Journal of Community Medicine & Health Education
Open Access

Gold nanoparticles; HPV; Cervical cancer; Two-photon imaging; Confocal microscopy; Multiphoton microscopy; Raman spectroscopy; SERS

According to the World Health Organization cervical cancer was the fourth most frequent cancer in women, with an estimated 530 000 new cases in 2012. In the same year, this disease was responsible for approximately 7.5% of all female cancer deaths worldwide. The mortality associated with cervical cancer could be significantly diminished if it were detected at the early stages of evolution or at the precancerous stage, denominated cervical intraepithelial neoplasia (CIN) [1]. It is well established that a long-term infection with human papillomavirus (HPV), especially types 16 and 18, is a major cause for the development of cervical pre-cancer and cancer [2,3]. The Papanicolaou (Pap) smear test is the most widely used method to detect cervical tissue damage; unfortunately, while this test has good specificity, as high as 95-98%, it also has low sensitivity of approximately 50% [4]. Techniques such as automated cytology and HPV testing have been used to reduce the false negative results [5-7]. When a Pap smear result is abnormal, the clinical diagnosis is complemented by colposcopy, biopsy and visual examination of the histological sections; however, the grading scale used by health professionals is subjective due to human intervention and sometimes pre-malignancy may not even be visible at all.

The search for new diagnostic tools with better specificity and sensitivity has led to the consideration of different spectroscopic techniques capable of detection at the molecular level. Over the past years, Raman spectroscopy has provided excellent results in the diagnosis of a wide range of cancers, including breast, prostate, esophageal, colon, lung, oral and cervical [8-14]. Raman spectroscopy can be implemented microscopically to study cellular components such as nucleus, nucleoli, or chromatin [10,15]; but due to its high spatial resolution, specific biomolecular complexes such as phenylalanine, tyrosine, and adenine can be analyzed [16-18]. The Raman effect can be greatly enhanced using Surfaced Enhanced Raman Scattering (SERS), this technique can detect analytes down to the single molecule level retaining molecular specificity [19-21]. The sensitivity of SERS spectroscopy could be dramatically improved by using either gold or silver nanostructures to obtain enhancement factors of several orders of magnitude, due to the local field effect induced by the surface plasmon resonance from the nanostructured surfaces [16,22-24]. Nevertheless, the local field enhancement is effective only when Raman tags are closely located (usually less than 100 nm) to the substrate surface.

One advantage of gold nanoparticles (Au NPs) for biomedical applications is that the plasmon resonance frequency could be tuned through control of the nanoparticle size, morphology, or geometry. In particular, it is possible to tune the gold nanoparticles absorption wavelength to the near infrared, region no absorbed by the tissue, allowing a true diagnosis [25]. In addition, it is possible to take advantage of the field enhancement effect from Au NPs with other techniques for example, with two-photon imaging (TPI). This powerful microscopic image acquisition tool is non-invasive and uses near infrared excitation, leading to large penetration depths of up to hundreds of microns [26-28]. It has been reported that TPI can be observed from roughened surfaces due to the resonant coupling of specific light frequencies to the surface plasmon resonances (SPR) that lead to local electric field enhancement [26,29,30]. Traditionally, samples for TPI have been marked with organic fluorophores [31] but recently other contrast agents have been tried, such as quantum dots [32], metallic nanoparticles [33-36], and gold nanorods [37,38].

In this work was evaluated the interaction of the gold nanoparticles with HPV and cervical cancer tissues. It was acquired microscopic images using SEM, chemical mapping confocal and two-photon imaging of the samples to demonstrate the introduction of gold nanoparticles to the tissues. We observed the Au NPs agglomerated in the tissues, promoting the local field effect, resulting in a favorable effect for SERS and also two-photon imaging (red region) excited at 900 nm was obtained. This preliminaries results allowed identification of differences between HPV and cancer cervical tissues, with the Au NPs offering for future alternative as markers and sensors.

Synthesis of gold nanoparticles: The Turkevich method was used to synthesize the gold NPs with some modifications [39]. Briefly, 250 μL of a 0.1 M HAuCl4 solution (prepared by dissolving 0.5 g of HAuCl4 (99.999%, Sigma-Aldrich) in 14.715 mL of deionized water) and 3.75 mL of 1% wt. sodium citrate solution (obtained by dissolving 0.0375 g of Na3C6H5O7 (Sigma-Aldrich) in 3.75 mL of deionized water) were added into 25 mL of boiling water under stirring (400 rpm). After a few minutes the solution turned ruby red, at this time the solution was cooled down to room temperature and the resulting colloid was filtered. The final product was kept in storage at 4°C.

Gold Nanoparticles in Cervix Tissue: Formalin-fixed paraffin preserved (FFPP) cervical tissue samples were obtained by an experienced breast pathologist from the Pathology Department of the Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE) in Guanajuato State (Mexico). A total of 15 samples with different grades of CIN were identified. Of those samples 5 presented low-grade squamous intraepithelial lesions (CIN1); other five showed medium grade squamous intraepithelial lesions (CIN2), and the final five presented high-grade squamous intraepithelial lesion (CIN3). These cases were randomly selected; each case represents a sample from an individual patient. For evaluation by the pathologist, the samples were dewaxed by immersion in baths of xylene (KARAL) and absolute ethanol (KEM). For the Au NPs study, two parallel 5-μm thick FFPP sections were cut from each block using a microtome, mounted on glass slides and dried.

To prepare the cervix tissue for impregnation with the Au NPs, the histological specimens embedded in paraffin and mounted on glass microscope slides were heated to 60° C during 1 hour. To deparaffinize the mounted tissues, they were immersed in xylene twice (15 minutes each); then the tissues were rehydrated by using different concentrations of absolute EtOH as follows: EtOH 100% (10 minutes), EtOH 90% (10 minutes), EtOH 70% (10 minutes) and EtOH 30% (10 minutes). Finally, the samples were immersed in PBS 1X (5 minutes) and rinsed in distilled water (5 minutes).

To stain the deparaffinized tissues, the water excess was removed, and the specimens outline was marked with a hydrophobic ink (Pappen ®), avoiding making contact with the tissue, this was made to maintain the colloidal solution concentrated only in the tissue area. The specimens were placed on a hot-bar and incubated with the Au NPs colloidal solution for 2 hours at 37°C. The tissue dryness was continuously checked; if required, more gold solution was added. Afterwards, the tissue was carefully rinsed with distilled water. For characterization using confocal microscopy, a few drops of DABCO (1,4-diazabicyclo[2.2.2]octane) were added. Finally, the samples were covered with a coverslip making sure that no bubbles were visible. Following a protocol used to preserve oral cells for chemical analysis by Raman spectroscopy [13], DABCO was not added to the samples studied by Raman micro-spectroscopy; instead, these tissues were dried at 90 °C for 3 minutes.

The morphology and size of the Au NPs in water solution were analyzed by transmission electron microscopy (TEM) using a FEI Titan 800-300 electron microscope with an accelerating voltage of 300 kV. Tissue was analyzed by a Scanning Electron Microscope (SEM) JEOL JSM-7800F and by a SEM-Energy Dispersive X-ray Spectrometer (EDS) from Oxford Instruments. UV-Vis absorption measurements were carried out using a Perkin Elmer Lamda 900 spectrometer with a spectral resolution of 2 nm. Optical characterization of cervix tissue was obtained by a Zeiss confocal microscope, model LSM-710-NLO, equipped with a Chameleon Vision-II ultrafast laser system from Coherent (tunable from 680 to 1040 nm, 140 fs, 80 MHz repetition rate). The excitation wavelength used for confocal images was 543 nm and 900 nm for TPI.

The Raman spectra were acquired using an inVia Raman microscope from Renishaw with a 20X objective and approximately 5 mW of excitation power at 785 nm wavelength. The integration time for each Raman spectrum was 20 seconds and the spectral range was from 700 to 1700 cm-1. The Raman data was obtained by mapping a minimum of 10 independent tissue regions in each sample, depending on the quality of each slide, each site was measured twice. The results represent the average of all the mapped regions for each sample.

The size and morphology of the Au NPs were studied by TEM; a typical micrograph of the particles is presented in Figure 1a), clearly, the shape of the nanoparticles is an elongated circle. The UV-VIS absorption spectra of Au NPs dispersed in aqueous solution is shown in Figure 1b). In all cases, the SPR was centered at 520 nm which is consistent with the particle size and the observed ruby-red color of the Au NPs obtained with the Turkevich method [40]. A lower magnification micrograph is shown in Figure 1c); the inset presents an histogram of the nanoparticles size distribution, the average size is approximately 13 ± 2 nm.

Figure 1: (a) High magnification TEM micrographs of gold nanoparticles; (b) absorption spectra of the gold nanoparticles; (c) TEM micrograph, inset shows the size distribution of gold nanoparticles.

In order to evaluate the presence of the Nps at the cellular level, we acquired SEM micrographs of the carcinogenic cervix cells and cells with HPV presence; representative images are presented in Figures 2a) and 2b), respectively. The size differences between cancerous, Figure 2a), and HPV infected, Figure 2c), cells is very clear; approximately a factor of three, this cell enlargement is common when cell damage is present [41]. Panels b) and d) in Figure 2 show the EDS maps of gold distribution in the same samples, cancerous and HPV, respectively. It was observed Au NPs distribution within the cells. Our hypothesis to explain this behavior is that the Au NPs have negative charge in their surface while the nucleus could be positively charged [42]. Once we observed the location and chemical identity of the Au NPs, we acquired optical microphotographs using confocal and two-photon microscopy. All images were taken with an epi-plan 50X/0.55 objective. Before acquiring the TPI images, we systematically tuned the laser wavelength from 800 to 1100 nm, in 50 nm increments, to determine the optimal excitation wavelength. We found that 900 nm yielded the brightest images; this wavelength corresponds to the frequency of the longitudinal plasmon resonance of the nanoparticles. Figure 3 displays some characteristic microscopic images from cancerous cervical tissue with and without Au NPs. The first column corresponds to confocal images, the second to two-photon imaging (TPI), and the third is presents compound images superimposing the corresponding panels from the previous columns. Panels a) - c) present images from cancerous tissue incubated with Au NPs; panels d) – f) show a close-up view of the red circled area in panel a); finally, panels g) – i) correspond to cancerous tissue with no Au NPs. Comparison of Figure 3) with the panels on the first row, we observe that the presence of Au NPs has two main consequences in the auto fluorescence signal from the cells, the signal from the cell nucleus quenches and the cell boundaries are blurred. The cells without NPs have relatively well-defined boundaries but after incorporation of the NPs the boundaries were no longer distinguishable in the confocal image. On the other hand, when we compared the second column in Figure 3, the TPI signal obtained at red emission region from the cell nuclei increased substantially after the incorporation of the Au NPs, as expected. The red shift emission of the Au NPs is possible due to the NPs aggregation inside of the tissue. On the contrary, the TPI signal from the cytoplasm, which does not have a significant concentration of Au NPs, is significantly weaker than the signal from the nucleus. The TPI signal from the nucleus is consistent with the observation of gold location from EDS. Thus, we can conclude that Au NPs could be used as marker for cell nucleus.

Figure 2: SEM and EDS micrographs of cancerous and HPV infected tissues impregnated with Au NPs a) SEM of carcinogenic cell; b) EDS map of gold (shown in green) in the same region as panel a); c) SEM of HPV infected cell; d) EDS map of gold in the same region as panel c). Note the difference in scale bars for each panel.

Figure 3: Micrographs of carcinogenic cervical tissue, a) confocal image of tissue with Au NPs, excitated at 543 nm; b) two-photon imaging of cervical tissue with Au NPs, excitated at 900 nm; c) superposition of images from panels a) and b); d) – f) zoom in of the red circled region in panel a); g) – i) confocal, TPI and superposition of both images, respectively of cervical tissue without Au NPs.

It has been reported that cervical tissue shows SERS peaks around 722, 755, 782, 849, 853, 921, 938 and 1245 cm^-1 [17,43-45]. Figure 4 shows three different micro-SERS spectra of our cervical tissue samples excited at 785 nm; the top panel corresponds to HPV infected tissue marked with Au NPs, the bottom spectra are from tissue with carcinoma, where the top line corresponds to tissue marked with Au NPs and the bottom line to unmarked tissue. The Raman spectrum signal level of the unmarked tissue is very weak, while the signal strength from the marked tissues is strong with well-defined peaks. The most prominent peaks we observed in HPV and carcinogenic tissues with Au NPs are listed in Table 1 [10,17,43].

Figure 4: SERS spectra of cervical tissues. Top panel, tissue infected with HPV and marked with Au NPs; bottom panel, tissues with carcinoma, one marked with Au NPs the other without NPs.

Table 1: Peaks assignments for SERS spectra of HPV and carcinogenic tissues with Au NPs.

In the comparison of SERS spectra between cervical cancer tissue with respect to the HPV ones, several differences in spectral intensities are reveled, principally in regions located in the ranges of 920-1140 cm^-1, 1160-1280 cm^-1 and 1290-1480 cm^-1 respectively. In Figure 5, it is shown a plot of the integrated signal of the 920-1140 cm^-1, 1160-1280 cm^-1 and 1290-1480 cm^-1 regions of vibrational bands, Figure 5a shows the integrated signals of HPV tissue and Figure 5(B) the Carcinogenic tissue ones. Each data point represents the average value from three SERS regions and error bars show the standard deviations. According with these results it could be observed that variations in Carcinogenic SERS spectra are lower than HPV ones, specially in the region of 920- 1140 cm^-1 that mostly corresponds to DNA bands, this can be due to the deformations involved in cell damage caused by HPV infection. Table 1 lists tentative assignments for the observed SERS bands, according to the literature [17,44,45].

Figure 5: A plot of the integrated SERS regions 920-1140 cm-1, 1160-1280 cm-1 and 1290-1480 cm-1 respectively. Each point represents the average value from three SERS spectra and error bars show the standard deviations. SERS spectra of cervical tissues. (A) panel, tissue infected with HPV and marked with Au NPs; (B) panel, tissues with carcinoma marked with Au NPs.

Comparing the signals from HPV infected tissue (Figure 4, top panel) and invasive carcinoma (Figure 4, lower panel), we observed the following spectral changes: (i) modification of lines associated with saccharides at 849, 889 and 917 cm⁻¹ due to glycogen skeletal deformation, CCH aromatic deformation and CCH deformation, respectively [46] marked by red arrows in Figure 4; (ii) collagen bands at 1277 and 1401 cm^-1 (green arrows) and CH₂CH₃ deformation at 1456 cm⁻¹ (black arrow) are present in the HPV infected tissue but disappear in the cancerous samples. These results corroborate previous reports stating the principal differences between normal cervical tissue and invasive cervical-cancer tissue are primarily on the basis of collagen bands and CH₂ stretching bands [42]. Cervical cells are unusual among other epithelial cells in that they accumulate large amounts of glycogen during the maturation process [46]. Glycogen is known to be linked to cellular maturation and disappears with the loss of differentiation during neoplasia.

Beside the aforementioned lines, invasive carcinoma also shows characteristic nucleic acid bands such as 970, 1084, 1181 and 1373 cm⁻¹ [17,44-46]. An increment in the intensity of the amide III bands at 1228 and 1254 cm^-1, and the amide II band at 1558 cm⁻¹ was observed in the spectra of carcinoma samples as compared to the HPV tissue samples, see magenta arrows in Figure 4. The increment in nucleic acids and proteins result from an increased proliferation of these substances in the tumor cells. In particular, the 1228 cm⁻¹ band associated with amide III of beta sheeted proteins could be used to differentiate normal and early stage cancerous cervix tissues. It has been reported that bands associated with high-grade squamous intraepithelial lesion tissue (HSIL) such as 738 and 1373 cm⁻¹ (thymine), 785 cm⁻¹ (cytosine) or 890 cm⁻¹ (saccharides) are associated with DNA; therefore, they indicate the presence of rapidly proliferating cells having high DNA content [44].

Some researchers have suggested that Raman spectra associated with the CIN2 lesion cluster with the signal of the basal epithelial cells of the normal squamous epithelium; therefore, this signal shares indistinctive biochemical profiles, such as the bands that are associated with the amide I and amide III bands [47]. In our case the Raman spectra of carcinogenic tissues show an increased contribution from amide III and phosphate stretching, at 1252 cm⁻¹ indicated by the second magenta arrow in Figure 4, and contributions from guanine, thymine and C–H deformation in proteins and carbohydrates at 1342 and 1373 cm⁻¹ as shown by the blue arrows in the 1300 cm^-1 region. In this work we propose that the increased presence of Au NPs in the nuclei of the cervix tissue cells lead to useful SERS spectra for possible aid in early cancer diagnostic. The agglomeration of Au NPs generates “hot-spots” which increase the signal of the molecules close to the nanoparticles.

In this study, the microstructural and SERS spectroscopic differences between HPV infected and carcinogenic tissues embedded with Au NPs were evaluated. SEM images showed changes in cell nuclei sizes of up to 300%, which is common when cellular damage occurs. EDS corroborated that Au NPs were located mostly in the cell nucleus. We hypothesize that this is due to the charge differences, Au NPs being negatively charged and the nucleus being positive. Two-photon imaging of the morphology of the tissue embedded in AuNPs was obtained in the red region, excited at 900 nm. SERS spectroscopy, enabled by the local field enhancement effect produced by the Au NPs, revealed several changes between HPV infected and carcinogenic tissues mainly in bands corresponding to DNA markers. A spectroscopic approach that yields compositional information of cell nuclei could be a powerful tool for rapid cell characterization and assessment of cellular activities at the sub-cellular level.

TLL is grateful to the 2019 UC-MEXUS-CONACyT Collaborative grants.

1. Buckley CH, Butler EB, Fox H (1982) Cervical intraepithelial neoplasia. J clin pathol 35: 1-13.
2. Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, et al. (2003) Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348: 518-527.
3. Naucler P, Ryd W, Törnberg S, Strand A, Wadell G, et al. (2007) Human papillomavirus and papanicolaou tests to screen for cervical cancer. N Engl J Med 357: 1589-1597.
4. Nanda K, McCrory DC, Myers ER, Bastian LA, Hasselblad V, et al. (2000) Accuracy of the papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: A systematic review. Ann Intern Med 132: 810-819.
5. Adams JM, Huang DC, Strasser A, Willis S, Chen L, et al. (2005) Subversion of the Bcl-2 life/death switch in cancer development and therapy. Cold Spring Harb Symp Quant Biol 70: 469-477.
6. Bolger N, Heffron C, Regan I, Sweeney M, Kinsella S, et al. (2006) Implementation and evaluation of a new automated interactive image analysis system. Acta Cytologica 50: 483-491.
7. Kyrgiou M, Tsoumpou I, Vrekoussis T, Martin-Hirsch P, Arbyn M, et al. (2006) The up-to-date evidence on colposcopy practice and treatment of cervical intraepithelial neoplasia: The cochrane colposcopy & cervical cytopathology collabortive group (C5 group) approach. Cancer Treat Rev 32: 516-523.
8. Ellis DI, Cowcher DP, Ashton L, O’Hagan S, Goodacre R, et al. (2013) Illuminating disease and enlightening biomedicine: Raman spectroscopy as a diagnostic tool. Analyst 138: 3871-3884.
9. Kendall C, Isabelle M, Bazant-Hegemark F, Hutchings J, Orr L, et al. (2009) Vibrational spectroscopy: A clinical tool for cancer diagnostics. Analyst 134: 1029-1045.
10. Lyng FM, Traynor D, Ramos IR, Bonnier F, Byrne HJ, et al. (2015) Raman spectroscopy for screening and diagnosis of cervical cancer. Anal Bioanal Chem 407: 8279-8289.
11. Nijsssen A, Koljenovic S, Schut TCB, Caspers PJ, Puppels GJ, et al. (2009) Towards oncological application of raman spectroscopy. J Biophotonics 2: 29-36.
12. Ceja-Fdez A, López-Luke T, Oliva J, Vivero-Escoto J, Gonzalez-Yebra AL, et al. (2015) Labeling of HeLa cells using ZrO2:Yb3+-Er3+ nanoparticles with upconversion emission. J Biomed Optics 20: 1-8.
13. Cepeda-Perez E, López Luke T, Plascencia Villa G, Perez Mayen L, Ceja Fdez A, et al. (2016) SERS and integrative imaging upon internalization of quantum dots into human oral epithelial cells. J Biophotonics 9: 683-693.
14. Cepeda-Pérez E, López-Luke T, Salas P, Plascencia-Villa G, Ponce A, et al. (2016) SERS-active Au/SiO2 clouds in powder for rapid ex vivo breast adenocarcinoma diagnosis. Biomed Opt Express 7: 2407-2418.
15. Ramos IR, Meade AD, Ibrahim O, Byrne HJ, McMenamin M, et al. (2016) Raman spectroscopy for cytopathology of exfoliated cervical cells. Faraday Discuss 187: 187-198.
16. Kneipp K, Haka SA, Kneipp H, Badizadegan K, Yoshizawa N, et al. (2002) Surface-enhanced raman spectroscopy in single living cells using gold nanoparticles. Appl Spectrosc 56: 150-154.
17. Movasaghi Z, Rehman S, Rehman IU (2007) Raman spectroscopy of biological tissues. Applied spectroscopy reviews 42: 493-541.
18. De Gelder J, De Gussem K, Vandenabeele P, Moens L (2007) Reference database of Raman spectra of biological molecules. J Raman Spectrosc 38: 1133-1147.
19. Kneipp K, Wang Y, Kneipp H, Perelman LV, Itzkan I, et al. (1997) Single molecule detection using surface-enhanced Raman scattering (SERS). Phys Rev Lett 78: 1667-1670.
20. Nie SM, Emery SR (1997) Probing single molecules and single nanoparticles by surface-enhanced raman scattering. Sci 275: 1102-1106.
21. Vlckova B, Pavel I, Sladkova M, Siskova K, Slouf M (2007) Single molecule SERS: Perspectives of analytical applications. J Mol Struct 834: 42-47.
22. Le Ru EC, Etchegoin PG (2006) Rigorous justification of the |E|4 enhancement factor in surface enhanced raman spectroscopy. Chem Phys Lett 423: 63-66.
23. Campion A, Kambhampati P (1998) Surface-enhanced raman scattering. Chem Soc Rev 27:241-250.
24. Xu H, Aizpurua J, Käll M, Apell P (2000) Electromagnetic contributions to single-molecule sensitivity in surface-enhanced raman scattering. Phys Rev E 62: 4318-4324.
25. Hu M, Chen J, Li ZY, Au L, Hartland GV, et al. (2006) Gold nanostructures: Engineering their plasmonic properties for biomedical applications. Chem Soc Rev 35: 1084-1094.
26. Konig K, Riemann I (2003) High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt 8: 432-439.
27. Masters BR, So PTC, Gratton E (1998) Multiphoton excitation microscopy of in vivo human skin - Functional and morphological optical biopsy based on three- dimensional imaging, lifetime measurements and fluorescence spectroscopy. New York Academy of Sciences 838: 58-67.
28. Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, et al. (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. PNAS 100: 7075-7080.
29. Boyd GT, Yu ZH, Shen YR (1986) Photoinduced luminescence from the noble- metals and its enhancement on roughened surfaces. Phys Rev B 33: 7923-7936.
30. Imura K, Nagahara T, Okamoto H (2005) Near-field two-photon-induced photoluminescence from single gold nanorods and imaging of plasmon modes. J Phys Chem B 109: 13214-13220.
31. Albota M, Beljonne D, Brédas JL, Ehrlich JE, Fu JY, et al. (1998) Design of organic molecules with large two-photon absorption cross sections. Science 281: 1653-1656.
32. Larson DR, Zipfel WR, Williams RM, Clark SW, Bruchez MP, et al. (2003) Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 300: 1434-1436.
33. Farrer RA, Butterfield FL, Chen VW, Fourkas JT (2005) Highly efficient multiphoton-absorption-induced luminescence from gold nanoparticles. Nano Lett 5: 1139-1142.
34. Huang XH, El-Sayed IH, Qian W, El-Sayed MA (2006) Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J Am Chem Soc 128: 2115-2120.
35. Sonnichsen C, Alivisatos AP (2005) Gold nanorods as novel nonbleaching plasmon-based orientation sensors for polarized single-particle microscopy. Nano Lett 5: 301-304.
36. Yelin D, Oron D, Thiberge S, Moses E, Silberberg Y (2003) Multiphoton plasmon-resonance microscopy. Opt Express 11: 1385-1391.
37. Mohamed MB, Volkov V, Link S, El-Sayed MA (2000) The ‘lightning’ gold nanorods: Fluorescence enhancement of over a million compared to the gold metal. Chem Phys Lett 317: 517-523.
38. Wang HF, Huff TB, Zweifel DA, He W, Low PS, et al. (2005) In vitro and in vivo two-photon luminescence imaging of single gold nanorods. PNAS 102: 15752-15756.
39. Turkevich J, Stevenson PC, Hillier J (1953) The formation of colloidal gold. J Phys Chem 57: 670-673.
40. Turkevich J, Garton G, Stevenson PC (1954) The color of colloidal gold. J Colloid Sci 9: 26-35.
41. Warburg O (1956) On the origin of cancer cells. Sci 123: 309-314.
42. Jung YK, Shin E, Kim BS (2015) Cell nucleus-targeting zwitterionic carbon dots. Sci 5:18807.
43. Lyng FM, Faoláin EO, Conroy J, Meade AD, Knief P, et al. (2007) Vibrational spectroscopy for cervical cancer pathology, from biochemical analysis to diagnostic tool. Exp Mol Pathol 82: 121-129.
44. Feng S, Lin J, Cheng M, Li YZ, Chen G, et al. (2009) Gold nanoparticle based surface-enhanced raman scattering spectroscopy of cancerous and normal nasopharyngeal tissues under near-Infrared laser excitation. Applied Spectroscopy 63: 1089-1094.
45. Huang H, Shi H, Feng S, Lin J, Chen W, et al. (2013) Silver nanoparticle based surface enhanced raman scattering spectroscopy of diabetic and normal rat pancreatic tissue under near-infrared laser excitation. Laser Physics Letters 10: 045603.
46. Chiriboga L, Xie P, Vigorita V, Zarou D, Zakim D (1998) Infrared spectroscopy of human tissue. II. A comparative study of spectra of biopsies of cervical squamous epithelium and of exfoliated cervical cells. Biospectrosc 4: 55-59.
47. Wong PT, Wong RK, Caputo TA, Godwin TA, Rigas B (1991) Infrared spectroscopy of exfoliated human cervical cells: evidence of extensive structural changes during carcinogenesis. PNAS 88: 10988-10992.