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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 22 — Nov. 4, 2013
  • pp: 27063–27073
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Second harmonic generation correlation spectroscopy for single molecule experiments

Jing Liu and Joseph Irudayaraj  »View Author Affiliations


Optics Express, Vol. 21, Issue 22, pp. 27063-27073 (2013)
http://dx.doi.org/10.1364/OE.21.027063


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Abstract

We demonstrate a single molecule detection approach to further extend the detection limit of correlation spectroscopic techniques through the Second Harmonic Generation Correlation Spectroscopy (SHGCS). SHG signals with high signal-to-noise ratio (SNR) were obtained from Barium titanium oxide, BaTiO3 (BTO) nanocrystals (NCs) upon excitation by a femto-second laser fitted to the scanning confocal bench. The fluctuation of SHG signals from BTO NCs in transparent and turbid media was examined and their diffusion time and particle concentration were evaluated by autocorrelation. Proof-of-concept measurements indicate that water-dispersed BTO NCs at different concentrations yield an average diffusion time of 6.43 ± 0.68 ms and the detection limit of SHGCS was found to be at 814 ± 41 fM, approximately 100 folds below the detection limit of fluorescence correlation spectroscopy (FCS). The dynamics of BTO NCs was demonstrated in serum with high SNR and selectivity to show its potential applicability in biomedicine. High SNR and the sub-picomolar detection limit positions SHGCS as an excellent technique for ultralow single particle or single molecule experimentation in a complex medium.

© 2013 OSA

1. Introduction

Fluorescence Correlation Spectroscopy (FCS) is a single molecule technique with demonstrated applicability to provide significant insights on diffusion dynamics, chemical thermodynamics, and kinetics of interaction [1

1. A. V. Orden and J. Jung, “Fluorescence correlation spectroscopy for probing the kinetics and mechanics of DNA hairbin formation,” Biopolymers 89(1), 1–16 (2008). [CrossRef]

10

10. J. Chen, S. Nag, P. A. Vidi, and J. Irudayaraj, “Single molecule in vivo analysis of Toll-like receptor 9 and CpG DNA interaction,” PLoS ONE 6(4), e17991 (2011). [CrossRef] [PubMed]

] of biomolecules in confined environments. The essence of this approach is to detect fluorescence fluctuations of molecules diffusing through a focal volume in a femto liter volume. Combined with confocal microscopy, FCS provides information on concentration, diffusion time, and binding constants of diffusers at the single molecule level. Since its invention in the 1970’s [11

11. D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system: measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972). [CrossRef]

,12

12. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974). [CrossRef] [PubMed]

], extensive efforts have been undertaken and several refinements have been proposed to improve the optics and data acquisition components to maximize the SNR and expand the applications. By cross-correlating signal fluctuation from two diffusing species, Fluorescence Cross-correlation Spectroscopy (FCCS) [5

5. M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two-beam cross-correlation: a method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999). [CrossRef] [PubMed]

] has been proposed to examine interactions between two or more molecules. Scanning/probe FCS [13

13. M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, “Zero-mode waveguides for single-molecule analysis at high concentrations,” Science 299(5607), 682–686 (2003). [CrossRef] [PubMed]

,14

14. K. Garai, M. Muralidhar, and S. Maiti, “Fiber-optic fluorescence correlation spectrometer,” Appl. Opt. 45(28), 7538–7542 (2006). [CrossRef] [PubMed]

] and total-internal-reflection FCS (TIR-FCS) [15

15. N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J. 33(3), 435–454 (1981). [CrossRef] [PubMed]

] have also been proposed to incorporate whole system analysis as well as in the analysis of selected optically active regions to extend the detection range to micro/milli Molar scales.

However, some limitations compound traditional FCS when probing ultralow concentrations (< 100 pM). First, the photon stability and cytotoxicity of fluorophores, such as fluorescent dyes [3

3. W. Al-Soufi, B. Reija, M. Novo, S. Felekyan, R. Kühnemuth, and C. A. M. Seidel, “Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: inclusion complexes of pyronines with cyclodextrin,” J. Am. Chem. Soc. 127(24), 8775–8784 (2005). [CrossRef] [PubMed]

], fluorescent nanoparticles (NPs) [16

16. J. Chen and J. Irudayaraj, “Quantitative investigation of compartmentalized dynamics of ErbB2 targeting gold nanorods in live cells by single molecule spectroscopy,” ACS Nano 3(12), 4071–4079 (2009). [CrossRef] [PubMed]

,17

17. Y. Wang, J. Chen, and J. Irudayaraj, “Nuclear targeting dynamics of gold nanoclusters for enhanced therapy of HER2+ breast cancer,” ACS Nano 5(12), 9718–9725 (2011). [CrossRef] [PubMed]

], and quantum dots [18

18. D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-soluble quantum dots for multiphoton fluorescence imaging in vivo,” Science 300(5624), 1434–1436 (2003). [CrossRef] [PubMed]

], pose some constraints on stability. When probing ultra-low concentrations, high excitation power is frequently required to obtain high fluorescence emission rates (counts-per-second per molecule), but photobleaching or blinking effect of fluorophores could interfere with the fluorescence fluctuation arising from the diffraction limited diffusion dynamics that might affect the SNR. Second, the background signal due to autofluorescence will interfere with the fluorescence signal, especially in biological fluids such as serum or blood and can easily overwhelm the signal from fluorophores. Correlation spectroscopic methods that have the potential to detect targets at ultra-low concentrations amidst a turbid background will have significant impact in in situ biodiagnostics.

In this report, we propose a Second Harmonic Generation (SHG) Correlation Spectroscopy (SHGCS) for label-free monitoring of the diffusion dynamics of structural components with second-order nonlinearity. In this proof of concept study, we replace the fluorescent probes with barium titanate, BaTiO3 (BTO) (nanocrystals, NCs) that can generate a second harmonic wave under ultra-fast laser excitation at low powers, to develop SHGCS for potential applications in biology. SHG [19

19. R. W. Boyd, Nonlinear Optics (Academic, 2003).

,20

20. R. L. Sutherland, D. G. McLean, and S. Kirkpatrick, Handbook of Nonlinear Optics (Dekker, 2003).

] can be construed as an optical nonlinearity resulting from the intrinsic asymmetric structure, where the dipole is proportional to the square of the incident electromagnetic field. SHG materials emit photons with energy doubling of its incident counterpart. When the phase-matching condition is fulfilled, SHG materials can generate coherent, non-bleaching, non-blinking signals at high emission efficiency [21

21. A. A. Gulamov, E. A. Ibragimov, V. I. Redkorechev, and T. Usmanov, “Maximum efficiency of generation of the second and third harmonics of neodymium laser radiation,” Sov. J. Quantum Electron. 13(7), 844–845 (1983). [CrossRef]

] (which can reach 90%) with a high SNR (SHG emission spectrum can be narrowed to within 10 nm). Typical inorganic crystals [20

20. R. L. Sutherland, D. G. McLean, and S. Kirkpatrick, Handbook of Nonlinear Optics (Dekker, 2003).

], such as barium titanate (BTO), lithium niobate (LiNbO3), and lithium triborate (LiB3O5) are used to generate second harmonic light. Some biological materials, such as collagen, microtubules, and muscle myosin, are also good candidates for SHG [22

22. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003). [CrossRef] [PubMed]

24

24. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003). [CrossRef] [PubMed]

] signal monitoring. The Second Harmonic Imaging Microscopy (SHIM) has been used to image collagen and membrane structure in live cells [24

24. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003). [CrossRef] [PubMed]

]. Interesting applications using amphiphilic porphyrins as probes for SHIM [25

25. J. E. Reeve, H. A. Collins, K. De Mey, M. M. Kohl, K. J. Thorley, O. Paulsen, K. Clays, and H. L. Anderson, “Amphiphilic porphyrins for second harmonic generation imaging,” J. Am. Chem. Soc. 131(8), 2758–2759 (2009). [CrossRef] [PubMed]

,26

26. J. E. Reeve, H. L. Anderson, and K. Clays, “Dyes for biological second harmonic generation imaging,” Phys. Chem. Chem. Phys. 12(41), 13484–13498 (2010). [CrossRef] [PubMed]

] have also been investigated. Few inorganic materials have also been used as probes for bio-imaging. Hsieh etc. utilized BaTiO3 (BTO) nanocrystals to realize regular and holographic SHIM [27

27. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, “Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes,” Biomaterials 31(8), 2272–2277 (2010). [CrossRef] [PubMed]

,28

28. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, “Second harmonic generation from nanocrystals under linearly and circularly polarized excitations,” Opt. Express 18(11), 11917–11932 (2010). [CrossRef] [PubMed]

]; recently Pantazis extended the application of BTO nanocrystals for in vivo imaging [29

29. P. Pantazis, J. Maloney, D. Wu, and S. E. Fraser, “Second harmonic generating (SHG) nanoprobes for in vivo imaging,” Proc. Natl. Acad. Sci. U.S.A. 107(33), 14535–14540 (2010). [CrossRef] [PubMed]

]. Significant efforts in biological imaging using SHG have also been expended; however, none address the implementation of SHG in the context of correlation spectroscopy except the recent report on realizing nonlinear correlation spectroscopy via high laser power (>200 mW) and small numerical aperture (N.A. = 0.22) objective with large size (700 nm polystyrene spheres) particles [30

30. M. Geissbuehler, L. Bonacina, V. Shcheslavskiy, N. L. Bocchio, S. Geissbuehler, M. Leutenegger, I. Märki, J. P. Wolf, and T. Lasser, “Nonlinear correlation spectroscopy (NLCS),” Nano Lett. 12(3), 1668–1672 (2012). [CrossRef] [PubMed]

].

In this work, we utilize BTO NCs as SHG probes to demonstrate second harmonic generation correlation spectroscopy (SHGCS) in a small focal volume (~femto liter) and to provide a theoretical and experimental basis for this concept. Spatial and temporal diffusion behaviors are observed from the experiments at ultra-low concentration with high SNR.

2. Theory

To derive a basis for SHGCS, we start from the SHG signal fluctuation calculation within a small detection volume. SHGCS detects the fluctuation of the total SHG signal, H(t), which is generated instantly without any lifetime delay under ultra-fast (~femtosecond) laser excitation and proportional to the number of nanoparticles N(t), as a function of time, t. H(t) fluctuation occurs as a function of the number of molecules N(t) diffusing in the focal volume. In our work, relatively low concentration, for example, sub-nanomolar (~10−10 M) concentration was considered to assure single diffusers.

The temporal auto-correlation function G(τ), under a constant laser excitation, can be defined by,
G(τ)=δH(t)·δH(t+τ)H(t)2
(1)
WhereδH(t)=H(t)H(t)denotes the signal fluctuation at time t, and δH(t+τ)=H(t+τ)H(t) is the fluctuation at timet+τ.H(t) is the time-averaged signal, andH(t)I(t)excitation2. A point to note here is that in traditional FCS, theoretical calculations are based on the assumption that the fluorescence emission of single molecule remains stable within the confocal volume; however some factors are known to influence signal fluctuation originating from diffusing molecules. One is the bleaching and blinking effect, especially for some slowly diffusing fluorophores the bleaching is more obvious. The other factor is the triplet dynamics [4

4. P. Schwille and E. Haustein, “Fluorescence correlation spectroscopy: an introduction to its concepts and applications,” Experimental Biophysics Group, University of Gottingen.

]. However, SHG signals are transient and stable enough for hours, thus in the SHGCS, the optical property of the diffusing SHG probes do not change when diffusing through the focal volume. Therefore consistent two-dimensional autocorrelation function can be derived as,
G(τ)=2N0·11+2τ/τD·1(1+(r0/z0)2(2τ/τD))
(2)
Where N0is the average number of molecules in the focal volume, τDis diffusion time, defined asτD=r02/4D, here r0is the beam radius at the focus plane, D is the diffusion coefficient of nanoparticle in solutions, z0is the diffraction length, which together with r0are determined by the system. Fitting Eq. (2) to the autocorrelation data, we can obtain the diffusion time as well as the concentration of molecules C0 fromN0=VeffC0;where Veff=π3/2r02z0is the effective focal volume. In our experiments, we set this value to 3.05 fL after calibrating with Rhodamine 123 [16

16. J. Chen and J. Irudayaraj, “Quantitative investigation of compartmentalized dynamics of ErbB2 targeting gold nanorods in live cells by single molecule spectroscopy,” ACS Nano 3(12), 4071–4079 (2009). [CrossRef] [PubMed]

].

3. Materials and methods

SHG material, Tetragonal structured BTO NCs, was gifted from Prof. Paul Bowen (Swiss Federal Institute of Technology, Lausanne, Switzerland) [31

31. A. Aimable, N. Jongen, A. Testino, M. Donnet, J. Lemaitre, H. Hofmann, and P. Bowen, “Precipitation of nanosized and nanostructured powders: process intensification using SFTR, applied to BaTiO3, CaCO3 and ZnO,” Chem. Eng. Technol. 34, 344–352 (2011). [CrossRef]

]. The asymmetric lattice structure ensures high efficiency of the second harmonic wave generation. Dry BTO NC powder (0.01mg/100ml) was dissolved in water and the solution sonicated for about 20 minutes to obtain mono-dispersed particles, and then filtered by a 0.2 um-pore membrane three times. Cover glass was immersed in aqua regia solution overnight to attain an ion-free surface and washed with nanopure water, and dried with Argon gas before spin-coating the coverslips with BTO NCs. SHG images and correlation spectroscopy data collection were obtained based on the experimental setup detailed below.

The SHG imaging and correlation spectroscopy apparatus was constructed based on a confocal microscopy bench (Fig. 1
Fig. 1 Schematic of the experimental setup. The objective lens is an Olympus water immersion OLYMPUS UPLANAPO 60X /1.20; 3-D scanner is a nanometer-precise PZT stage controller where the objective is mounted; dichroic mirror is the 600 dcxr (Chroma Inc.); confocal pinhole size is 50um; different filters were inserted before the detector (APD: avalanche photodiode).
). A Chameleon Ultra Ti-Sapphire tunable laser (Coherent Inc., CA.) operating in the 690-1020 nm wavelength range was utilized as the excitation source. The pulse width of the two-photon laser beam was set to 140 fs, the repetition rate was 80 MHz and the output laser power was tunable in the 0-2 W range using a tunable neutral density filter. In our experiments average laser power used was 0.64 mW, resulting in 8.93 GW/cm2 in laser intensity; while in ref [30

30. M. Geissbuehler, L. Bonacina, V. Shcheslavskiy, N. L. Bocchio, S. Geissbuehler, M. Leutenegger, I. Märki, J. P. Wolf, and T. Lasser, “Nonlinear correlation spectroscopy (NLCS),” Nano Lett. 12(3), 1668–1672 (2012). [CrossRef] [PubMed]

], mean laser power was greater than 200 mW, which equals 107 GW/cm2 in intensity; and for the regular two photon FCS, excitation laser intensity is usually more than 100 GW/cm2. The modulated laser beam is expanded and then delivered by a water-immersion objective, OLYMPUS UPLANAPO 60X /1.20 (Olympus Inc.), into an inverted microscopy, Olympus IX71. Although the SHG signal from the traditional second order bulk nonlinear crystals is forward directionally favored, due to the nano-sized crystals and asymmetric morphology, the forward and backward signals are both favored for BTO NCs. In this apparatus, SHG signal was collected using the same objective in the backward detection mode, a 50 um pinhole was used to reject the background noises and the SHG was filtered using appropriate band pass filters before redirecting the signals into single photon avalanche photodiodes (SPAD) (SPCM-AQR-14, PerkinElmer Inc.).

SHG signal was collected using the time-correlated single photon counting (TCSPC) mode (Time Harp200, PicoQuant GmbH Berlin, Germany), and fitted with a single exponential function using the SymphoTime software (version, 5.13, PicoQuant).

4. Discussion

SHG microscopy of BTO NCs on cover slips under femtosecond laser excitation is shown in Fig. 2
Fig. 2 TEM (a) and Scanning SHG microscopy (c-f) of BTO nanocrystals deposited on a glass substrate using different band-pass filters (Chroma), (c), 460-50 filter; (d), 400-40 filter; (e), 480-10 filter; (f), 520-40 filter. Cross section distribution of below two BTO NCs in (c) is shown in (b); inset in (b) shows the excitation power dependent SHG signal. Unit of scale bar: counts/ms. Pixel dwell time is 0.6 ms.
from different band-pass filters. The wavelength of the irradiated laser beam was set at 880 nm; therefore the theoretical wavelength of the generated SHG signals accordingly should be ~440 nm. In our experiments, considering the high efficiency of SHG, and the single photon counting module used in measurements, we set the laser power at the focal plane as 0.64 mW to avoid detector saturation. The emission filter used for SHG is 460-50 (Chroma), where 460 is the center wavelength with 50 nm as the open range for the filter. Figure 2(c) is the image of BTO NCs on cover slip, where a three-particle cluster is illustrated. A cross sectional distribution of the SHG imaging in Fig. 2(c) is illustrated in Fig. 2(b) and fitted by Gaussian peaks. Gaussian peak fitting indicates that the half width-half maximum (HWHM) of each peak is around 250 nm, which is almost at the diffraction limit of the 440 nm light. TEM images of BTO nanocrystals, Fig. 2(a), clearly show that the size of BTO nanocrystals is in the range between 30 nm and 100 nm, beyond the diffraction limit of the 440 nm light. Furthermore, the excitation intensity, IExcitationand the SHG intensity ISHGwill follow the relation, ISHGIExcitation2,as shown in Fig. 2(b) (inset), the slope of ln(ISHG)with respect to ln(IExcitation)is around 2, confirming the generation of the second harmonic signal.

Except for the 460-50 filter which is used to collect the SHG signal, other filters were also used to check the spectrum and high SNR of SHG. Three other different band-pass filters, 400-40, 480-10, and 520-40 (Chroma) were used to obtain scanning images. The center wavelength of these filters was 400 nm, 480 nm, and 520 nm, with band pass widths of 40 nm, 10 nm, and 40 nm, respectively. As shown in Fig. 2(d)-2(f), minimum SHG signal can pass through these filters, with intensities three to four orders of magnitude less than the SHG signal. A point to note here is that, after the signal passes through the 400-40 filter a small portion of the SHG signal remains, which yield a maximum of 4 counts/ms. In theory, the SHG signal should be in the close vicinity of twice the excitation wavelength, however, due to the relatively large size of BTO nanocrystals, which are about 30-100 nm in effective diameter, the scattering effect would be efficient enough to couple with the second harmonic generation, namely the second order hyperscattering effect, which can broaden the region of second harmonic generation [32

32. K. Clays and A. Persoons, “Hyper-Rayleigh scattering in solution,” Phys. Rev. Lett. 66(23), 2980–2983 (1991). [CrossRef] [PubMed]

].

Based on the analysis of SHG imaging, it is easy to conclude that SHG signals can be collected with high efficiency, with no saturation at a high SNR. The maximum emission of BTO NCs in Fig. 2(c) is ~1100 counts/ms, while the background counts is only 0.200 counts/ms, indicating that the SNR of SHG can reach up to 10000. Although much less signal is detected 20 nm away in the spectrum (Fig. 2(d)), the SHG signal is narrowed around 440 nm, which can be easily selected by signals from fluorophores when mixed together. Thus SHG and fluorescence spectroscopy could be integrated for imaging/sensing as a dual-mode technique. Furthermore, the high emission efficiency and SNR of BTO NCs shows that the BTO nanocrystals to be an ideal tool for performing SHG correlation spectroscopy in the single molecule realm.

Due to the unique optical properties of BTO NCs, it is easy to obtain well-shaped correlation curves from SHGCS. In SHGCS measurements, the signal collection time from the detector is set to 2 minutes for correlation analysis; the focus points are set to measure at a depth of 30 um in solution and SHG signal is collected backwardly by SPAD with respect to time. Figure 3
Fig. 3 Time traced SHG signal intensity and correlation spectroscopy of BTO nanocrystals dispersed in nanopure water at different concentrations. 25 pM: (a), (f); 12.5 pM: (b), (g); 5 pM: (c), (h); 2.5 pM: (d), (i); 0.5 pM: (e), (j). circle: experimental curve, solid line: theoretical fitting.
shows the time-traced SHG signals and correlation curves from BTO NCs at the sub-nanoMol to sub-picoMol concentration range.

By correlating the time-traced SHG signals in Fig. 3(a)-3(e), we can obtain an auto-correlation function of SHG signals, shown in Fig. 3(f)-3(j). By fitting these experimental curves using Eq. (2) (solid red line in Fig. 3(f)-3(j)) one can estimate the diffusion time and average number of nanocrystals in the focal volume. It should be noted that the current theoretical model cannot perfectly fit the correlation curve, which can be attributed to two major reasons; one is that the particle size variation can lead to the distortion of correlation curve, since the size of BTO NCs is around 30~100 nm, and the hydrodynamic diameter is about 57.4 ± 3 nm from Einstein equation (D=kBT6πηRh;kB,Boltzmann constant; T, temperature;η,viscosity of the solvent; Rh,hydrodynamic radius); the other reason is that the gradient force from the excitation laser can induce a bias on the diffusers, although it is difficult to “trap” the nanocrystals. Theoretical fitting indicates that diffusion times of BTO NCs in water solutions at different concentrations are similar, and the estimated value was 6.43 ± 0.68 ms; and the estimated concentration of BTO NCs at the different levels were 23.8 ± 5.53 pM, 13.7 ± 3.08 pM, 4.96 ± 0.88 pM, 1.98 ± 0.55 pM, and 0.81 ± 0.04 pM, respectively (Fig. 4
Fig. 4 Estimated concentration of BTO NCs by SHGCS from theoretical fitting.
). The estimated concentrations are in good agreement with the experimental concentrations, which are 25 pM, 12.5 pM, 5 pM, 2.5 pM, and 0.5 pM respectively. The concentration level can be directly obtained from the time-traced SHG signals in Fig. 3(a)-(e). The number of BTO NCs passing through the focal volume decreases with concentration. At a concentration of 0.5 pM, less than 10 nanocrystals pass through the focal volume within 2 min. However, upon further decreasing the BTO concentration to beyond 100 fM, the number of BTO NCs in the focal volume decreases, while the detected concentration from theoretical fitting did not change, at this point the noise begins to influence the signal from nanocrystals, limiting the system to higher amplitude values in the correlation curve. This indicates that the detection limit of SHGCS can be extended to hundreds of femto Molar, a much smaller than the regular value of dyes or nanoparticles observed from FCS measurements.

To further illustrate the robustness of SHGCS, the diffusion dynamics of BTO NCs was demonstarted in a turbid media with high selectivity. The dynamics of BTO NCs were investigated in fetal bovine serum (Atlanta Biologicals, GA), shown in Fig. 5(a)
Fig. 5 (a) Time traced SHG signal intensity (black) of BTO in serum and background signal intensity (red) of serum solution. (b) Normalized autocorrelation curve of BTO NCs in serum with different concentration. (c) Averaged diffusion time of BTO NCs in serum indicates that SHGCS is not affected by the turbid environment. (d) Time traced fluorescent intensity of serum, 10 nM Alex488, and a mixture. (e) Normalized autocorrelation curve of serum as well as Alex488 in serum at different concentration. (f) Averaged diffusion time obtained from (e) indicates that in FCS a turbid media will affect the fluorophore dynamics.
-5(c). The time-traced SHG intensity from BTO NCs in Fig. 5(a) shows that a signal with very high SNR can be recorded against the serum background. It should be noted under 880 nm ultrafast laser illumination, that high fluorescence from serum can be recorded in the wavelength range 550-700 nm. However, the use of the emission filter located at half the wavelength of 880 nm will prevent interference from media fluorescence and enable the acquisition of SHG signals for estimation of the diffusion time of BTO NCs in serum at different concentrations (Fig. 5(b), (c)). As a control for SHGCS, the dynamics of Alexa 488 in serum was investigated at different concentration by two photon FCS (Fig. 5(d)-5(f)). Under the same laser irradiation, serum can provide similar fluorescent intensity as a 10 nM Alexa 488 (Fig. 5(d)) in the wavelength range 500 nm-540 nm. From autocorrelation, the diffusion time for autofluorescing serum molecules was 0.3243 ± 0.05ms. The diffusion time of pure Alexa 488 in water was about 0.046 ± 0.002ms, however when investigating the dynamics of Alexa 488 in serum, the viscosity and background noise of serum molecules will dominate the correlation curve when the concentration of Alexa 488 is decreased (Fig. 5(e), 5(f)). Due to the narrow emission band of SHG signals and minimum fluorescence background in the emission band, SHGCS can provide unique selectivity to monitor SHG material/molecules in a turbid media.

Another key aspect of the experiment is the observation that SHGCS does not give rise to triplet states, thus the conventional triplet state dynamics observed in fluorescence correlation, does not exist. In our experiment, rhodamine 123 at 5 nM concentration was used as a control to compare FCS and SHGCS results. In the derivation of FCS theory, there is an assumption that fluorescence signals from molecules diffusing through the laser focal volume is unaltered. Although average fluorescence information is obtained from these diffusing molecules for FCS, when single molecule experiments are performed, photobleaching and triplet state transitions of the diffusers will affect the autocorrelation curve. When analyzing the fluorescence dynamics of diffusing molecules through a confocal limited spot, the emitted fluorescent photons are from radiative electron transitions originating from singlet excited state S1 to ground state S0, shown in Fig. 6(a)
Fig. 6 (a). Energy transition schematic of fluorescence (left) showing triplet state and SHG (right). (b). Time correlated single photon counting of fluorescence from rhodamine 123 (black) and SHG signals from BTO NCs (red). (c). Normalized FCS (black) of rhodamine 123 in water showing a triplet state effect under the 465 nm laser irradiation, and SHGCS (red) curve of BTO NCs in water showing the diffusion dynamics.
. For most fluorophores, a triplet excited state T1 exists so that some electrons on S1 can easily transit to T1, while the transition between T1 and S0 is quantumly forbidden, thus longer time is needed for electrons at T1 to relax back to S0. Usually the time needed for transition between S1 and S0 is a few nanoseconds (Fig. 6(b)), usually referred to as the fluorescence lifetime. A single exponent decay fitting of the time-correlated single photon counting (TCSPC) data indicates that the lifetime of rhodamine 123 is 4.04 ns. While time in the microseconds scale is required for the T1-S0 transition, during which the fluorophore remains in the ‘dark’ state, and in the correlation curve a triplet dynamics effect appears, as shown in Fig. 6(c). However, this ‘triplet dynamics’ does not exist in the SHGCS due to the transient SHG emission, as shown in Fig. 6(a) and 6(b). Due to the coherent and phase-matching properties, lattice relaxation is not necessary (intra-state transition kintra in Fig. 6(a)) to compensate for the momentum mismatching during the inter-state transition k21, therefore no time is needed for electrons to relax back to the ground state. Thus no lifetime is observed expect for the instrument response time (Fig. 6(b)). Compared to FCS, SHGCS can provide a better explanation of diffusive behavior of nanoparticles in solutions under turbid conditions or when significant autofluorescence exists, because SHG signals are stable and intense, hence accurate estimation of concentration and diffusion times of the target species is possible.

Our experiments indicate that SHG using BTO NCs enabling a SHGCS technique offers exciting possibilities in turbid deep tissue and in vivo imaging and detection because of its strong signal in addition to the excitation wavelength which is in the range between 700 nm and 1000 nm. Therefore SHGCS could be an excellent tool for detecting proteins in ultra-low concentrations and even single particle detection. Besides, when functionalized with relevant biomolecules, for example, antibody targeting specific proteins in blood or specific cell surface markers, the SHG BTO probes can potentially be used to study targeting of specific cells or delivery of drugs in in vivo models.

5. Conclusion

In summary, we propose a second order harmonic generation correlation spectroscopy for monitoring subpicomolar concentrations. Due to the coherent property of SHG, the signal generation efficiency is high and so is the intensity. In our experiments, BTO nanocrystals were used as signaling probes and its diffusion characteristics in water was studied from the innate SHG signal. Theoretical fitting of SHG signals indicates that the characteristic diffusion time is about 6.43 ± 0.68 ms and the lowest detected concentration is about 0.81 ± 0.04 pM, defining the detection limit of SHGCS. Our study also demonstrated that SHG signals with hign SNR can be collected and its dynamics monitored even in a turbid media. Materials such as BTO nanocrystals, with second order nonlinearity can serve as ideal candidates for single molecule detection due to its excellent signal generation efficiency.

Acknowledgments

This work was supported in part by the National Science Foundation (Grant no. 0945771 and 0754740), the CTSI (IUPUI-Purdue) grant, and the Purdue Center for Cancer Research Innovative grant.

References and links

1.

A. V. Orden and J. Jung, “Fluorescence correlation spectroscopy for probing the kinetics and mechanics of DNA hairbin formation,” Biopolymers 89(1), 1–16 (2008). [CrossRef]

2.

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002). [CrossRef]

3.

W. Al-Soufi, B. Reija, M. Novo, S. Felekyan, R. Kühnemuth, and C. A. M. Seidel, “Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: inclusion complexes of pyronines with cyclodextrin,” J. Am. Chem. Soc. 127(24), 8775–8784 (2005). [CrossRef] [PubMed]

4.

P. Schwille and E. Haustein, “Fluorescence correlation spectroscopy: an introduction to its concepts and applications,” Experimental Biophysics Group, University of Gottingen.

5.

M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two-beam cross-correlation: a method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999). [CrossRef] [PubMed]

6.

K. M. Berland, P. T. So, and E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68(2), 694–701 (1995). [CrossRef] [PubMed]

7.

P. Schwille, E. L. Elson, and R. Rigler, eds., Fluorescence correlation spectroscopy. Theory and applications (Springer, 2001).

8.

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. 36(1), 151–169 (2007). [CrossRef] [PubMed]

9.

L. Varghese, R. Sinha, and J. Irudayaraj, “Single molecule kinetic investigations of protein association and dissociation using fluorescence cross-correlation spectroscopy,” Anal. Chim. Acta 625, 103–109 (2008). [CrossRef] [PubMed]

10.

J. Chen, S. Nag, P. A. Vidi, and J. Irudayaraj, “Single molecule in vivo analysis of Toll-like receptor 9 and CpG DNA interaction,” PLoS ONE 6(4), e17991 (2011). [CrossRef] [PubMed]

11.

D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system: measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972). [CrossRef]

12.

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974). [CrossRef] [PubMed]

13.

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, “Zero-mode waveguides for single-molecule analysis at high concentrations,” Science 299(5607), 682–686 (2003). [CrossRef] [PubMed]

14.

K. Garai, M. Muralidhar, and S. Maiti, “Fiber-optic fluorescence correlation spectrometer,” Appl. Opt. 45(28), 7538–7542 (2006). [CrossRef] [PubMed]

15.

N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J. 33(3), 435–454 (1981). [CrossRef] [PubMed]

16.

J. Chen and J. Irudayaraj, “Quantitative investigation of compartmentalized dynamics of ErbB2 targeting gold nanorods in live cells by single molecule spectroscopy,” ACS Nano 3(12), 4071–4079 (2009). [CrossRef] [PubMed]

17.

Y. Wang, J. Chen, and J. Irudayaraj, “Nuclear targeting dynamics of gold nanoclusters for enhanced therapy of HER2+ breast cancer,” ACS Nano 5(12), 9718–9725 (2011). [CrossRef] [PubMed]

18.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-soluble quantum dots for multiphoton fluorescence imaging in vivo,” Science 300(5624), 1434–1436 (2003). [CrossRef] [PubMed]

19.

R. W. Boyd, Nonlinear Optics (Academic, 2003).

20.

R. L. Sutherland, D. G. McLean, and S. Kirkpatrick, Handbook of Nonlinear Optics (Dekker, 2003).

21.

A. A. Gulamov, E. A. Ibragimov, V. I. Redkorechev, and T. Usmanov, “Maximum efficiency of generation of the second and third harmonics of neodymium laser radiation,” Sov. J. Quantum Electron. 13(7), 844–845 (1983). [CrossRef]

22.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003). [CrossRef] [PubMed]

23.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003). [CrossRef] [PubMed]

24.

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003). [CrossRef] [PubMed]

25.

J. E. Reeve, H. A. Collins, K. De Mey, M. M. Kohl, K. J. Thorley, O. Paulsen, K. Clays, and H. L. Anderson, “Amphiphilic porphyrins for second harmonic generation imaging,” J. Am. Chem. Soc. 131(8), 2758–2759 (2009). [CrossRef] [PubMed]

26.

J. E. Reeve, H. L. Anderson, and K. Clays, “Dyes for biological second harmonic generation imaging,” Phys. Chem. Chem. Phys. 12(41), 13484–13498 (2010). [CrossRef] [PubMed]

27.

C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, “Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes,” Biomaterials 31(8), 2272–2277 (2010). [CrossRef] [PubMed]

28.

C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, “Second harmonic generation from nanocrystals under linearly and circularly polarized excitations,” Opt. Express 18(11), 11917–11932 (2010). [CrossRef] [PubMed]

29.

P. Pantazis, J. Maloney, D. Wu, and S. E. Fraser, “Second harmonic generating (SHG) nanoprobes for in vivo imaging,” Proc. Natl. Acad. Sci. U.S.A. 107(33), 14535–14540 (2010). [CrossRef] [PubMed]

30.

M. Geissbuehler, L. Bonacina, V. Shcheslavskiy, N. L. Bocchio, S. Geissbuehler, M. Leutenegger, I. Märki, J. P. Wolf, and T. Lasser, “Nonlinear correlation spectroscopy (NLCS),” Nano Lett. 12(3), 1668–1672 (2012). [CrossRef] [PubMed]

31.

A. Aimable, N. Jongen, A. Testino, M. Donnet, J. Lemaitre, H. Hofmann, and P. Bowen, “Precipitation of nanosized and nanostructured powders: process intensification using SFTR, applied to BaTiO3, CaCO3 and ZnO,” Chem. Eng. Technol. 34, 344–352 (2011). [CrossRef]

32.

K. Clays and A. Persoons, “Hyper-Rayleigh scattering in solution,” Phys. Rev. Lett. 66(23), 2980–2983 (1991). [CrossRef] [PubMed]

OCIS Codes
(070.4550) Fourier optics and signal processing : Correlators
(160.4330) Materials : Nonlinear optical materials
(190.2620) Nonlinear optics : Harmonic generation and mixing

ToC Category:
Spectroscopy

History
Original Manuscript: July 3, 2012
Revised Manuscript: August 7, 2012
Manuscript Accepted: August 14, 2012
Published: October 31, 2013

Virtual Issues
Vol. 9, Iss. 1 Virtual Journal for Biomedical Optics

Citation
Jing Liu and Joseph Irudayaraj, "Second harmonic generation correlation spectroscopy for single molecule experiments," Opt. Express 21, 27063-27073 (2013)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-22-27063


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References

  1. A. V. Orden and J. Jung, “Fluorescence correlation spectroscopy for probing the kinetics and mechanics of DNA hairbin formation,” Biopolymers89(1), 1–16 (2008). [CrossRef]
  2. O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys.65(2), 251–297 (2002). [CrossRef]
  3. W. Al-Soufi, B. Reija, M. Novo, S. Felekyan, R. Kühnemuth, and C. A. M. Seidel, “Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: inclusion complexes of pyronines with cyclodextrin,” J. Am. Chem. Soc.127(24), 8775–8784 (2005). [CrossRef] [PubMed]
  4. P. Schwille and E. Haustein, “Fluorescence correlation spectroscopy: an introduction to its concepts and applications,” Experimental Biophysics Group, University of Gottingen.
  5. M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two-beam cross-correlation: a method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem.71(3), 609–616 (1999). [CrossRef] [PubMed]
  6. K. M. Berland, P. T. So, and E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J.68(2), 694–701 (1995). [CrossRef] [PubMed]
  7. P. Schwille, E. L. Elson, and R. Rigler, eds., Fluorescence correlation spectroscopy. Theory and applications (Springer, 2001).
  8. E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct.36(1), 151–169 (2007). [CrossRef] [PubMed]
  9. L. Varghese, R. Sinha, and J. Irudayaraj, “Single molecule kinetic investigations of protein association and dissociation using fluorescence cross-correlation spectroscopy,” Anal. Chim. Acta625, 103–109 (2008). [CrossRef] [PubMed]
  10. J. Chen, S. Nag, P. A. Vidi, and J. Irudayaraj, “Single molecule in vivo analysis of Toll-like receptor 9 and CpG DNA interaction,” PLoS ONE6(4), e17991 (2011). [CrossRef] [PubMed]
  11. D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system: measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett.29(11), 705–708 (1972). [CrossRef]
  12. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers13(1), 29–61 (1974). [CrossRef] [PubMed]
  13. M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, “Zero-mode waveguides for single-molecule analysis at high concentrations,” Science299(5607), 682–686 (2003). [CrossRef] [PubMed]
  14. K. Garai, M. Muralidhar, and S. Maiti, “Fiber-optic fluorescence correlation spectrometer,” Appl. Opt.45(28), 7538–7542 (2006). [CrossRef] [PubMed]
  15. N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J.33(3), 435–454 (1981). [CrossRef] [PubMed]
  16. J. Chen and J. Irudayaraj, “Quantitative investigation of compartmentalized dynamics of ErbB2 targeting gold nanorods in live cells by single molecule spectroscopy,” ACS Nano3(12), 4071–4079 (2009). [CrossRef] [PubMed]
  17. Y. Wang, J. Chen, and J. Irudayaraj, “Nuclear targeting dynamics of gold nanoclusters for enhanced therapy of HER2+ breast cancer,” ACS Nano5(12), 9718–9725 (2011). [CrossRef] [PubMed]
  18. D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, “Water-soluble quantum dots for multiphoton fluorescence imaging in vivo,” Science300(5624), 1434–1436 (2003). [CrossRef] [PubMed]
  19. R. W. Boyd, Nonlinear Optics (Academic, 2003).
  20. R. L. Sutherland, D. G. McLean, and S. Kirkpatrick, Handbook of Nonlinear Optics (Dekker, 2003).
  21. A. A. Gulamov, E. A. Ibragimov, V. I. Redkorechev, and T. Usmanov, “Maximum efficiency of generation of the second and third harmonics of neodymium laser radiation,” Sov. J. Quantum Electron.13(7), 844–845 (1983). [CrossRef]
  22. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A.100(12), 7075–7080 (2003). [CrossRef] [PubMed]
  23. E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med.9(6), 796–801 (2003). [CrossRef] [PubMed]
  24. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol.21(11), 1356–1360 (2003). [CrossRef] [PubMed]
  25. J. E. Reeve, H. A. Collins, K. De Mey, M. M. Kohl, K. J. Thorley, O. Paulsen, K. Clays, and H. L. Anderson, “Amphiphilic porphyrins for second harmonic generation imaging,” J. Am. Chem. Soc.131(8), 2758–2759 (2009). [CrossRef] [PubMed]
  26. J. E. Reeve, H. L. Anderson, and K. Clays, “Dyes for biological second harmonic generation imaging,” Phys. Chem. Chem. Phys.12(41), 13484–13498 (2010). [CrossRef] [PubMed]
  27. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, “Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes,” Biomaterials31(8), 2272–2277 (2010). [CrossRef] [PubMed]
  28. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, “Second harmonic generation from nanocrystals under linearly and circularly polarized excitations,” Opt. Express18(11), 11917–11932 (2010). [CrossRef] [PubMed]
  29. P. Pantazis, J. Maloney, D. Wu, and S. E. Fraser, “Second harmonic generating (SHG) nanoprobes for in vivo imaging,” Proc. Natl. Acad. Sci. U.S.A.107(33), 14535–14540 (2010). [CrossRef] [PubMed]
  30. M. Geissbuehler, L. Bonacina, V. Shcheslavskiy, N. L. Bocchio, S. Geissbuehler, M. Leutenegger, I. Märki, J. P. Wolf, and T. Lasser, “Nonlinear correlation spectroscopy (NLCS),” Nano Lett.12(3), 1668–1672 (2012). [CrossRef] [PubMed]
  31. A. Aimable, N. Jongen, A. Testino, M. Donnet, J. Lemaitre, H. Hofmann, and P. Bowen, “Precipitation of nanosized and nanostructured powders: process intensification using SFTR, applied to BaTiO3, CaCO3 and ZnO,” Chem. Eng. Technol.34, 344–352 (2011). [CrossRef]
  32. K. Clays and A. Persoons, “Hyper-Rayleigh scattering in solution,” Phys. Rev. Lett.66(23), 2980–2983 (1991). [CrossRef] [PubMed]

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