, wherein forming one or more channels within the poly dimethylsiloxane base, comprises: positioning, one or more pins, at one or more ends of the mold;forming a polydimethylsiloxane solution, wherein the polydimethylsiloxane solution is formed on contacting the elastomer and the curing agent; pouring a two-part system into the mold, wherein the two-part system comprises an elastomer and a curing agent; curing the polydimethylsiloxane solution; forming a poly dimethylsiloxane slab; removing the one or more pins from the mold; removing the poly dimethylsiloxane slab from the mold; and outputting the poly dimethylsiloxane base comprising the one or more channels.

10. The method of claim 1, comprising culturing one or more cells on a surface of the sample imaging chamber.

11. A method comprising: positioning a sample imaging chamber on an optical setup; and performing an imaging operation, wherein the imaging operation images a sample disposed within the sample imaging chamber; and providing one or more solutions to the sample imaging chamber, wherein the one or more solutions may be exchanged.

12. The method of claim 11, comprising: forming the sample imaging chamber, via a method comprising: forming a three dimensional nanoprinted metalized insert; forming, via photolithography, a polydimethylsiloxane base, wherein the poly dimethylsiloxane base comprises one or more channels; inserting the three dimensional nanoprinted metalized insert into the polydimethylsiloxane base; and binding the three dimensional nanoprinted metalized insert disposed within the poly dimethylsiloxane base to a coverslip.

13. The method of claim 11 , wherein the sample imaging chamber is biocompatible.

14. The method of claim 11, wherein the sample imaging chamber comprises: a three dimensional nanoprinted metalized insert; a polydimethylsiloxane base: and one or more channels configured to specify a tilt angle of a light sheet generated by the optical setup.

15. The method of claim 11, wherein the sample imaging chamber is configured to allow imaging in one or more illumination geometries, wherein the one or more illumination geometries comprise epi illumination, transmission illumination, and / or light sheet illumination.

16. A sample imaging chamber comprising: a three dimensional nanoprinted metalized insert; a poly dimethylsiloxane base; and one or more channels configured to specify a tilt angle of a light sheet generated by the optical setup within the sample imaging chamber.

17. The sample imaging chamber of claim 16, wherein a sample is positioned in the one or more channels.

18. The sample imaging chamber of claim 17, wherein the sample is a plurality of cells.

19. The sample imaging chamber of claim 16. wherein the microfluidic chip is biocompatible.

20. The sample imaging chamber of claim 1 , wherein the one or more channels is permeable to gas.

Images