Background-Free Imaging of a Viral Capsid Proteins Coated

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Background-Free Imaging of Viral Capsid Proteins-Coated Anisotropic Nanoparticle on Living Cell Membrane with Dark-Field Optical Microscopy Zhongju Ye, Lin Wei, Xuyao Zeng, Rui Weng, xingbo shi, Naidong Wang, Langxing Chen, and Lehui Xiao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03762 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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Analytical Chemistry

Background-Free Imaging of Viral Capsid ProteinsCoated Anisotropic Nanoparticle on Living Cell Membrane with Dark-Field Optical Microscopy Zhongju Ye,1 Lin Wei,2 Xuyao Zeng,1 Rui Weng,3 Xingbo Shi,4 Naidong Wang,5 Langxing Chen,1 and Lehui Xiao*,1,2 1

State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing

and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China; 2

Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research,

Ministry of Education, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, 410081, China; 3

Key Laboratory of Agro-food Safety and Quality of Ministry of Agriculture, Institute of

Quality Standards and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 4

Hunan Provincial Key Laboratory of Food Science and Biotechnology, College of Food

Science and Technology, Hunan Agricultural University, Changsha, 410128, China; 5

*

College of Veterinary Medicine, Hunan Agricultural University, Changsha, 410128, China. Corresponding author

Email: [email protected] Fax: +86-022-23500201

ACS Paragon Plus Environment

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ABSTRACT

Exploring the diffusion dynamics of viral capsid proteins (VCP) functionalized nanocarrier on living cell membrane could provide plenty of kinetic information for the better understanding of their biological functionality. Gold nanoparticles are excellent core material of nanocarrier because of the good bio-compatibility as well as versatile surface chemistry. However, due to the strong scattering background from subcellular organelles, it is a grand challenge to selectively image individual nanocarrier on living cell membrane. In this work, we demonstrated a convenient strategy to effectively screen the scattering background from living cells for single particle imaging with a polarization-resolved dual channel imaging module. By taking the polarization advantage of anisotropic gold nanoparticles (gold nanorods, GNRs), the signals from cell components could be counteracted after subtracting the sequential images one by one while those transiently rotating GNRs on cell membrane still exist in the processed image. In contrast to the previously reported methods, this method doesn’t require complicated optical setup alignment and sophisticated digital image analysis process. According to the single particle imaging results, the majority of VCP-GNRs were anchoring on the cell membrane with confined diffusion. Interestingly, further inspection of the diffusion trajectories, the particles displayed anomalous confined diffusion with randomly distributed large walking steps during the whole track. Non-Gaussian step distribution was noted, indicating heterogeneous binding and desorption processes on the cell membrane. As a consequence of the robust background screening capability, this approach would find broad applications for single particle imaging under noisy environment, e.g. living cells.

KEYWORDS


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Dark-field optical microscopy; Gold nanorods; Cell membrane; Single particle tracking; Viral capsid proteins

INTRODUCTION Over the past decades, with the rapid development of nanotechnology, advances in material science are offering great promises to revolutionize medicine. New nanocarrier platforms exhibiting great potentials in improving drug packaging, delivery, and targeting efficiencies are currently emerging.1-3 In contrast to regular drug carriers, these new nanoparticles typically exhibit high loading efficiency, enhancement of drug releasing to the target tissue, prevention of premature drug degradation or interaction with undesired biological environment. Examples of extensively explored nanomaterials include synthetic polymeric and liposomal nanoparticles.4-8 These nanocarriers, however, still have some limitations such as wide size distributions, difficulty in site-specific functionalization, biocompatibility and instability.9

The advances in protein engineering represent a new class of nanomaterial, i.e. caged protein, which may address the majority of these concerns.9-12 Caged protein complexes are hollow structures comprised of self-assembled protein subunits that produce nano-capsules with a nearly monodispersed size distribution. The structure can be chemically or biologically engineered in an extremely precise way, making them uniquely attractive platforms for drug, gene and protein delivery. An extensively studied example is the self-assembled virus-like particles (VLPs), which have found broad applications as vehicles for gene therapy, drug translocation and vaccines for infectious agents.13-16 As an emerging and important nanocarrier platform, VLPs offer the great advantages of morphological uniformity, biocompatibility, and easy functionalization.

However,

in

biological

milieu, these self-assembled

hollow


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nanostructures are typically not stable upon storage. Incorporation of rigid nanoparticles (i.e., gold nanoparticles) to the inner cavity of the VLPs provides a perfect solution to this challenge.17,18

According to the earlier studies, caged proteins with dimension from several nanometers to micrometers could all be internalized by living cells with distinctive translocation efficiency owing to the grand barrier formed by cell membrane.19-21 On the aspect of developing robust nanocarrier platform, it is of great interest to explore the translocation dynamics of these hybrid nanoparticles on the cell membrane at single particle level. Until now, several kinds of materials have been encapsulated inside the protein cage, e.g. quantum dots, magnetic nanoparticles and gold nanoparticles.22-24 Among these nanoparticles, gold nanoparticles exhibit great advantages because of the versatile surface chemistry, well-defined size and morphology controllability, and excellent biocompatibility.25,26 Meanwhile, the large scattering cross-section enables it to be a good candidate as an imaging contrast probe.27-37 However, under living cell environment, the strong scattering light from subcellular organelles makes it difficult to differentiate the signal of individual gold nanocarrier with conventional dark-field optical microscope when the size of the nanoparticle is small, i.e. tens of nanometers.31,38 To address this issue, several interesting methods have been developed.28-32,35,39 For example, by detecting the photo-thermal induced refractive index change around the nanoparticle, gold nanoparticles down to several nanometers could be detected by using photothermal heterodyne imaging.28 One of the limitations of this method for the extensive applications of single particle tracking in living cells might be the complicated optical setup and high excitation intensities to obtain high signal-to-noise ratios (SNRs), which can be detrimental to the cell.


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In this work, we demonstrated a convenient and robust scattering-based background-free method to track the diffusion dynamics of VCP-coated anisotropic nanoparticles on living cell membrane. The interfering scattering background inside the cell from sub-cellular organelles was effectively screened by subtracting sequential images from a polarization-resolved dual channel detector to distinguish the specific scattering signal from individual anisotropic nanoparticles (i.e. GNRs) that transiently rotating on the cell membrane. Herein, porcine circovirus type 2 (PCV2) was used as a model protein to directly image the diffusion process of VCP-GNRs on living HepG2 cell membrane. PCV2 is a widespread pathogen, causes various diseases and syndromes, which is also possible to infect human hepatic cells.40 Several commercial vaccines have used PCV2-VLPs as the active component. Exploring the interaction of PCV2 capsid protein-functionalized nanocarriers on the cell membrane would then afford valuable information for the understanding of translocation efficiency as well as improvement of the biological functionality. By using this technique, the continuous diffusion trajectory of the target VCPGNRs on the cell membrane was then elucidated by integrating the time-dependent polarizationresolved images together. Interestingly, temporal confinement for random waiting times between surface displacements produced by excursions through the weak binding sites on the living cell membrane was resolved. This observation was noticeably distinct from the confined twodimensional random walk and Gaussian statistics that are commonly assumed for small biomolecule recognition process.

Experimental section Chemicals and Materials. HAuCl4•3H2O, NaBH4, NaOH, ascorbic acid (AA), cetyltrimethylammonium bromide


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(CTAB), sodium oleate (NaOL), NH2OH•HCl, AgNO3, NaCl, KCl, MgCl2, NaH2PO4•2H2O, and Na2HPO4 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). O-[2(3-MercaptopropionylaMino)ethyl]-O′-Methylpolyethylene glycol (SH-PEG-CH3, MW: 6000), Polyvinylpyrrolidone (PVP, MW: 10K), Bis-(p-sulfonatophenyl)phenylphosphine dehydrate dipotassium (BSPP), 11-mercaptoundecanoic acid (MUA), 1-(3-Dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were purchased from Sigma Aldrich (St. Louis, MO, U.S.A.).

Fabrication of GNRs

The GNRs used in this experiment (69±3 nm in length and 24±1.7 nm in width) were synthesized based on a seed-mediated growth method that is similar to the procedures described before with minor modifications.41 The seed was fabricated as below, 1 mL of 0.2 M CTAB was gently mixed with 20.6 µL of 24.28 mM HAuCl4 and 1 mL H2O. Then, 150 µL of 0.01 M icecold NaBH4 was rapidly injected into the mixture with vigorously stirring for 2 min until the color of the seed solution turned into pale brown-yellow. The seed solution was aged at room temperature for 30 min before usage. The growth solution was prepared as below: 0.7 g CTAB and 0.1234 g NaOL were dissolved in 25 mL water (~50 °C) with gently stirring. The solution was allowed to cool down to 30 °C and then 0.18 mL of 40 mM AgNO3 solution was added to the solution. The sample was kept quiet at 30 °C for 15 min. 1.03 mL of 24.28 mM HAuCl4 and 23.97 mL H2O were added into the mixture until it became colorless. 0.19 mL of 37 wt. % HCl was then introduced to adjust the pH. Finally, 125 µL of 64 mM AA was added into the mixture. To fabricate GNRs, 70 µL of seed solution was quickly added into the growth solution, followed by rapid shaking of the test tube for 30 s. The reaction solution was left undisturbed for 4 h at


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30 °C. The excess reagents were removed by centrifugation at 7000 rpm for 10 min. The resulted GNRs were washed with DI water three times. The longitudinal resonance peak in the UV-vis absorption spectrum of these GNRs is around 842 nm.

To generate GNRs with larger size, we further coated a thin silver and gold layer to the surface of GNRs. In brief, 500 µL of 0.1 M CTAB solution was added into 1 mL of freshly synthesized GNRs solution. 20 µL of 0.1 M AA, 80 µL of 0.01 M AgNO3 and 20 µL of 2 M NaOH were injected to the seed solution sequentially. Then 15 µL of 242.8 µM HAuCl4 was slowly added to the reaction solution after the addition of 40 µL 0.4 M NH2OH•HCl. The longitudinal resonance peak of the resulted GNRs was then shifted to 670 nm which exhibited much stronger scattering signal for single particle imaging. Morphological characterization of GNRs was performed on a transmission electron microscopy (TEM, JEM1230, JEOL).

Expression of PCV2 Capsid Proteins The method to express PCV2 protein was similar to the protocol reported before.40 In detail, PCV2 cap gene (GenBank: KF700357) was firstly synthesized (GenScript Biotech Co., Nanjing, China) and subcloned into a pET100/D-TOPO plasmid vector (Life Technologies, Inc., Rockville, MD, USA). Recombinant plasmids were introduced into E. coli BL21 (DE3) cells by transformation according to the manufacturer’s instructions, and PCV2 Cap was expressed in E. coli as described in the earlier work.40 An harvested E. coli BL21 cell pellet was suspended in 30 mL buffer A (0.1 M NaH2PO4•2H2O, 0.1 M Na2HPO4, 20 mM imidazole, 10 mM Tris base, 300 mM NaCl, 50 mM KCl, 2 mM MgCl2, 0.1 M ammonium citrate and 5% glycerol that pH is 8.0) with 0.5% Triton X-100, 5 mM b-mercaptoethanol and protease inhibitors (0.1 mM PMSF and 1 U of leupeptin per mL). The E. coli was disrupted by sonication and purified by centrifugation.


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Then the supernatant was loaded on a prepacked HisTrapTM HP column (GE Healthcare Life Sciences, New York, USA) in an automated FPLC system (AKTA, GE-Healthcare Life Sciences) according to the manufacturer’s instructions. The column was washed with buffer B including 50 mM NaH2PO4•2H2O, 20 mM imidazole and 500 mM NaCl. PCV2 VCPs were eluted with buffer C (300 mM imidazole and 300 mM NaCl, pH 6.0).

Conjugation of PCV2 VCPs onto the Surface of GNRs

To conjugate PCV2 VCPs onto the surface of GNRs, VCPs modification solution was prepared first. Briefly, 1 µL of 0.05 mg/mL MUA, 4.8 µL 0.1 mg/mL EDC, 2.9 µL 0.01 mg/mL NHS and 2 µL of 0.3 mg/mL VCPs were mixed with 40 µL of borate buffer (50 mM, pH 8.0) for 3 h. 1 mL freshly synthesized GNRs solution was centrifuged at 7000 rpm for 10 min to remove the extra chemicals in the solution. The pellet was dispersed in 100 µL DI water. After that, 4 µL VCPs modification solution was gradually added to the concentrated GNRs solution and left to react for additional 3 h. In order to increase the colloidal stability of the GNRs, 2 µL 0.1 mg/mL SH-PEG-CH3 was further added to the solution for 3 h. Those unreacted chemicals were removed by centrifugation for three times. The VCP-GNRs were suspended in 100 µL of DI water and stored at 4 °C prior to usage. ζ-potential (Nanozs90, Malvern, U.K.) was then measured to further ascertain the modification process.

Cell Culture and Imaging of Single VCP-GNRs

Hepatoma (HepG2) cells were obtained from American Type Culture Collection (ATCC, U.S.A.). HepG2 cells were cultured on a cleaned glass slide in a plastic cell culture dish. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Thermo Fisher


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Scientific Inc., U.S.A.) supplemented with 10% fetal bovine serum at 37 oC, 5% CO2 in a humidified atmosphere. For the single particle imaging experiments, 20 µL of VCP-GNRs was added into the cell culture dish and incubated for 30 min at 4 oC. The cells were washed with DMEM three times to remove those unreacted particles.

The dark-field optical microscopic imaging experiments were performed on a Nikon Eclipse Ni-U upright optical microscope (Japan) with a polarization-resolved dual channel module. The halogen lamp was focused onto the sample via an oil immersion dark-field condenser (NA 1.43-1.20). Scattered light from the sample was collected using a 60× objective (NA 0.5-1.25) and then were captured by a sCMOS camera (Orcaflash 4.0, Hamamastu, Japan) that was mounted on the front port of the microscope. To measure the polarization dependent scattering signal from single particles, the GNRs were firstly immobilized on the glass slide surface. Then a polarizer was put below the oil dark-field condenser. Through rotating the optical axis of the polarizer, the orientation dependent scattering signal from individual GNRs immobilized on the glass slide surface were recorded by the sCMOS camera simultaneously. For single particle imaging on living cell membrane, typically, 2000 frames were acquired to record the diffusion dynamics of individual VCP-GNRs on the cell membrane. The pixel size of the sCMOS camera is 6.5 µm × 6.5 µm. The images were processed with ImageJ (http://rsbweb.nih.gov/ij/).

RESULTS AND DISCUSSION The optical microscopic imaging experiments were performed on a commercial upright dark-field optical microscope coupled with a polarization-resolved dual channel module. An iris adjustable 60× oil immersion objective was used to collect the scattering signal from the sample


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and then was split into two images with perpendicular polarizations (i.e. horizontal H and vertical V polarizations) by a polarization beam splitter. The ratio of transmission efficiency between V and H channels under the wavelength of 400-800 nm is around 0.9. All of the images from the H channel prior to the data analysis were multiplied by this parameter.

Due to the localized surface plasmonic resonance effect, GNRs exhibit two resonance peaks in the UV-vis absorption spectrum, which are sensitive to the polarization direction of the excitation light, Figure 1. When the polarization direction of the light is parallel to the long axis of the nanorod, the longitudinal resonance is the dominant mode and typically falls in the red wavelength region. Figure 1c shows the representative dark-field image of individual GNRs immobilized on the glass slide surface. Excellent polarization dependent scattering intensity variation from single GNR was observed in the polarization modulation experiments which can be well fitted by a squared cosine function (i.e. I ∝ , where I is the scattering intensity of GNR, and is the angle of GNR relative to the optical axis of the polarizer), Figure 1d.41 These results illustrate that the scattering signal from individual GNRs could be decomposed into two images with distinctive scattering intensity by a polarization beam splitter when the long axis of the nanorod is not permanently parallel to the optical axis of the beam splitter. Even a slight angle deviation, the intensity variation between the two channels is substantial because of the squared cosine relationship between the scattering intensity and the angle relative to the optical axis of the beam splitter.

For the particles lacking of an optical axis or rigid crystal structure (i.e. the majority of vesicles from living cells), the scattered signal is typically randomly polarized in each direction. That is, from the H and V channels, the intensity difference from the same particle is minor,


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Figure 2a. However, in the case of GNRs, noticeable intensity difference could be observed between these two channels when the particles are randomly adsorbed on the glass slide surface, Figure 2b.These unique optical features from the cellular components and GNRs together afford an efficient way to discriminate the target signal from the noisy background. The principle to screen the interfering background from cellular organelles is shown in Scheme 1. Firstly, a stack of time-dependent sequential images was taken to record the diffusion process of the nanocarrier on the noisy environment. Due to the negligible fluctuation of the cellular components as well as the polarization insensitive character, the scattering intensity between two adjacent images (e.g. N(i) and N(i+1)) from the same channel is almost the same. On this basis, it is expected that the scattering background could be effectively counteracted through subtracting the images sequentially, i.e. N’(i)=N(i+1)-N(i). As a proof-of-concept experiment, we firstly adopted fluorescent micro-bead (400 nm) as a model to mimic the vesicles from cell component. As illustrated in Figure 3a, a set of dark-field optical microscopic images from single micro-bead in solution trapped by a thin layer of PVP molecules coated on the glass slide surface was recorded. No obvious intensity difference was noted from the H and V channels. Meanwhile, the scattering intensity from the same channel between two adjacent images was nearly identical. In order to quantitatively analyze the correlation in intensity and location between two images, we calculated the Pearson’s correlation coefficient (PCC) with the function of =

∑ × ̅ , ∑ ×∑ ̅

where Ri and Gi refer to the

intensity values from two separate images of pixel i, and R̄ and Ḡ refer to the mean intensities of the entire image. PCC is a simple yet robust way to measure the intensity dependent pixel overlap between two images. An exact co-localization of the point-spreading-function (PSF) from the micro-bead will result in a PCC close to 1. Any difference in intensity will result in the


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decrease of this coefficient. In this case, the calculated PCC between the PSFs from the microbead marked with green arrow in the image of N(i) and N(i+1) is 0.98. A more discernable presentation of this good correlation relationship is shown in the 3D surface plots (Figure 3b) and the 2D intensity profiles from two sequential images (Figure 3c).

Owing to the excellent co-localization effect, the scattering signal from the micro-bead could then be perfectly removed through subtracting the image one by one (N(i+1)-N(i)), i.e. the new stack of N’, Figure 3a. This approach should be applicable in living cell system because the cell is relatively immobilized. In the case of GNRs, due to the thermal-induced rotation within the polymer layer, the signals between two adjacent images changed greatly. Bright spot still displayed in the new generated image stack, i.e. N’’(i)=N’H(i)+N’V(i), Figure 3d. Depending on the angle of the GNRs, the signal of the particle could either locate in the channel of H, V or both of them. The PCC of PSF from the GNRs is then degenerated. For example, the PCC is only 0.58 from the particle noted with green arrow. It is worth to point out that the decrease of PCC is mainly due to the rotation induced intensity change. A continuous translocation track as a function of time from GNRs could be reconstructed, Figure s1. As a more evident presentation of removing the interfering background from the micro-bead, GNRs were mixed with microbeads together. The images were then analyzed with the method elaborated above. Under regular dark-field imaging mode, all of the particles could be observed readily, Figure 4a. However, the GNRs show distinctive scattering intensity between the H and V channels. From the fluorescent mode, the micro-beads close to the GNRs could be confidently identified, Figure 4b. In the processed image, only the target objects exist in the reconstructed image, Figure 4c. It is worth to note that, even though a single channel imaging modality is enough to screen the scattering background, a polarization-based dual channel detection scheme was compulsory to establish a


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continuous diffusion track from the GNRs.

To further demonstrate the applicability of this approach in living cells, a representative dark-field optical microscopic image from single cell without GNRs is presented in Figure 4d. Evidently, strong scattering signal could be noted from the whole cell. As expected, in the processed image, the scattering background was perfectly removed, Figure 4e. To further validate the background screening capability for single particle imaging, GNRs were loaded on to the cell membrane for 30 min. Due to the electrostatic interaction of the GNRs with lipid membrane, some GNRs were slightly anchoring on the cell membrane with continuous rotational movement. With the same image processing method, individual bright spots could be recognized readily on the cell membrane, Figure 4g. As a consequence, the method presented herein affords a promising platform for single anisotropic nanoparticle imaging on living cell membrane.

Exploration of the diffusion dynamics of the nanocarrier on living cell membrane at single particle level would provide ample valuable information for the detailed understanding of the cellular translocation mechanism.42-48 These messages would help to further improve the performance of the nanocarrier in a rational approach. By using fluorescent optical microscopy, earlier pioneer studies have explored the cellular translocation process of individual virus real time. It has been demonstrated that most of viruses infect the host cells through endocytosis. The attachment of virus capsid proteins with the cell membrane components (e.g. membrane proteins, carbohydrates, and lipids) plays an essential role in concentrating virus on the cell membrane and subsequently regulates the pathway of how they enter into the cell. Differing from one to one, several interesting mechanisms have been discovered based on single particle tracking results.19,49-51 For example, hepatitis B virus enters the cell via a caveolin-mediated endocytic


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pathway.49

Despite those interesting observations, in the case of VLP or VLP-caged nanocargos, little information was deduced because the particle size, morphology as well as the surface charge of the nanocargo may different from that of native viruses. In this regard, it is of great interest to explore the diffusion dynamics of individual VCP-GNRs on the living cell membrane. In this work, we chose porcine circovirus type 2 (PCV2) as a model protein to directly image the diffusion process of VCP-GNRs on living HepG2 cell membrane.

Instead of physical coating, we cross-linked VCPs to the surface of GNRs via amide bond. CTAB molecules on the surface of GNRs were firstly replaced with MUA, rending a shell of carboxyl groups for the subsequent protein conjugation via the linker of EDC/NHS. Zeta potential analysis after the protein conjugation showed that around 40.7 mV (CTAB-coated GNRs 44.7 mV, VCP-GNRs 4.0 mV) surface charge decrease was found, indicative of the successful cross-linking process. Prior to the single particle tracking experiments, the stability of these particles in the biological milieu was also explored as demonstrated in Figure s2. According to the UV-vis absorption spectra, no obvious difference was noted from the shape as well as the resonance peak position when the nanoparticles were dispersed in DI water and cell culture medium. This is also confirmed in the microscopic image where the scattering intensity from the particles dispersed in cell culture medium were almost the same. No aggregated clusters were noted.

In order to specifically monitor the diffusion dynamics of VCP-GNRs on the cell membrane, the particles were firstly incubated with HepG2 cells at 4 oC for 30 min. This treatment guarantees the anchoring of VCP-GNRs on the cell membrane without endocytosis


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process. Those particles randomly diffusing in the solution were then washed away. Figure 5a illustrates a set of representative diffusion trajectories of individual VCP-GNRs on the cell membrane. The majority of them displayed confined diffusion with mean 2D diffusion coefficient of 0.004±0.004 µm2/s. Further inspection of the diffusion trajectories from these nanoparticles, we noticed that some of the particles exhibited occasional large jumps during the diffusion process, indicative of weak association force under those time points, Figure 5b. Through statistically analyzing the step size distribution with a lag time of 0.1 s (changing of the lag time doesn’t results in a significant variation of the mode of distribution), non-Gaussian distribution was noted, Figure 5d. Basically, two models have been proposed to depict the intermittent hopping diffusion at the interface, i.e. desorption mediated diffusion as described by the O’Shaughnessy mode and the continuous time random walk (CTRW) model.52 The first model is based on an essential assumption that a fixed characteristic desorption time dominates the whole process, which obviously cannot well delineate the process as noted above. While in the latter case, the model basically describe that the walker will spend random waiting time immobilized between each instantaneous displacement, which is more consistent with the results presented above. It is worth to note that, further extending the incubation time at 37 oC (40 min), the majority of the particles were confined on the membrane without noticeable hopping diffusion, Figure s3. In the earlier work, we also observed similar kind of intermittent hopping diffusion from cell penetrating peptide-modified nanoparticle on artificial lipid membrane.46 Due to the interaction of peptide with lipid membrane, the particles could be confined on the lipid membrane with random waiting time and followed with evident bulk-mediated confined diffusion on a 2D surface. Non-Gaussian distributed step length with a stretched power law like tail was observed. Taking together, according to the results in this work and those reported


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before,52 it is expected that this kind of intermittent hopping diffusion would be a common phenomenon adopted by biomolecules at various scales, for example, the localization of enzymes to a specific DNA sequence inside the cell, the active transportation of cargos on microtubule by motor proteins.

CONCLUSION In conclusion, in this work, we proposed a convenient method for single particle imaging under the noisy environment, living cell, with dark-field optical microscope. The essential merit of this method is that the majority of scattering background from cellular components could be readily screened based on a polarization-based dual-channel imaging module. Different from the previous interesting strategies, this approach doesn’t require convoluted optical path alignment and data analysis.29,46,49,51 The translocation trajectories of individual nanoparticles on the cell membrane could be efficiently deduced. By tracking the diffusion dynamics of PCV2 capsid proteins-functionalized GNRs on the cell membrane, most of the particles showed confined diffusion due to the strong interaction with the cell membrane, e.g. the interaction of VCP with glycosyl proteins which have been clarified as a common approach for virus anchoring on the cell membrane. Interestingly, we found that some of the VCP-GNRs displayed anomalous confined diffusion with randomly distributed large walking steps during the whole track. By analyzing the distribution of step size with a time resolution of 0.1 s, non-Gaussian distribution was observed which is distinctively different from those particles displaying strong interaction with the cell membrane. This kind of anomalous confined diffusion might be an efficient approach for VCP-GNRs distinguishing a suitable penetration site on the living cell membrane.


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ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. All author reviewed the manuscript. ACKNOWLEDGMENT This work was supported by national natural science foundation of China (NSFC, Project no. 21405045 and 21522502). Supporting Information The Supporting Information is available free of charge on the website. The more detailed presentation of the image analysis process and supporting figures are available in the supporting Information.


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FIGURES AND CAPTIONS

Figure 1. a) The UV-vis absorption spectrum of GNRs in DI water with longitudinal resonance located at 670 nm. b) The TEM image of GNRs. c) Representative dark-field optical microscopic image of the GNRs. d) The polarization dependent scattering response (green circles) from single GNR as a function of the angle relative to the optical axis of the polarizer. The red line is the fitted curve based on the relationship of I ∝ .


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Figure 2. Polarization-resolved dual channel (horizontal and vertical polarizations) dark-field optical microscopic images of the cell lysates a) and GNRs b). The corresponding 3D intensity profiles from individual particles were shown in the lower panels.


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Scheme 1. The schematic diagram of the optical path for the polarization-resolved dual channel dark-field optical microscopic imaging. To selectively observe the anisotropic nanoparticles, a stack of polarization-resolved dual channel dark-field images was recorded. A new generated image stack N’ without interfering particles was then obtained by subtracting the images sequentially, i.e. N’(i)=N(i+1)-N(i). The continuous track from individual GNRs could then be recovered based on the equation of N’’(i)=N’H(i)+N’V(i).


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Figure 3. a) The polarization-resolved dual channel dark-field optical microscopic images of fluorescent micro-beads trapped on glass slide surface with PVP molecules and the corresponding processed images based on the method as noted in Scheme 1. b) The 3D intensity profiles of single micro-bead (marked with green arrow in Figure 3a)) from the images of NH(i), NH(i+1), N’H(i) and N’’(i). c) The corresponding 2D intensity profiles of the green line (in Figure 3a)) from the images as noted in the figure. d), e) and f) are the same set of images from the sample of GNRs.


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Figure 4. a) The polarization-resolved dual channel dark-field optical microscopic images of fluorescent micro-beads and GNRs trapped on glass slide surface with PVP molecules. b) Fluorescence image from the same area. c) The processed dark-field image N’’. d) and f) are the dual channel dark-field optical microscopic images of living HepG2 cell without and loaded with GNRs respectively. e) and g) are the corresponding processed images N’’ with the method described in Scheme 1 respectively.


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Figure 5. a) Representative single particle diffusion trajectories of individual VCP-GNRs on HepG2 cell membrane. b) The time dependent diffusion track along the x axis. c) The double logarithmic plot of mean squared displacement (MSD) from those particles as a function of lag time (∆t). The statistical analysis of the 2D diffusion coefficient of VCP-GNRs on the cell membrane is shown in the inserted histogram. d) The measured step size distribution (green circles) of the particles with a lag time of 0.1 s. The fitted distribution with Gaussian decay is plotted with red circles.


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Background-Free Imaging of a Viral Capsid Proteins Coated

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