Nano-engineering of materials for nonlinear optical

Invited Paper

Nano-engineering of materials for nonlinear optical imaging Koen Claysa,c, Jos Vanderleydenb,c, Harry L. Andersond Department of Chemistry, University of Leuven, Celestijnenlaan 200D, BE-3001 Leuven, Belgium b Centre of Microbial and Plant Genetics, University of Leuven, Kasteelpark Arenberg 20, BE-3001 Leuven, Belgium c Institute for Nanoscale Physics and Chemistry (INPAC), BE-3001 Heverlee, Belgium d Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, OX1 3TA, UK

a

ABSTRACT We discuss the use of genetically modified fluorescent proteins (FPs) and a specially engineered chromophore for nonlinear optical imaging. While it is clear that FPs can be used for two-photon fluorescence (TPF) microscopy, we show that they also exhibit a large second-order nonlinear optical response, useful for second-harmonic generation (SHG) imaging. The relation between the linear and nonlinear optical properties in a small series of FPs will be discussed. We also present a new optimized chromophore for combined TPF and SHG microscopy and we show imaging results obtained on this chromophore on a water droplet model system. Keywords: fluorescent proteins, chromophores, second-harmonic generation, two-photon fluorescence, imaging

1. INTRODUCTION Nonlinear optical (NLO) imaging is a new imaging technique that takes advantage of the enhanced spatial resolution offered by the well-known two-photon fluorescence (TPF) detected in reflection, ideally in combination with the enhanced structural information offered by second-harmonic generation (SHG) detected in transmission. TPF can provide information on the location of the target molecules, while SHG can provide additional complementary information on orientation and symmetry. For this dual imaging technique, the optical probe has to combine optimized odd-order (for TPF) and even-order (for SHG) nonlinear optical properties. While all materials exhibit odd-order nonlinear properties, only non-centrosymmetric probes can be used for SHG imaging (SHIM).1 Because of the high peak intensity that is needed to produce appreciable levels of both the second-order NLO SHIM and the third-order NLO TPF, SHIM technically requires the same pulsed near-infrared laser as is needed for TPF. Only a detection path in the forward direction is additionally needed for the coherently generated SHIM signal with separate optical filtering around the second-harmonic.2 Nonetheless the additional structural information that is obtained is highly valuable. While TPF, being a third-order NLO effect, is originating from all structures irrespective of symmetry and, hence, provides information about the location only of the chromophores, the second-harmonic signal, being a secondorder NLO effect, is observed specifically for non-centrosymmetric arrangements of the chromophores.3,4 Several studies have reported on the qualitative use of second-harmonic imaging from Green Fluorescent Protein (GFP).5,6 We have recently reported a quantitative value for the second-order nonlinear optical properties for enhanced Green Fluorescent Protein (EGFP) and for the photoswitchable variant Dronpa.7 Based on these observations, we have evaluated variants of these fluorescent proteins (FPs) to elucidate strategies to obtain other FPs with different linear and third-order nonlinear optical properties (variations in absorption, one-photon emission and two-photon emission spectrum) but in combination with good second-order nonlinear optical properties for combined TPF microscopy and SHIM.

Linear and Nonlinear Optics of Organic Materials IX, edited by Theodore G. Goodson, III, Proc. of SPIE Vol. 7413, 74130J · © 2009 SPIE · CCC code: 0277-786X/09/$18 · doi: 10.1117/12.824517

Proc. of SPIE Vol. 7413 74130J-1

We have also combined our expertise in molecular nonlinear optics of organic chromophores in the field of porphyrin dyes as molecular scaffold8,9 with that in the field of N-alkyl pyridinium acceptors10 to engineer a new amphiphilic porphyrin dye 1 which exhibits a strong SHG signal in lipid membranes. Previous work has shown that related donoracceptor meso-ethynyl porphyrins exhibit strong second-order NLO activity.8,9 We have modified the structure of these dyes by using polar pyridinium electron-acceptors,10 and by omitting the orthogonal meso-aryl substituents, to create a molecule with high affinity for biological membranes. Our SHG dye can be viewed as a hybrid between donor-acceptor porphyrins and commercially available membrane-binding dyes, which can be used to probe potential changes via SHG despite their weak NLO activity. We report on the use of this engineered chromophore (highly polarizable porphyrin scaffold, N,N-dialkyl-amino donor group, N-alkyl pyridinium acceptor group, long alkyl groups for hydrophobic moiety, polar head group for hydrophilic group, resulting in combined amphiphilic property for membrane intercalation) for combined TPF and SHIM.

2. EXPERIMENTAL PART 2.1 Expression and purification for engineering of recombinant fluorescent proteins The fluorescent proteins EGFP, EYFP and DsRed (for active chromophore structure, see Figure 1) were obtained by heterologous overexpression in Escherichia coli and purification.11 The fluorescent proteins were expressed in E. coli Top-10 cells which were cultured in 4 l LB-medium supplemented with ampicillin (100 mg/l). Expression of the proteins was induced by 0.2% arabinose at an optical density of 0.5 at 600 nm. The cells were harvested after 12 hours and a cell lyste was obtained. The fluorescent proteins, carrying an N-terminal His-tag (His = histidine), were purified under native conditions by Ni-affinity chromatography (1 ml HisTrap columns, GE Healthcare) according to the manufacturers recommended protocol and concentrated using a Vivaspin concentrator (cutoff 5000 Da). The final pH was 7.3. The proteins were checked for purity by SDS-PAGE and mass spectrometry. Concentration of the samples was determined by means of absorption / extinction coefficient.

Fig. 1. Models of the isolated EGFP, EYFP, and DsRed chromophores


2.2 General materials and methods for molecular engineering of optimized chromophore. The manipulation of all air and/or water sensitive compounds was carried out using standard high vacuum techniques. Dichloromethane and THF were obtained either by distillation or by passing through a column of activated alumina. Triethylamine was distilled from CaH2 under nitrogen before use and pyrrole was distilled from CaH2 under reduced pressure. All other reagents were used as supplied by commercial agents. Analytical thin layer chromatography (TLC) was carried out on Merck® aluminum backed silica gel 60 GF254 plates and visualization when required was achieved using UV light or I2. Column chromatography was carried out on silica gel 60 GF254 using a positive pressure of nitrogen. Size exclusion chromatography (SEC) was carried out using Bio-Beads S-X1, 200-400 mesh (Bio-Rad). NMR spectra were recorded at ambient probe temperature using either a Brucker DPX400 (400 MHz) or Brucker AVANCE AV400 (400 MHz). UV/Vis spectra were recorded on a Perkin Elmer Lambda 20 UV-Vis and fluorescence spectra were recorded on a FluoroMax-2. Mass spectra were carried out using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-ToF) and ElectroSpray Ionization (ESI). The details of the chemical synthesis of the chromophore 1 have been reported.1 The molecular engineering towards a highly optimized chromophore results in the chromophore shown in Figure 2. Its structure consists of a highly polarizable porphyrin core (blue), an ethynyl-linked electron-donating N,N-dialkylanilino group (green) and an electronaccepting N-alkyl pyridinium group (red). The long alkyl chains impart hydrophobic properties to the left part of the molecule, while the right polar pyridinium group is hydrophilic, resulting in an amphiphilic global molecular structure.

Fig. 2. Molecular structure of the optimized chromophore 1

2.3 Hyper-Rayleigh Scattering Quantitative information on the second-order nonlinear optical behavior of EGFP, EYFP, DsRed and the porphyrin dye 1 was obtained by hyper-Rayleigh scattering (HRS).12 This experiment allowed us to measure the first hyperpolarizability β at selected wavelengths, which is the main factor determining the efficiency of second-harmonic generation in the SHG imaging experiments. The intensity of the SHG image is determined by three parameters: (1) the individual molecular second-order nonlinear optical response, or the first hyperpolarizability, dominated by βzzz, along the molecular z-axis of the chromophore; (2) the relative orientation of the chromophore with respect to the polarization of the incoming laser light; and (3) the overall symmetry of the ensemble of chromophores. The first, microscopic, condition is the most fundamental: At the molecular level, a centrosymmetric dye is not amenable to SHG imaging. The other two dependences of the SHG intensity provide more information than TPF: The orientation of the dipolar chromophore with respect to the polarization of the light can be deduced, and centrosymmetric arrangements of dipolar molecules can be extracted from the difference image with TPF. The relationship between the hyperpolarizability βzzz and the efficiency of SHG can be quantified in terms of the macroscopic second-order nonlinear optical susceptibility along the molecular axis χzzz = Nf βzzz where N is the number density of the chromophores, f is the global local field factor, βzzz is the major hyperpolarizability tensor element and is an orientation factor. From this susceptibility, the nonlinear polarization P induced by the electric field E along the z-axis can be given as Pzzz2ω= χzzz(Ez)². This shows that a large first hyperpolarizability is a crucial factor for a large SHG image intensity.


HRS is an incoherent second-order nonlinear scattering technique. Hence, TPF can contribute to the HRS signal and result in an overestimation of the β for probes specifically designed for combined SHG and TPF microscopy. Therefore, we employed high amplitude-modulation (AM) frequency-resolved HRS, to test whether or not there is a multiphoton fluorescence contribution at the second-harmonic wavelength in the first place, and if so, to obtain accurate, fluorescence-free hyperpolarizability values. Our approach is the Fourier-transform in the frequency-domain of the more intuitive separation between immediate (hyper-Rayleigh) scattering and time-delayed (two-photon) fluorescence in the time domain.12 We use the intrinsic high harmonic content of an ultrashort laser pulse and cross-correlation electronics to extract the amplitude and phase of the HRS response at successively higher AM frequencies. Any fluorescence contribution, χflu, characterized by a finite response time, shows demodulation for high frequencies. This shows up as a reduction of the apparent frequency-dependent hyperpolarizability, with an inflection point at the AM frequency corresponding with the inverse of the fluorescence lifetime, τflu. The high-frequency limit of the apparent β is the accurate, fluorescence-free value. However, if no multiphoton fluorescence contributes to the HRS signal, no demodulation is observed for successively higher AM frequencies, and a simple average of the β values over the different measurement frequencies provides an estimate for the precision of the value. The experimental set-up and data analysis for femtosecond HRS measurements at these wavelengths has been described in detail.12 For all the chromophores used in this study, we assume dipolar symmetry, resulting in the relation ² = 6/35 ².

2.4 TPF and SHG Imaging Imaging was performed using a Leica TCS SP2 (Leica Microsystems) two-photon laser scanning microscope with a Tisapphire laser at 840 nm (Mai Tai Broadband, Spectra-Physics; 100 fs pulse width; 80 MHz repetition rate). The typical laser power was 21 mW on the sample (15% power setting). Images were recorded using a 10x immersion objective with a numerical aperture of 0.3. The isotropic fluorescence and forward propagating SHG signals were collected with external photomultiplier tubes (Leica Microsystems). The fluorescence detector was mounted in the descanned detection channel, that is, the emitted light traveled back through the pinhole following the same path as the incident excitation light, and was subsequently separated using a dichroic (525/50) and a short-pass filter (900 nm). The SHG signal, which radiates primarily in the direction of the incident light, was collected using a forward mounted non-descanned detector with narrow band-pass filters, 300–450 nm, BG3 and BG40 (Thorlabs). Images were acquired in frame scan mode with 8-line averaging and a pixel dwell time of 2.4 μs. The water droplets were prepared using an adaptation of a literature procedure: Palmitoyl oleoyl phosphatidyl choline (POPC, 7.6 mg, 10 μmol) and the dye under investigation (200 nmol) were sonicated in dodecane (1 mL) until a homogeneous solution was achieved. This solution was added to a 1 mm path length cuvette, and KClaq (2 μL of 0.5 M) was injected and the cuvette was shaken to form small droplets.

3. RESULTS AND DICUSSION 3.1 Recombinant fluorescent proteins The linear optical properties of the fluorescent proteins show the gradual batochromic shift from the green (for EGFP) over yellow (for EYFP) to red (for DsRed). The UV/Vis spectral characteristics (wavelength of maximal absorption and wavelength of maximal emission) are given in Table 1. The experimental absorption spectra are shown in Figure 3. Table 1. Linear and nonlinear optical properties of the 3 fluorescent proteins: wavelength of maximal absorption, λmax,abs (nm); wavelength of maximal emission, λmax,em (nm); dynamic first hyperpolarizability at 800 nm, βHRS,800 (10-30 esu); static first hyperpolarizability, βHRS,o (10-30 esu); fluorescence contribution to the apparent dynamic first hyperpolarizability, χflu,800 (10-30 esu); and fluorescence lifetime of the two-photon excited fluorescence at the second-harmonic wavelength, τflu (ns).

protein λmax,abs (nm) λmax,em (nm) βHRS,800 (10-30 esu) βHRS,o (10-30 esu) χflu,800 (10-30 esu) τflu (ns)

EGFP 488 507 107 +/- 17 33 +/- 5 137 +/- 12 1.0 +/- 0.4

EYFP 513 527 37 +/- 5 14 +/- 2 40 +/- 6 1.5 +/- 0.6


DsRed 558 583 81 +/- 8 39 +/- 4 103 +/- 7 1.2 +/- 0.4

Fig. 3. Experimental (solid line) and simulated (dashed line) absorption spectra for EGFP (green lines), EYFP (yellow lines) and DsRed (red lines).

As for the nonlinear optical properties determined by hyper-Rayleigh scattering, all three fluorescent proteins exhibit the very typical demodulation of an apparent, modulation frequency-dependent β value, together with the phase shift between signal and pure scattering (Figure 4). From the simultaneous analysis of phase and amplitude data,12 it has been possible to deduce the nonlinear optical parameters presented in Table 1, together with the linear optical properties.

Fig. 4. The apparent, AM frequency-dependent (or apparent), dynamic first hyperpolarizability βHRS,800 for EGFP (■), EYFP (▲) and DsRed (●), together with the growing phase difference between the total HRS signal and a scattering reference (shown in the inset).


An optical property is always dependent on the wavelength at which it has been determined. Because of electronic resonances between ground and excited electronic states, an enhancement of the optical properties occurs near such resonances. This is well-known for the linear optical properties, where there is a one-photon enhancement of both refractive index and absorption near such a resonance. The same occurs in nonlinear optics, both in the one-photon and in the two-photon regime. An enhancement of the first hyperpolarizability βHRS,800 is observed due to the proximity of the second-harmonic wavelength (400 nm) with the wavelength of maximal absorption. Since the latter is different for the three proteins, the degree of enhancement is different. This is factored out in the intrinsic, or static first hyperpolarizability, βHRS,o, which is calculated based on the two-level model.11 A larger static first hyperpolarizability is expected for a smaller energy difference between HOMO and LUMO for the chromophore, congruent with a longer wavelength of maximal absorption. Hence, the largest static hyperpolarizability for DsRed with the longest wavelength of maximal absorption and emission was expected. Congruently, an intermediate hyperpolarizability value is expected for the EYFP with the intermediate wavelength of absorption and emission. However, the smallest value is observed, even smaller than for our benchmark. This is rationalized in terms of the strategy that was invoked to induce the red-shift of the emission from green to yellow in EYFP. This strategy is not the simple enlargement of the conjugation in the chromophore, since the chromophore itself is identical for EGFP and EYFP. The shift to longer absorption and emission wavelength has been attributed to the ππ stacking between the chromophore phenolic ring of Tyr66 and the phenolic ring of Tyr203 (Tyr = tyrosine). While this induces a larger effective polarizable system, a sufficient condition for longer wavelength absorption and emission, it also introduces an inversion centre for part of the effective chromophore, namely in between the two phenolic moieties. Since for second-order nonlinear effects, centrosymmetric structures do not contribute, this results in an effectively shorter chromophore as far as any second-order nonlinear effects are considered. This hypothesis is shown in Figure 5.

Fig. 5. Presentation of the chromophore of EYFP situated in the protein barrel structure shown as a grey cartoon (PDB-code 1YFP). The chromophore and tyrosine 203 are shown as yellow sticks and show a clear inversion centre between the two phenolic moieties (indicated by dashed lines). This suggests that the remaining effective chromophore for secondorder nonlinear optics is limited to the small imidazolinone moiety only. The picture was generated using Pymol.11

It is interesting to observe that, while GFP has been used in second-harmonic imaging,5,6 YFP has been reported not to produce a detectable second-harmonic signal.2 Our experimental results confirm that at 800 nm, the second-order nonlinear optical polarizability of EYFP is less than half that of EGFP which would result in a signal that is more than four times weaker. While this is only very qualitative, both the previous report2 and our recent results do show that the yellow emitting chromophore in YFP and EYFP is not optimal for second-harmonic imaging.


3.2 Optimized porphyrin-based chromophore The absorption spectrum for porphyrin 1 (Figure 6) shows the typical B- and Q-bands in the high- and low-energy region of the visible part of the spectrum. These linear optical properties are going to be determinant for the wavelength dependence of the second-order NLO properties.

60000 50000 40000 30000

e

20000 10000 0 350 450 550 650 750 850 wavelength (nm) Fig. 6. Absorption spectrum of chromophore 1

We performed frequency-resolved experiments at 800, 840 and 1300 nm on 1 in chloroform. We did not observe any TPF at the second-harmonic wavelength in any of these experiments, as evinced by the constant value for the hyperpolarizability for increasing AM frequencies (Figure 7). This is extremely convenient, since it allows for the simultaneous excitation with one laser wavelength of the SHG and TPF image that can then be separately viewed at the appropriate detection wavelength (the second harmonic for SHG and the emission maximum for TPF).

Fig. 7. Apparent hyperpolarizability βHRS as a function of amplitude modulation frequency for HRS measurement at 800 nm (blue solid diamonds); 840 nm (green squares) and 1300 nm (red open diamonds) for porphyrin 1 in CHCl3. These frequency dependences result in βzzz values for 1 of (2300 +/- 150) x 10-30 esu at 800 nm; (5800 +/- 200) x 10-30 esu at 840 nm; and (320 +/- 10) x 10-30 esu at 1300 nm, using the relation ² = 6/35 ².


The results show that 1 has particularly attractive second-order nonlinear optical properties, which trace their origin to the large porphyrin moiety with strong (second and third-order) nonlinear polarizability, and the strong noncentrosymmetry, imposed by the efficient electron donating and withdrawing substituents, resulting in a well-expressed second-order hyperpolarizability. The frequency-dispersion of the first hyperpolarizability of porphyrin-elaborated molecules has been studied and the proximity of the B or Q-band to the second-harmonic wavelength is pivotal for a large response.8,9 Here, the closeness of the B-band maximum explains the large values at 800 and 840 nm, while the bimodal nature of the Q-band with the dip at the second-harmonic wavelength of 650 nm points to two resonance enhancement effects with opposite sign at this wavelength, resulting in an effective partial cancellation. The conclusion of this HRS study is that this porphyrin 1 is extremely well suited for the combined SHG-TPF microscopy study with laser excitation from the generic femtosecond laser system (mode-locked Ti:sapphire laser), since it has a large first hyperpolarizability value at the wavelength of the fluorescence excitation (800 and 840 nm), while the symmetry-sensitive second-order nonlinear SHG image at the harmonic wavelength (400 and 420 nm) is not convolved with the third-order two-photon emission. The hyperpolarizability values at 840 nm of 1 in chloroform (5800 +/- 200 x 10-30 esu) and DMF (8000 +/- 250 x 10-30 esu) show the expected dependence on solvent polarity, suggesting that these probes are ideal candidates as membrane potential sensors for electrophysiological processes. 3.3 TPF and SHG imaging Initial SHG imaging experiments were carried out using water droplets, coated with lipid monolayers, in dodecane. This protocell model provided a stable asymmetric interface for optimizing imaging protocols. Dye 1 was introduced into the oil layer, and rapidly adsorbed onto the surfaces of the water droplets. Confocal laser-scanning microscopy (840 nm; Ti:sapphire laser; 100 fs pulse width), revealed a TPF signal from the isotropic dye dissolved in the oil, and a SHG signal from dye molecules oriented in the surfaces of the water droplets (see Figure 8). As expected, the TPF signal is observed in both the reflection and transmission detection channels, whereas the SHG signal is only detected in the transmission channel (i.e. in the forward direction of the incident light). The SHG signal at exactly 420 nm was collected with a wide aperture condenser and passed through a (450 ± 50) nm bandpass filter to remove the TPF component centered around 800 nm. Under these conditions, the SHG signal from this porphyrin dye can be clearly observed using a dwell time of 4.9 microseconds per pixel which is much less than the time span of an action potential. The SHG signal is not uniform around the circumference of the water droplets: a significant signal is only generated in those regions where the molecular βzzz tensor is aligned with the polarization of the incident laser light.

Fig. 8. SHG (red) and TPF (green) images of a droplet of aqueous potassium chloride (0.5 M) in a solution of palmitoyl oleoyl phosphatidyl choline (POPC, 10 μM) in dodecane containing 1 (scale bar: 75 μm);


4. CONCLUSIONS We have experimentally evaluated the potential of 3 fluorescent proteins for use in second-harmonic imaging. EGFP, EYFP and DsRed form a small subset of the available rainbow of fluorescent polymers, engineered to have red-shifted absorption and emission spectra, i.e. linear optical properties. We have shown that the different engineering strategies for red-shifted linear optical properties have drastically different consequences for second-order nonlinear optical properties. For DsRed, the extended conjugation path leads to both a strong red-shift and a larger first hyperpolarizability. However, the centrosymmetric stacking interaction in YFP, resulting in a weak red-shift to the yellow, is counterproductive for second-order NLO properties, where non-centrosymmetry is a crucial prerequisite. We have also shown that amphiphilic donor-acceptor meso-ethynyl porphyrins with polar pyridinium acceptor head groups and hydrophobic dialkyl-amino donors have strong second order NLO activity and high affinities for biological membranes, making them promising probes for SHG imaging.

5. ACKNOWLEDGEMENTS INPAC is acknowledged for financial support for part of this research.

REFERENCES [1] [2] [3] [4]

[5]

[6] [7]

[8]

[9]

[10]

[11]

[12]

Reeve, J. E., Collins, H. A., De Mey, K., Kohl, M. M., Thorley, K. J., Paulsen, O., Clays, K. and Anderson, H. L., “Amphiphilic Porphyrins for Second Harmonic Generation Imaging,” J. Am. Chem. Soc. 131, 2758-2759 (2009). Nikolenko, V., Nemet, B. and Yuste, R., “A two-photon and second-harmonic microscope,” Methods 30, 3-15 (2003). Moreaux, L., Sandre, O., Blanchard-Desce, M. and Mertus, J., “Membrane imaging by simultaneous secondharmonic generation and two-photon microscopy,” Opt. Lett. 25, 320-322 (2000). Wampler, R. D., Kissick, D. J., Dehen, C. J., Gualtieri, E. J., Grey, J. L., Wang, H. F., Thompson, D. H., Cheng, J. X. and Simpson, G. J., “Selective Detection of Protein Crystals by Second Harmonic Imaging,” J. Am. Chem. Soc. 130, 14076-14077 (2008). Lewis, A., Khatchatouriants, A., Treinin, M., Chen, Z. P., Peleg, G., Friedman, N., Bouevitch, O., Rothman, Z., Loew, L. and Sheres, M., “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. Elegans,” Chem. Phys. 245, 133-144 (1999). Khatchatouriants, A., Lewis, A., Rothman, Z., Loew, L. and Treinin, M., “GFP is a Selective Non-linear Optical Sensor of Electrophysiological Processes in C. Elegans,” Biophys. J. 79, 2345-2352 (2000). Asselberghs, I., Flors, C., Ferrighi, L., Botek, E., Champagne, B., Mizuno, Z., Ando, R., Miyawaki, A., Hofkens, J., Van der Auweraer, M. and Clays, K., “Second-Harmonic Generation in GFP-like Proteins,” J. Am. Chem. Soc. 130, 15713-15719 (2008). Zhang, T. G., Zhao, Y., Asselberghs, I., Persoons, A., Clays, K. and Therien, M. J., “Design, Synthesis, Linear and Nonlinear Optical Properties of Conjugated (Porphynato)zinc(II)-based Donor-Acceptor Chromophores Featuring Nitrothiophenyl and Nitrooligothiophenyl Electron-Accepting Moieties,” J. Am. Chem. Soc. 127, 9710-9720 (2005). Zhang, T. G., Zhao, Y., Song, K., Asselberghs, I., Persoons, A., Clays, K. and Therien, M. J., “Electronic Modulation of Hyperpolarizable (Porphynato)zinc(II) Chromophores Featuring Ethynylphenyl-, Ethynylthiophenyl-, Ethynylthiazolyl-, and Ethynylbenzothiazolyl-Based Electron-Donating and –Accepting Moieties,” Inorg. Chem. 45, 9703-9712 (2006). Coe, B. J., Harris, J. A., Hall, J. J., Brunschwig, B. S., Hung, S. T., Libaers, W., Clays, K., Coles, S. J., Horton, P. N., Light, M. E., Hursthouse, M. B., Garin, J. and Orduna, J., “Syntheses and Quadratic Nonlinear Optical Properties of Salts Containing Benzothiazolium Electron-Acceptor Groups,” Chem. Mater. 18, 5907-5918 (2006). Demeulenaere, E., Asselberghs, I., de Wergifosse, M., Botek, E., Spaepen, S., Champagne, B., Vanderleyden, J. and Clays, K., “Second-order nonlinear optical properties of fluorescent proteins for second-harmonic imaging,” J. Mat. Chem. (2009) DOI:10.1039/b907789h. Wostyn, K., Binnemans, K., Clays, K. and Persoons, A., “Hyper-Rayleigh Scattering in the Fourier domain for higher precision: Correcting for multiphoton fluorescence with demodulation and phase data,” Rev. Sci. Instrum. 72, 3215-3220 (2001).
