Three Dimensional Orientation of Anisotropic Plasmonic Aggregates

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Three Dimensional Orientation of Anisotropic Plasmonic Aggregates at Intracellular Nuclear Indentation Sites by Integrated Light Sheet Super-Resolution Microscopy Suresh Kumar Chakkarapani, Yucheng Sun, Seungah Lee, Ning Fang, and Seong Ho Kang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b00025 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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Three Dimensional Orientation of Anisotropic Plasmonic Aggregates at Intracellular Nuclear Indentation Sites by Integrated Light Sheet SuperResolution Microscopy

Suresh Kumar Chakkarapani†, Yucheng Sun†, Seungah Lee‡, Ning Fang*,§, Seong Ho Kang*,†,‡ †

Department of Chemistry, Graduate School, Kyung Hee University, Yongin-si, Gyeonggi-do

17104, Republic of Korea ‡

Department of Applied Chemistry and Institute of Natural Sciences, Kyung Hee University,

Yongin-si, Gyeonggi-do 17104, Republic of Korea §

Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, United States

-------------------------------------------------------------------------------------------------------------------------------

*Corresponding authors: Ning Fang: Phone, +1-404-413-5513; e-mail, [email protected]. Seong Ho Kang: Phone, +82-31-201-3349; e-mail, [email protected].

KEYWORDS: integrated 3D nanoscopy, gold nanorod, anisotropy, super-localization, polarization

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ABSTRACT. Three-dimensional (3D) orientations of individual anisotropic plasmonic nanoparticles in aggregates were observed in real time by integrated light sheet super-resolution microscopy (iLSRM). Asymmetric light scattering of a gold nanorod (AuNR) was used to trigger signals based on the polarizer angle. Controlled photo-switching was achieved by turning the polarizer and obtaining a series of images at different polarization directions. 3D sub-diffraction limited super-resolution images were obtained by super-localization of scattering signals as a function of the anisotropic optical properties of AuNRs. Varying the polarizer angle allowed resolution of the orientation of individual AuNRs. 3D images of individual nanoparticles were resolved in aggregated regions, resulting in as low as 64 nm axial resolution and 28 nm spatial resolution. The proposed imaging setup and localization approach demonstrates a convenient method for imaging under a noisy environment where the majority of scattering noise comes from cellular components. This integrated 3D iLSRM and localization technique was shown to be reliable and useful in the field of 3D non-fluorescence super-resolution imaging.


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Plasmonic nanoparticles (NPs) have emerged as an important bio-labelling probe for high sensitivity detection with optical microscopes.1-4 High photostablity5 and large extinction cross sections6,7 make them reliable for obtaining high spatial and temporal resolution images. Despite the super-resolution achieved with fluorescence-based optical microscopy,8,9 challenges still remain for NP-labelled biomolecules. Factors such as diffraction limitations,10 dielectric constants,11 and coupling of nearby plasmonic NPs make single-particle studies with optical microscopes diffficult.12-15 Imaging single gold nanorods (AuNRs) becomes increasingly important with the developments of AuNRs as cancer drug carrier,16,17 RNA delivering agent,18 selective oligonucleotide DNA releasing agent,19 and Ferritin-Encapsulated nanodrug carrier.20 So far, single-molecule studies of plasmonic NPs have been reported by super-localization based wavelength modulated enhanced dark field microscopy,21 polarization based differential interference contrast microscopy (DIC),22,23 hyperspectral enhanced dark field microscopy,24 wavelength-dependent total internal reflection (TIR) microscopy,25,26 and light sheet microscopy (LSM).27 In most of the above reports, the NPs of different scattering wavelengths were resolved by super-localizing their center coordinants.28 However, resolving NPs with the same scattering wavelength at subdiffraction limit resolution remains a challenge for optical microscopes. Previous reports on super-localization of AuNRs suggested that it is still difficult to separate individual NPs at their aggregates.29 The anisotropic shape of AuNRs makes them polarization sensitive, allowing tracking of a single particle intracellularly.7,18,30 Polarization-based super-localization can provide super-resolution imaging of AuNRs.31 However, single-particle imaging of intracellular AuNRs’ three-dimensional (3D) position and aggregated orientation has not yet been reported.


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In this study, an integrated light sheet super-resolution microscope (iLSRM) setup comprised of a dove-type prism-based LSM coupled to a differential interference contrast (DIC) microscope was used for polarization-based 3D super-localization of anisotropic AuNRs (Figure 1), giving insight into the intracellular position of NPs. Similar to stochastic photo-switching of fluorescence molecules, the scattered signal of AuNRs could also be switched on and off by changing the polarization of the incident beam with respect to the orientation of the AuNRs, or vice versa.31 The polarization effect was coupled with astigmatism for 3D localization of AuNRs.

RESULTS AND DISCUSSION Prism-based 3D Integrated Light Sheet Super-resolution Microscopy. Although conventional microscopic techniques have been used extensively for single particle imaging, they have drawbacks and limitations such as the diffraction limitation,10 dielectric constants,11 and coupling of nearby plasmonic NPs.12-15 To overcome these issues, we designed a dualwavelength 3D integrated light sheet super-resolution microscope (i.e., 3D iLSRM), which combines LSM and DIC microscopy (Figure 1 and Supporting Information, Figure S1). LSM has been proposed with inverted selective-plane illumination microscopy (iSPM),32 using a polished AFM cantilever as a mirror for sub-cellular imaging applications.33 These techniques still left a gap (2 m) that could not be imaged within live cell samples.34 To address this issue, Hu et al. designed a prism-based LSM setup for intracellular imaging.34 However, due to use of a prism that was horizontal to the focal plan of the imaging objective lens, this technique suffered from PSF artifacts, especially when using astigmatism for 3D imaging. Therefore, we designed a micro dove-type prism-based LSM with a plan perpendicular to the plane of the imaging objective lens. The micro dove-type prism-based LSM provided the additional advantage of 4 ACS Paragon Plus Environment

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using DIC microscopy for imaging a single cell simultaneously by both techniques. The design was also more compatible with cover glass imaging of cell cultures without requiring a special sample holder (Supporting Information, Figure S1c). Additionally, the design enhanced imaging of NPs by using a high magnification lens with a numerical aperture (NA) of 0.95. The virtual light sheet for LSM was generated by cylindrical optics35 on the surface of the micro dove-type prism (Figure 1a). To reduce the thickness of the light sheet, the cylindrical lens angle was changed from perpendicular to the direction of the laser beam, to horizontal to the direction of the laser beam. The astigmatism of the cylindrical lens stretched the light sheet, making it narrower and longer, resulting in a wider light sheet (Supporting Information, Figure S1b). The light sheet was focused on a prism surface for characterization (Supporting Information, Figure S2). The light sheet was ~1.2 µm thick and ~10 µm long and gave higher S/N (Supporting Information, Figure S2e). The thickness of the light sheet could be further reduced by turning the cylindrical lens more horizontal to the plan of illumination from the beam expander. However, this resulted in a large amount of lost light and poor S/N. The proposed light sheet thickness was therefore optimum for imaging a single cell. HeLa cell thicknesses were measured to be 10 µm, which was quite large for normal TIR imaging, and can typically reach imaging depths up to 200 nm. Single-Particle Scattering. The surface plasmon resonance of the AuNR was split into the transverse and longitudinal resonance modes using its asymmetric structure and the polarization of the incident light.36 The extinction spectra of 40  80 nm AuNR (an aspect ratio of 2) therefore exhibited two extinction maximum in the visible region (Supporting Information, Figure S3). The peak at the shorter wavelength with a low extinction intensity corresponded to the transverse resonance mode, and the peak at the longer wavelength with a high extinction 5 ACS Paragon Plus Environment

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intensity corresponded to the longitudinal resonance mode.37,38 For single-molecule studies of AuNR, a AuNR was immobilized on a cover glass and illuminated with a 671 nm laser light sheet. Images were recorded with a 100× objective lens with adjustable NA (Supporting Information, Figure S4). The S/N of a single emitter was observed to be as high as 0.95, which was considered optimum for imaging NPs with iLSRM. Due to the large scattering cross section of the AuNR, the spectrum was broad (Supporting Information, Figure S3). To resolve the scattering signal wavelength of a 40  80 nm AuNR, two laser sources at different wavelength (473 nm and 671 nm) were illuminated with various bandpass filters ranging from 420 nm to 680 nm (Supporting Information, Figure S5). Scattering signals were observed to be higher with the 671 nm laser than with the 473 nm laser, with signals at 420/10 nm, 575/10 nm, and 680/10 nm filters. There was no scattering signal with either 485/15 nm or 520/10 nm band pass filters. The filter ranges at which no signal was detected could be utilized for other labels for multi-label imaging. For single-molecule super-resolution imaging of NPs, NPs of different wavelength were imaged simultaneously with a dual-view setup. The scattering wavelength of 80 nm silver NPs (AgNP) was around 480 nm, which did not interfere with the scattering wavelength of 40  80 nm AuNRs. Therefore, when these NPs were near each other, NPs were imaged simultaneously with a dual-wavelength setup using suitable bandpass filters (Figure 2 and Supporting Information, Figure S6). With 473 nm and 671 nm lasers, the scattering signal of both NPs were inseparable without bandpass filters. Theoretically, the full width at half maximum (FWHM) in xy of 80 nm AgNPs at a scattering wavelength of 473 nm under a 0.95 NA 100× objective lens was around 280 nm, compared to 350 nm for a 40  80 AuNR at a scattering wavelength of 680 nm. Therefore, the scattering overlap could not be resolved when NPs were within hundreds of 6 ACS Paragon Plus Environment

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nanometers of each other. To resolve NPs with sub-diffraction limit resolution, each NP position was localized by taking over 1000 frames simultaneously with a dual-view setup (the detailed procedure on super-localization is given in the experimental section). The localization precision of 80 nm AgNP was around 2.48 nm and that of AuNR was around 4.86 nm. The spectral shifts from plasmon coupling was unavoidable when NPs were close to each other; however, by using a dual-view setup with band-pass filters specific to the scattering maxima of each NP, the negative effect on NP localization was minimized. To confirm the effect of plasmon scattering on localization precision, 80 nm AgNPs were imaged individually and their localization precision was calculated to be 2.06 nm, which was similar to 2.48 nm. (Supporting Information, Figure S7). Therefore, the dual-view setup almost completely removed the influence of plasmon coupling on localization precision of plasmonic NPs. To localize NPs in the z-direction in 3D images, a cylindrical lens was inserted into the optical path of the microscope to induce astigmatism. The PSF of the sphere-shaped and rodshaped NPs were similar under optical microscopes, but with a cylindrical lens, the PSF in the zdirection of AuNRs was different than that of AgNPs (Supporting Information, Movie M1). In zslices, the deviation in FWHM of AuNRs was 5.6 times higher than that of AgNPs (Supporting Information, Figure S8). The differences in PSF were largely due to the anisotropic intensity distribution of AuNRs in the z-direction. An axial resolution of around 63 nm was achieved using this method (Supporting Information, Figure S6f). However, the method was only applicable when two nearby NPs had different scattering wavelengths. The proposed imaging setup and localization approach demonstrates the convenient method for imaging under the noisy environment with the majority of scattering noise from cellular components.


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Super-localization with Polarization. For single, isolated 40  80 nm AuNRs, images were collected with a 680/10 nm bandpass filter using an electron multiplied charged cooled device (EMCCD) camera. Theoretically, the FWHM of the PSF for a single emitter with a NA = 0.95 objective is around 400 nm (λ = 675 nm), whereas that of a single isolated AuNR image under LSM conditions was 350 nm (Figure 3a). Over one thousand frames were recorded and localization precision was calculated for the AuNR in the x and y coordinates by fitting the true position of the emitter multiple times. Localization precision was calculated with the following equation39

𝑛

1 𝑥 = √ ∑(𝑥 − 𝑥̅ )2 , 𝑛−1 𝑖=1

where n is the number of the estimated fitting of the true position, x is the spread of the true position, and 𝑥 is the mean value of the true position. The same applies for y coordinates, with localization precision in x and y calculated by 𝑥𝑦 = √(𝑥 2 + 𝑦 2 ).39 Localization precision of an isolated AuNR was measured to be around 4.86 nm (Figure 3b). An additional advantage of using plasmonic NPs as labeling probes is the absence of photo bleaching, allowing large numbers of photons to be detected for a single plasmonic NP with a localization precision lower than 1 nm.40 However, due to the asymmetric distribution of intensity of AuNRs, the localization precision was found to be larger than that of a typical single NP. Single AuNR images at various angles were acquired by turning the polarizer (Supporting Information, Figure S9). The significant intensity changes with respect to the AuNR orientation (Supporting Information, Movie M2) suggests the possibility of anisotropy-based photo-switching. Figure 3c shows the super-localization track of a AuNR with respect to changes in the polarizer angle. The polarizer 8 ACS Paragon Plus Environment

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was turned to different angles and the localization precision was calculated at each angle. Localization precision was inversely related to the intensity of the emitter, which depends on the polarizer angle and the AuNR orientation (Figure 3d). Localization precision was the best at the maximum scattering intensity when the polarization direction and AuNR orientation were parallel, and poorly localized points were filtered out during reconstruction. The polarization cycle was repeated several times and reconstructed to form a single image that could resolve a AuNR at the sub-diffraction limit. The image was reconstructed with the least square algorithm used for conventional fluorescence super-resolution images.41 After reconstruction, the PSF was localized at each angle, resulting in a composite image based on images from each angle (Supporting Information, Figure S10). During post processing, events with poor localization are filtered by local density based filtering, resulting in images only at angles with the best localization precision (Figure 3e). As the intensity of the emitter for AuNR was highest at two angles (Supporting Information, Movie M2), the reconstructed super-resolution image contains two points in a single image that show AuNR orientation. The FWHM of localized events was calculated to be approximately 41 nm. Fluorescence-free iLSRM Imaging. Our goal was to image AuNR aggregates to resolve individual AuNRs orientations with fluorescence-free imaging at sub-diffraction limited resolution. Differences in localization precision were taken into account to resolve plasmon coupling of AuNRs. Nearby NPs were chosen, and images were taken at different polarizer angles (Figure 4a). The polarizer was turned repeatedly to obtain enough photons for localization of individual AuNRs, and super-localization tracking showed distinct NPs depending on the orientation of polarizer (Supporting Information, Movie M3). The pink circle indicates two identical spots with maximum intensities at different angles, which resulted in excellent


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localization precision. Conversely, lower intensities gave poor localization precision. Following the same strategy used with isolated AuNRs, poorly localized regions were filtered out from the super-localization images. (Figure 4c). The distance between the resolved NPs was 48 nm, which is well below the diffraction limit of optical microscopes. Pattern matching was used to study the orientation of two nearby AuNRs.41 Figure 4e shows the scattering spectra of nearby AuNR with a change in the polarizer orientation. Spectral peaks were split into either three peaks or one peak, depending on the angle. To ascertain the angle between two AuNRs, a finite difference time domain (FDTD) simulation was carried out with angles between 0 and 180. Simulation results suggested that when two AuNRs were parallel to each other, the scattering wavelength was around 560 nm, which was not within the detection range for the bandpass filter used in this study. However, spectra at a 670 nm were intensified as the angle between AuNRs approached 180. By comparing the simulated result (Supporting Information, Figure S11) with experimental scattering spectra (Figure 4e), the angle between the AuNRs could be resolved. For example, the orientation angle had a single peak at 120 (Figure 4e) and showed the angle between the AuNR at that point was approximately 45. The feasibility of our proposed super-resolution imaging method was extended to an orientation analysis of NP aggregates. A region with adjacent NPs was chosen and imaged with TEM microscopy and analyzed with 3D iLSRM (Figure 5). The difference in AuNR orientations in aggregates produces different scattering signals as a function of polarization. Scattering signals are super-localized and can be resolved at sub-diffraction limit resolution using localization filtering (Figure 5c). However, NPs on a copper grid were parallel on the surface (Figure 5a). The 3D iLSRM image shows that the orientation of NPs numbered 1, 2 and 3 were parallel to the surface and had similar axial positions (Figure 5e).


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3D iLSRM Imaging in Live Single Cells. Based on Chithrani et al. and Xiao et al., AuNRs suspended in DMEM can be taken up by a cell via receptor mediated endocytosis.42,43 After 4 h of incubation with HeLa cells, NPs were imaged in iLSRM (Figure 6b). The orientation of AuNRs in cells was different than on cover glass: on cover glass, AuNRs were positioned on the surface and were approximately parallel to the surface, while in live cells, AuNRs interactions with intracellular organelle and the incubation site affected their orientations. 3D imagining was therefore required to analyze AuNRs in live cells. In conventional 3D super-resolution fluorescence imaging, a cylindrical lens is placed in the optical path to create an astigmatism for localization in z direction.44 By following the same method with the proposed orientationdependent super-localization, a 3D fluorescence-free super-resolution image could be obtained. The cell was identified with a DIC microscope, which is an advantage of using an integrated setup (Figure 6a). Over 1000 frames of iLSRM image were acquired at 100 ms frame rate that took about 3 min for the overall acquisition. The iLSRM image with polarizer rotation was reconstructed into a single frame, super-resolution image (Figure 6c and Supporting Information, Figure S12). A portion of the image was expanded to show the resolution difference between the images. Figure 6c shows the resolved AuNRs, which were not resolved with a conventional microscope (Figure 6b). The distance between isolated points was measured to be approximately 28 nm laterally and 68 nm axially (Figure 6c), indicating the reliability of the method. Localized AuNRs were resolved in 3D with a lateral resolution of 40 nm (Figure 6). The two PSF of a single AuNR were resolved in 3D (Supporting Information, Movie M4) with respect to the orientation of the AuNR in a HeLa cell. The orientation of each resolved AuNR aggregate is illustrated in Figure 6 (1-4). While axial positions of individual AuNR aggregates were similar, their orientations varied (Supporting Information, Movie M4). By z slicing with a cylindrical


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lens, AuNRs give rise to different anisotropic PSFs at opposite angles. The feasibility of distinguishing opposite angles varied from 30–50 nm. 3D-astigmatism based localization of the PSF generated in opposite directions resulted in a localized point in 3D. Oppositely orientated points were matched to calculate the orientation angle of AuNRs.

CONCLUSION A 3D dual-view DIC integrated LSRM (3D iLSRM) was tested for analysis of 3D orientation of AuNRs in single cells. For multi-wavelength SRM imaging of AuNR, a dual-view setup was combined with a cylindrical lens for simultaneous 3D imaging. The system was proposed to overcome the diffraction limit of light and achieve fluorescence-free imaging of single particles and NPs aggregates. The wavelength-resolved 3D super-resolution approach gave a lateral resolution of 28 nm and an axial resolution of 64 nm. However, to resolve individual AuNRs at aggregates, a polarizer had to be added to the 3D iLSRM system to exploit orientation-dependent photoswitching of AuNRs. By changing the orientation of the polarizer, individual NPs were super-localized and tracked dynamically. Using the difference in localization precision of AuNRs with respect to the orientation angle, individual AuNRs were resolved even in NP aggregates. Signals were brighter (higher) in opposite directions, corresponding to the polarizer angle matching that of the AuNR. Brighter signals were localized while poorly localized spots for the same AuNR were filtered with a localization filter. Thus, individual AuNR signals were localized, and by combining this approach with a cylindrical lens, the 3D orientation of AuNR was measurable in live cells. Astigmatism provided different ellipsoidal PSFs of the same AuNR at different angles. By localizing and matching PSF in opposite directions, the orientations of AuNRs were framed in NPs aggregates at the intranuclear 12 ACS Paragon Plus Environment

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indentation site. The 3D iLSRM system and super-localization technique could be widely applicable in fields such as optics, biomedicine and materials science.

MATERIALS AND METHODS Sample Preparation. AuNRs used in this study were purchased from Nanopartz as a colloidal suspension (Salt Lake City, UT, USA). For single particle studies, 50 µL of the AuNR colloid were centrifuged at 5500 rpm for 10 min, resuspended in 15 mL of ultrapure water, and sonicated for 20 min, which removed excess surfactant from the solution. For nanoparticle incubation with cells, colloids were suspended in 15 mL of indicator-free Dulbecco’s modified Eagle’s medium (DMEM, pH 7.4, GIBCO, Gaithersburg, MD, USA). Cervical cancer HeLa cells (PromoCell, Heidelberg, Germany) were cultured on a cleaned glass slide in a plastic cell culture dish. The cells were maintained in DMEM supplemented with 10% fetal bovine serum (GIBCO) and 1% antibiotics and antimitotic (GIBCO) at 37 °C, 5% CO2 in a humidified atmosphere. For single-particle tracking, 20 µL of SH-PEG protected AuNR suspended in DMEM was added to the cell culture dish and incubated for 4 h. Lab-built 3D iLSRM System. The system was constructed from an Olympus BX-51 upright microscope (BX-51, Olympus, Tokyo, Japan) equipped with a CytoViva EDF illumination device (CytoViva Inc., Auburn, AL, USA) and a 100× objective lens (UPLANFLN, adjustable NA from 0.6 to 1.3, Olympus). A dual-wavelength module (DV2, Photometrics, Tucson, AZ, USA) was mounted between the objective lens and the EMCCD camera (QuantEM, 512 SC, Photometrics, Tucson, AZ, USA), alongside a cylindrical lens and a polarizer (Thorlabs Inc., Newton, New Jersey, USA). A beam expander and a cylindrical lens with a 500 mm focal 13 ACS Paragon Plus Environment

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length were used for generating a light sheet. The light sheet was illuminated on the surface of a micro dove-type prism (0.5 cm 2.11 cm, PS990, Thorlabs Inc.) placed on a prism holder by a 10× objective lens (Olympus). For 3D information, z-axis sectioning was achieved with a zmotor (LEP MAC 5000, LUDL Electronic Products Ltd., Hawthorne, NY, USA). Data were acquired using MetaMorph (Universal Imaging, Sunnyvale, CA, USA). 3D Super-localization of Images. Images were reconstructed in 2D by a least square algorithm and in 3D by a lab-made least cubic algorithm. The centroid coordinates in the x, y, and z directions were measured using astigmatism-based 3D super-localization. Poorly localized PSFs were filtered out by a localization filter and then center coordinates were fit using the 3D least cubic algorithm. Briefly, the following 3D Gaussian function is the intensity distribution of the PSF.

1

𝐼(𝑥, 𝑦, 𝑧; 𝐼0 , 𝐴, 𝑥0 , 𝑦0 , 𝑧0 , 𝜎𝑥 , 𝜎𝑦 , 𝜎𝑧 ) = 𝐼0 + 𝐴 exp [− [( 2

𝑥−𝑥0 2 𝜎𝑥

) +(

𝑦−𝑦0 𝜎𝑦

2

𝑧−𝑧0 2

) +(

𝜎𝑧

) ]] (1)

In addition, let 𝐼exp (𝑥, 𝑦, 𝑧) be the intensity at location (𝑥, 𝑦, 𝑧) obtained from the experiment. Assuming that intensities are observed in a rectangular parallelepiped volume, 𝑉 = {(𝑥, 𝑦, 𝑧)| 𝑎 ≤ 𝑥 ≤ 𝑏, 𝑐 ≤ 𝑦 ≤ 𝑑, 𝑒 ≤ 𝑧 ≤ 𝑓}. We found constants (I0, A, x0, y0, z0, σx, σy, and σz) to minimize the objective function 𝐹(𝐼0 , 𝐴, 𝑥0 , 𝑦0 , 𝑧0 , 𝜎𝑥 , 𝜎𝑦 , 𝜎𝑧 ) when p was 2 or 3: 𝐹(𝐼0 , 𝐴, 𝑥0 , 𝑦0 , 𝑧0 , 𝜎𝑥 , 𝜎𝑦 , 𝜎𝑧 ) = ∑(𝑥,𝑦,𝑧)∈𝑉|𝐼(𝑥, 𝑦, 𝑧; 𝐼0 , 𝐴, 𝑥0 , 𝑦0 , 𝑧0 , 𝜎𝑥 , 𝜎𝑦 , 𝜎𝑧 ) − 𝑝

𝐼exp (𝑥, 𝑦, 𝑧)| . (2)


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Here, 𝑝 = 2 signifies the conventional least square algorithm and 𝑝 = 3 signifies the least cubic algorithm, which is useful for finding constants with considerable noise and asymmetry. In this study, the least cubic algorithm was implemented after its validity was verified by testing a Gaussian function with known constants with Gaussian noise up to 21% of the maximum intensity. In addition, to avoid the influence of noise, we removed low-level intensities less than an empirically established threshold (w) when using experimental images:

𝐼exp (𝑥, 𝑦, 𝑧) = Max (

𝐼exp (𝑥, 𝑦, 𝑧) − 𝐼Min 𝑤 ,𝑤 ) × . 𝐼Max − 𝐼Min 1−𝑤

(3)

Here, 𝐼exp (𝑥, 𝑦, 𝑧) is the intensity at (𝑥, 𝑦, 𝑧) in the obtained image, 𝐼Max is the maximum intensity in the obtained image, 𝐼Min is the minimum intensity in the obtained image, and 𝐼exp (𝑥, 𝑦, 𝑧) is the normalized intensity at (𝑥, 𝑦, 𝑧) after the cut off. We substituted 𝐼exp (𝑥, 𝑦, 𝑧) for 𝐼exp (𝑥, 𝑦, 𝑧) in Eq. (2).


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Figure 1. iLSRM setup and super-localization approach. Schematic illustration of (a) full 3D iLSRM system. Abbreviations: EMCCD, electron-multiplying cooled charge-coupled device camera; DV, dual-view; RP, rotatory polarizer; CL, cylindrical lens; RF, rotatory filter; PNP, polarizer Nomarski prism; CAD, condenser aperture diaphragm; CNP, condenser Nomarski prism; DOL, detecting objective lens; IOL, illumination objective lens; HL, halogen lamp; M, mirror; MS, mechanical shutter; L, laser; BE, beam expander; DM, dichroic mirror; z, z-stage controller. (b) Combinational approach including cylindrical lens and dual-view setup for 3D multi-wavelength SRM imaging of gold nanorods (AuNRs) and silver nanoparticles (AgNPs). (c) 3D SRM approach for resolving two AuNRs by combining RP, CL, and super-localization approach. (d) Illustration of the integrated LSM, DIC combined super-localization for iLSRM.


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Figure 2. Wavelength-dependent 3D iLSRM (a) TEM and (b) LSM images of a 80 nm AgNP and 40  80 nm AuNR near each other. (c) 2D raw LSM merged image and (d) super-localized merged image of the two channels (CH 1 and CH 2). (e) 3D raw LSM image and (f) 3D superlocalized merged image of the two channels.


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Figure 3. Super-localization of AuNR. (a) TEM (upper left inset), DIC (bottom left inset) and iLSRM images of an isolated AuNR. (b) Localization precision of isolated AuNR. (c) Superlocalization tracking of single emitter with change in orientation angle of polarizer. (d) Superlocalization tracking of the position of AuNR with respect to the orientation angle of the polarizer. (e) Super-localized images of AuNR with schematic representation of the orientation in the inset. (f) Relative scattering normalized intensity (RSI) profile with distance of AuNR with respect to the line region in (d).


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Figure 4. Sub-diffraction limit resolution images. (a) LSM raw image of AuNR. (b) Superlocalization tracking of the position of AuNR with respect to the orientation angle of the polarizer. (c) Resolved AuNR iLSRM images at sub-diffraction limit resolution. (d) iLSRM images at different angles and (e) relative scattering intensity with respect to various angles.


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Figure 5. 3D super-resolution of AuNR aggregates. (a) TEM, (b) raw LSM, and (c) SRM images of the AuNR aggregates. (d) Raw LSM 3D image and (e) 3D super-resolution images of AuNR aggregates. The numbers 1, 2, and 3 represent the AuNR in (a). The yellow mark indicates the orientation of the AuNR and the blue line shows the axial difference in localization of each NPs.


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Figure 6. 3D super-resolution image of AuNR in live single HeLa cell. (a) DIC and (b) LSRM images of HeLa cell labeled with AuNRs. (c) Reconstructed 3D iLSRM image of AuNR in a live HeLa cell. (b1) and (c1) are the insets in (b) and (c). (b2) and (c2) are the insets in (b1) and (b2). (1-4) 3D super-localized images of the AuNR numbered in (c1). The yellow mark indicates the orientation of the AuNR and the blue line shows the axial difference in localization of each NPs.


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ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Additional twelve figures (PDF) and four movies (AVI), as referenced in the main text including figures of 3D iLSRM setup physical layout, laser light sheet characterization, AuNR spectra, Numerical aperture analysis, Wavelength-dependent scattering analysis of AuNRs, Wavelengthdependent 3D localization, Photo-switching of an isolated single AuNR, Super-localization AuNR, FDTD simulation scattering spectra, Photo-switching of an isolated AuNR, Real time super-localization track of AuNR, 3D orientation of AuNRs in a live single HeLa cell.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT


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This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2015R1A2A2A01003839). N.F. was supported by National Science Foundation (CBET1604612).


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Three Dimensional Orientation of Anisotropic Plasmonic Aggregates

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