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View Article Online Chemical View Journal Science Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: H. Gao, P. Wu, P. Song, B. Kang, H. Chen and J. Xu, Chem. Sci., 2021, DOI: 10.1039/D0SC04764C. Volume 9 Number 1 This is an Accepted Manuscript, which has been through the Chemical 7 January 2018 Pages 1-268 Royal Society of Chemistry peer review process and has been accepted for publication. Science Accepted Manuscripts are published online shortly after acceptance, rsc.li/chemical-science before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard ISSN 2041-6539 Terms & Conditions and the Ethical guidelines still apply. In no event EDGE ARTICLE Xinjing Tang et al. Caged circular siRNAs for photomodulation of gene shall the Royal Society of Chemistry be held responsible for any errors expression in cells and mice or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/chemical-science
Page 1 of 10 PleaseChemical do not adjust margins Science View Article Online Chemical Science DOI: 10.1039/D0SC04764C ARTICLE This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Received 00th January 20xx, Video-rate imaging of sub-10 nm plasmonic nanoparticles in Chemical Science Accepted Manuscript cellular medium free of background scattering Open Access Article. Published on 04 January 2021. Downloaded on 1/12/2021 12:01:29 PM. He Gao, a Pei Wu,a Pei Song,a Bin Kang,*a Jing-Juan Xu*a and Hong-Yuan Chena Accepted 00th January 20xx Plasmonic nanoparticles (e.g gold, silver) have attracted extensive attentions in biological sensing and imaging as DOI: 10.1039/x0xx00000x promising nanoprobes. Practical biomedical applications demand small gold nanoparticles with comparable size to quantum dots and fluorescent proteins. While too small nanoparticles with size below Rayleigh limit (usually
PleaseChemical do not adjust margins Science Page 2 of 10 ARTICLE Journal Name nm) since the scattering cross-section of nanoparticles scale down promote the study of using small gold nanoparticlesView as Article biological Online with six power of their size.23, 24 Worse, cellular medium is a probes for dynamic optical tracking of biological process within DOI: 10.1039/D0SC04764C heterogeneous environment with many strong scattering organelles, living cells. resulting in a strong scattering background. The weak scattering signal, in somewhat, could be compensated by using a more sensitive camera. However, the scattering background from cellular Results and discussion components is hard to fully eliminate since the Rayleigh scattering from subcellular components usually have a wide spectra. Even This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. some background scavenging reagents, background smoothing algorithms, etc., have been tried to reduce the intracellular scattering background, however, such problem has not been fully Chemical Science Accepted Manuscript solved.25-28 To detect small plasmonic nanoparticles, several types of photothermal imaging methods were developed based on Open Access Article. Published on 04 January 2021. Downloaded on 1/12/2021 12:01:29 PM. plasmonic resonance absorption instead of scattering, since the absorption cross section of nanoparticles scales down with three power of particle size (slower than scattering). Laser scanning photothermal imaging is able to detect nanoparticles down to a few nanometers, and enable high resolution imaging of cellular structure. In this type of imaging, the photothermal signals, defined by the relative change of probe beam with and without heat beam, Figure 1. Scheme of the absorption modulating scattering still scale down with absorption cross-section.29-31 Thus small microscopy （AMSM）. (a) Principle of pump-probe imaging of a nanoparticles below 10 nm demand a tightly focused and high single gold nanoparticle. (b) Time sequence of pump pulse (20 Hz), frequency modulated heat beam and a lock-in amplifier to extract probe pulse and camera gate (40 Hz). (c) Schematic diagram of the weak photothermal signal.32 For imaging of living cells, such working time window of probe pulse and camera gate in one time strong and focused laser beam might cause irreversible cell period of imaging. damage.33 Also, this type of laser scanning based imaging method usually needs minutes to hours to obtain a frame of image, which The absorption modulating scattering microscopy (AMSM) is based seems too slow for living cell imaging since many dynamic biological on a pump-probe detection technique,39 which is realized by using a events occurs in seconds or even faster. Besides laser scanning self-built setup (Figure 1 and Figure S1, Supporting Information). A photothermal imaging method, photothermal imaging could also be 532 nm pulse laser (~5 ns) was used as pump beam to heat the gold realized based on surface plasmon resonance (SPR). 34SPR imaging nanoparticles, and pulsed white light (~6 µs) was used as probe enable to detect photothermal signal with very fast speed, however, beam to detect the scattering light signal from gold nanoparticles it seems hardly to image nanoparticles inside of cells since the SPR (Figure 1a, Figure S2). Without pump beam, temperature of the effect highly relies on the interface of gold film.35 Recently, there liquid medium around gold nanoparticles is uniform and the are also some new photothermal imaging methods, like widefield intensity of scattering light is defined as IOFF. Once the nanoparticle photothermal sensing (WPS), which aimed to break the obstacle of is irradiated by pump beam, it would be heated to a ‘hot’ state in imaging speed36. Such photothermal method is still based on the hundreds picoseconds through photon-phonon interaction.40-42 absorption characteristics of the objects. Beside absorption-based After a very short time (~ns), the nanoparticle transfers a part of principle, interferometric optical detection can also be used to heat to the surrounding medium, which resulting in a localized image 2-5 nm gold nanoparticles based on their scattering thermal field in the medium. As refractive index of the medium properties.37 In cells, it usually requires larger nanoparticles to depends on temperature, a local refractive index change is formed achieve a good image quality because the cellular enviroment in the medium surrounding the nanoparticles, which is often called contains a lot of scattering objects and contribute a strong “nano-thermal lens”.31, 32 Assisted by the effect of nano-lens, more scattering background. 38 light scattered from gold nanoparticles was captured by the imaging Here, we demonstrated a method, named absorption modulated unit. Under this condition, the scattering light intensity is defined as scattering microscopy (AMSM). This AMSM imaging method applied ION. Hence, absorption modulated scattering signal Ф was defined both the resonance absorption and scattering properties of as Ф = (ION − IOFF)/ IOFF, which represented the change of the plasmonic nanoparticles, rather than each of single effect. Thus the scattering light of nanoparticles caused by absorption of pump AMSM method exhibited a remarkable ability on removal of energy. After stopping pump, the nanoparticle is cooling down and scattering background. Compared to regular dark-field microscopy the “nano-thermal lens” disappeared in about a few microsecond.43 that typically can only detect 30-50 nm gold nanoparticles, our Then the system returns to the initial state without pump and the AMSM method was able to detect much smaller nanoparticles with scattering light intensity return back to IOFF until next time of heat. size down to ~9 nm. Moreover, the imaging speed of AMSM To achieve video rate imaging, time sequence control was method is much faster than regular laser-scanning photothermal introduced in above system. The pump laser worked at 20 Hz and a microscopy, thereby allows for real-time video rate (20 fps) imaging delay generator was used to synchronize frequency and generate a of sub-10 nm nanoparticles in living cells. This AMSM method might 2 | Chem. Sci., 2019, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins
Page 3 of 10 PleaseChemical do not adjust margins Science Journal Name ARTICLE transistor-transistor logic (TTL) signal sequence with 40 Hz, which View Article Online acted on the probe beam source and imaging module (Intensified DOI: 10.1039/D0SC04764C Camera, ICCD) to collect the scattering light signal (Figure 1b). The time interval of probe sequences was 25 ms, in one cycle of pump, Where C is a constant, ∂ /∂ is the rate of refractive index (n) the scattering intensity was recoded as ION, and 25 ms later, the changes with temperature, P is the power of pump beam, ρAu and scattering intensity was recorded as IOFF after the nanoparticle was cAu are the density and specific heat of nanoparticles. Following this fully cooled. By subtracting IOFF from ION in chronological order, formula, Φ is closely related to many factors, but not absorption absorption modulated scattering signal Ф varying with time would and scattering cross sections of the nanoparticles (see Supporting This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. be obtained. As illustrated in Figure 1c, about 6 μs was needed for Information for details). According to Mie theory, the scattering probe beam (pulse xenon lamp) to reach the maximum brightness cross sections decreases sharply with reduce of particle size, so that and the half-width of time duration was about 6 μs as well. Thus, the traditional dark field microcopy is failed to detect too small Chemical Science Accepted Manuscript the ICCD gate time was set to a duration of 10 μs to ensure nanoparticles. Fortunately, the Φ signal of our AMSM, in theory, is capturing most of the scattering light (Figure. 1c) with a very short independent on the nanoparticle size, which makes it possible to start time of 19 ns. detect very small nanoparticles. Notable that even Φ does not Open Access Article. Published on 04 January 2021. Downloaded on 1/12/2021 12:01:29 PM. As mentioned above, the signal Ф originates from the change of depend on particle size in principle, the practical imaging still suffer refractive index in the medium surrounding the gold nanoparticles a particle size limit because of the limitation of laser power and caused by light absorption. Thus, the Φ signal value can be sensitivity of camera. ultimately expressed as (see Supporting Information for details):44, 45 n P C T AucAu (1) This journal is © The Royal Society of Chemistry 20xx Chem. Sci., 2019, 00, 1-3 | 3 Please do not adjust margins
PleaseChemical do not adjust margins Science Page 4 of 10 ARTICLE Journal Name Figure 2. Colocalization imaging analysis of 80 nm gold nanoparticles (a) color dark field image, (b) ICCD image, and (c) Φ signal image of View Article Online nanoparticles in glass substrate. (scale bars: 10 μm). (d) Scatter plot of the Φ values versus R/G values of each nanoparticle and statistical DOI: 10.1039/D0SC04764C histograms of the Φ distribution of monomeric (green) and aggregated (yellow) particles. (e) Scattering light intensity and (f) Φ signal intensity of different size nanoparticles. Red arrow in (a) (b) indicates a dust particle. The feasibility of this imaging method was demonstrated firstly in objects must be considered, especially in complex environment like vitro (Figure 2). Gold nanoparticles coated with polyethylene glycol cells. Therefore, we then investigated the capability of our AMSM This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. (Au-PEG) with size of 80 nm was immerged into glycerol medium to method on eliminating such strong scattering background signals, simulate the environment of cytoplasm with similar refractive index. using dust and polystyrene sphere particles as models (Figure S3). Figure 2a showed an example of raw image captured by ICCD, and As expected, since the absorption characteristics of dust or Chemical Science Accepted Manuscript its corresponding Ф signal image was shown in figure 2b. All the polystyrene sphere particles are significantly different to gold gold nanoparticles in figure 2a were recognized in figure 2b except nanoparticles, then they could not be heated by the pump laser and of the super bright dust particle. Dust particles have a much larger thereby cannot be detected in final AMSM image. These results Open Access Article. Published on 04 January 2021. Downloaded on 1/12/2021 12:01:29 PM. size (in micrometer scale) and a huge scattering cross section, thus suggested the signal extraction capabilities of our system from they scatter much more light than gold nanoparticles. For scattering complex scattering background. 29 imaging, the background scattering from this type of super bright Figure 3 Colocalization imaging of gold nanoparticles in single cell. (a) Color dark field image, (b) ICCD image, and (c) Φ signal image of HeLa cell incubated with 80 nm gold nanoparticles. (d) Scatter plot of the Φ signal versus R/G ratio and Φ distribution histogram of monomeric (green) and aggregated (yellow) nanoparticles. (e) Localization of monomeric (green) and aggregated (red) nanoparticles in HeLa cell. (f) ICCD dark field image and (g) Φ image of 39 nm gold nanoparticles in HeLa cell. (h) The corresponding Φ distribution histogram of monomeric and aggregated nanoparticles. (i) Localization of monomeric (green) and aggregated (red) 39 nm gold nanoparticles in HeLa cell. (j-k) Splitting localization images of monomeric (j) and aggregated (k) nanoparticles. (scale bars: 5 μm). In cell imaging using gold nanoparticles as probe, particle and aggregates usually could be distinguished under true color dark aggregation is almost inevitable. The states of particle monomers field image, since aggregates show different color with monomers 4 | Chem. Sci., 2019, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins
Page 5 of 10 PleaseChemical do not adjust margins Science Journal Name ARTICLE (Figure. 2d). We then splited the RGB channels of color dark field counting, the Φ distribution histogram of monomeric and View Article Online image, and calculated the Red/Green (R/G) value of each aggregated nanoparticles were shown out (Figure 3h). Compared to DOI: 10.1039/D0SC04764C nanoparticles. Then R/G values and corresponding Ф values of large 80 nm nanoparticles, more aggregated particles were observed for number of nanoparticles in many frames of images were plotted 39 nm nanoparticles. The position of all monomeric and aggregated into figure 2d and figure S4. The populations of monomers and gold nanoparticles in cells were recognized and marked (Figure 3i- aggregates were clearly distinguished according to either R/G 3k). Usually, nanoparticles with size below 40 nm were hard to values or Ф signal intensity, since the particle aggregates exhibited capture via regular dark field imaging. Here we can still use the Φ a red-shift color and a higher Ф signal. Then the Ф signal values of value intensity to identify the status of gold nanoparticles. A This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. these two populations were statistically analyzed and shown in complete set of signal analysis processes for cell imaging is right histogram. This result verified the ability of this method to described in details in Supporting Information (Figure S13, S14). identity the monomeric and aggregated state of nanoparticles, After that, we gradually reduced the size of gold nanoparticles to Chemical Science Accepted Manuscript which is an important issue in imaging of nanoparticles within cells. explore the limit of our home-built AMSM system. The 20 nm The detailed procedure for data processing was presented in nanoparticles still can loom through the scattering background cell Supporting Information (Figure S5). in ICCD image (Figure 4a), but 14 nm and 9 nm nanoparticles were Open Access Article. Published on 04 January 2021. Downloaded on 1/12/2021 12:01:29 PM. According to equation 1, the Ф signal is independent to particle size totally drowned in the background scattering (Figures 4b and 4c). in theory. To prove this concept, the intensity of scattering light and Fortunately, small nanoparticles with size down to 9 nm could still Ф signal of nanoparticles with size range from 80 to 9 nm were be clearly imaged using our equipment. Some strong scattering light measured (Figure 2e and 2f, Figure S6–S10). From the dark field from dust particles or cellular components were successfully images, we can see that most of particles are dispersed monomers filtered. on glass, because the surface of gold nanoparticles is wrapped by Similar to large size particles, the Φ signals of the small size gold PEG. The intensity of scattering signal doped sharply with the nanoparticles also have two distinct distributions. Particle particle size, which is consistent well with the Mie theory (Figure community with relative small Φ values indicated monomeric 2e). With the decrease of particle size, the scattering signal particle and the community with relative large Φ values indicated decreased sharply by a power of 5.73, which is close to the Mie the particle aggregation. When we separated intracellular theory that scattering cross section drops with particles size by the monomeric and aggregated gold nanoparticles and get the similar power of 6 (Figure S10). While the Φ signals of different size overlay image as before, the position of gold nanoparticles can be nanoparticles almost remain in the same range, only with a slight accurately pointed out. We further statistically analyzed the drop (~8%) along with the size decreasing from 80 nm to 9 nm scattering intensity of different size nanoparticles with in cellular (Figure 2f). This tendency is basically consistent with theory of medium, the scattering signal dropped sharply with the decrease of equation 1, the slight drop on Φ signals of very small nanoparticles particle size by a power of 5.68 (Figure 4d), which approximately could be attribute to the response linearity of camera. We further complies with the Mie theory. However, the intensity of Φ signal explored the influence of pump energy, surface coating, and decreased only about 14 % although the particle size was reduced surrounding medium on the intensity of Φ signal (Figure S11). For a to more than one-eighth of the beginning (Figure 4e). This tendency given medium, Φ signal intensity is proportional to the pump laser is also consistent with the theory of AMSM. The universality of power. However, different surface modifications on nanoparticles AMSM was also verified on imaging of small size gold nanoparticles did not alter the intensity of their Φ signals. in MCF-7 cells (Figure. S15). We then demonstrated AMSM imaging of nanoparticles within cells. Dynamic tracking technology is an urgent demand in cell imaging to For the convenience of observation and also minimization of reveal intracellular events in real time. Benefit from the principle of adverse effect on cells, we controlled the incubation concentration time sequence control of our AMSM method, video-rate or even of gold nanoparticles to ensure only a small amount of fast imaging is feasible (see Supplementary Video). To achieve this, nanoparticles were internalized into cells. According to the dark 40 frames of HeLa cells with 9 nm gold nanoparticles were recorded field images of cells, the numbers of nanoparticles within each cell (Figure 5a) in one second, and finally 20 frames of Ф signal images were around ~102. Gold nanoparticles incubated with HeLa cells were obtained with a frame rate of 20 fps (Figure 5b). Four particles were captured in color dark field image and Ф signal image (Figure were extracted as examples and their Ф signals over time were 3a-c). Through the population in the scatter plot of R/G ratio and Ф exhibited in Figure 5c. The particles 1 and 2 show relatively stable Ф signal intensity, monomeric and aggregated gold nanoparticles values around 0.15, thus they are monomeric gold nanoparticles could be separated (Figure 3d). Thus the positions of each according to the previous discussion. The particles 3 and 4 are nanoparticle could be located within cells (Figure 3e). The identified as aggregates from the Ф signal, which shows a stronger assignment of monomeric or aggregated nanoparticles well intensity and a wider variation range than monomers. Then we matched color dark field image. HeLa cell without incubating with extracted the position information of these four nanoparticles at gold nanoparticles was also checked (Figure. S12). Compared to each frame to track their motion states (Figure 5d). The two dark field image (Figure. 3a), the Ф signal image (Figure 3c) showed monomeric gold nanoparticle 1 and 2 exhibit a fast movement with very tiny background scattering from cellular components, because a long journey of 1736 and 1438 nm. However, the aggregated cellular components did not have a strong resonance absorption particles 3 and 4 show a slower movement and a shorter journey like gold nanoparticles. within 500 nm. Apparently, the moving speed of aggregates within Then the same imaging procedure was applied by using 39 nm gold cellular medium is much slower than the monomers. Here we just nanoparticles (Figure 3f, g). After signal extracting and particle showed the dynamics imaging ability of our method, the moving This journal is © The Royal Society of Chemistry 20xx Chem. Sci., 2019, 00, 1-3 | 5 Please do not adjust margins
PleaseChemical do not adjust margins Science Page 6 of 10 ARTICLE Journal Name states of gold nanoparticles within cells could be tracked at video View Article Online rate (20 fps) for a long term if necessary. DOI: 10.1039/D0SC04764C This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Chemical Science Accepted Manuscript Open Access Article. Published on 04 January 2021. Downloaded on 1/12/2021 12:01:29 PM. Figure 4 Imaging of small gold nanoparticles within cells. (a-c) ICCD image, corresponding Φ image, Φ distribution histogram and the spatial position of monomeric and aggregated gold nanoparticles of (a) 20 nm, (b) 14 nm, and (c) 9 nm gold nanoparticle. (d) Scattering 6 | Chem. Sci., 2019, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins
Page 7 of 10 PleaseChemical do not adjust margins Science Journal Name ARTICLE light intensity of nanoparticles with different sizes in Hela cells. Inset: Linear fitting of the logarithm of scattering light intensityView versus Article Online particle size. (e) Φ signal value of gold nanoparticles with different sizes in HeLa cell. (scale bars: 5 μm) DOI: 10.1039/D0SC04764C This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Chemical Science Accepted Manuscript Open Access Article. Published on 04 January 2021. Downloaded on 1/12/2021 12:01:29 PM. Figure 5 Dynamic tracking of 9 nm gold nanoparticles in cells. (a) A series of dark field image of HeLa cell incubated with gold nanoparticles captured by ICCD in sequence. (scale bars: 5 μm) (b) The corresponding Φ signal images of intracellular gold nanoparticles in 1 second with a time interval of 50 ms. (c) The variation of Φ signal of four typical particles extracted in the dynamic Ф signal image. (d) Movement tracking of the four particles in 1 second. A videography of gold nanoparticles tracking was vividly shown in Supporting Information. photobleaching, the AMSM imaging method makes great potential for real-time cell imaging by using small size gold Conclusion nanoprobes for unfolding dynamics molecular processes within living cells. We have demonstrated an absorption modulated scattering Experimental microscopy (AMSM) for imaging small gold nanoparticles within scattering cellular medium. Since the AMSM utilized Reagents both absorption and scattering rather than a single one, only subjects with characteristic features of resonance absorption O-[2-(3-Mercaptopropionylamino)ethyl]-O'- and scattering were detected, and the scattering background methylpolyethylene glycol (Mw= 5000, SH-PEG), and glycerol from cellular component could be almost fully removed. The (GC, ≥99.5%) were obtained from Sigma-Aldrich Co. (U.S.A.). AMSM signal was very sensitive to the nanoparticle states, Human cervical cancer (HeLa) cells, Michigan Cancer which enable to distinguish monomers or aggregates in Foundation-7 (MCF-7) cells, paraformaldehyde (4 %), cellular environment. Compared to regular darkfield phosphate buffer solution (PBS, 10 mM, pH=7.4) were microcopy, the AMSM could detect much smaller provided by KeyGEN Biotech. Co. (Nanjing, China). The nanoparticles with size far beyond Rayleigh limit, with a polystyrene spheres used in this work were purchased from sensitivity comparable to that of a photothermal microscopy. Zhejiang Tianke high tech Development Co., Ltd (Zhejiang, The imaging speed of AMSM could be close to video rate or China). Gold nanoparticles (Au NPs) with an average diameter even faster in principle, if with a high frequency pulsed laser of 9 nm ,14 nm, 20 nm, 39 nm and 80 nm were purchased and a fast camera. On account of stable signal intensity, fast imaging speed, background removal ability, and without This journal is © The Royal Society of Chemistry 20xx Chem. Sci., 2019, 00, 1-3 | 7 Please do not adjust margins
PleaseChemical do not adjust margins Science Page 8 of 10 ARTICLE Journal Name from Ted Pella Inc. (U.S.A.) (Figure. S16). Ultra-pure water from 1. H. C. Hulst and H. C. van de Hulst, Light scattering by View Article Online Millipore Milli-Q (18 MΩ·cm−1) was used in the experiments. small particles, Courier Corporation, 1981. DOI: 10.1039/D0SC04764C 2. G. Mie, Annalen der physik, 1908, 330, 377-445. Sample 3. S. Schultz, D. R. Smith, J. J. Mock and D. A. Schultz, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 996. The preparation process of Au-PEG particles with different particle sizes is basically the same. Gold nanoparticles of 4. W. Eck, G. Craig, A. Sigdel, G. Ritter, L. J. Old, L. Tang, different sizes (9 nm 9.5nM, 14 nm 2.3nM, 20nm 1.2nM, 40 M. F. Brennan, P. J. Allen and M. D. Mason, ACS Nano, 2008, 2, 2263-2272. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. nm 150 pM) were mixed with 1 mmol/L SH-PEG of the same volume to prepare Au-PEG particles of different sizes. At the 5. E. Oh, R. Liu, A. Nel, K. B. Gemill, M. Bilal, Y. Cohen same time, the desired Au-PEG was obtained by mixing the 80 and I. L. Medintz, Nat. Nanotechnol., 2016, 11, 479- nm gold nanoparticles (18 pM) with an equal volume of 486. Chemical Science Accepted Manuscript 0.1mmol/L PEG solution. The mixture was maintained at 37 C 6. N. Chen, Y. He, Y. Su, X. Li, Q. Huang, H. Wang, X. and stirred continuously for 24 hours. By using the appropriate Zhang, R. Tai and C. Fan, Biomaterials, 2012, 33, speed, the resulting Au-PEG were then centrifuged and 1238-1244. Open Access Article. Published on 04 January 2021. Downloaded on 1/12/2021 12:01:29 PM. washed several times. Au NPs, Au-PEG or Au-DNA was absorbed on positively 7. C. Rosman, S. Pierrat, A. Henkel, M. Tarantola, D. charged glass slides for 30 minutes, respectively. Then Au- or Schneider, E. Sunnick, A. Janshoff and C. Sönnichsen, Au-complex coated glass slides were washed three times and Small, 2012, 8, 3683-3690. placed on an objective for detection. Moreover, water or 8. X. Nan, P. A. Sims and X. S. Xie, ChemPhysChem, glycerol was dropped on each glass slides acted as surrounding 2008, 9, 707-712. medium. 9. I. H. El-Sayed, X. Huang and M. A. El-Sayed, Nano HeLa cells, MCF-7 cells were routinely cultured in DMEM Lett., 2005, 5, 829-834. medium with 10% fetal bovine serum, 2% antibiotics, and 5% 10. J. A. Yang, H. T. Phan, S. Vaidya and C. J. Murphy, CO2 in a 37 C incubator. When making samples, HeLa cells, Nano Lett., 2013, 13, 2295-2302. MCF-7 cells were seeded on a glass coverslip. After cultured for 24 h, proper Au-PEG solution was added to incubate. Five 11. G. A. Craig, P. J. Allen and M. D. Mason, in Cancer hours later, the HeLa, MCF-7 cells coated glass coverslips were Nanotechnology: Methods and Protocols, eds. S. R. washed with PBS for several times to remove excess Grobmyer and B. M. Moudgil, Humana Press, nanoparticles and ready for living detection. The fixed cell Totowa, NJ, 2010, DOI: 10.1007/978-1-60761-609- samples were prepared by incubation with 4% 2_12, pp. 177-193. paraformaldehyde for additional 15 min, and then the 12. W. Qian, X. Huang, B. Kang and M. El-Sayed, J. coverslips coated with fixed cells were washed and infiltrated Biomed. Opt., 2010, 15, 046025. in PBS solution for detection. The temperature of the glass 13. C. Sönnichsen, B. M. Reinhard, J. Liphardt and A. P. slides and glass coverslips were controlled by a thermal Alivisatos, Nat. Biotechnol., 2005, 23, 741-745. platform equipped with a sensitive temperature control system. For all cell experiments on microscope, at least three 14. L. Zhang, Y. Li, D.-W. Li, C. Jing, X. Chen, M. 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