TIME RESOLVED AND STEADY STATE EXPERIMENTS

WITH PRODAN AND LAURDAN SOLUTIONS

by

Matthew J. Phillips

A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Chemistry and Biochemistry

Spring 2019

© 2019 Phillips All Rights Reserved

TIME RESOLVED AND STEADY STATE EXPERIMENTS

WITH PRODAN AND LAURDAN SOLUTIONS

by

Matthew J. Phillips

Approved: ______Lars Gundlach, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee

Approved: ______Brian Bahnson, Ph.D. Chair of the Department of Chemistry and Biochemistry

Approved: ______John Pelesko, Ph.D. Interim Dean of the College of Arts and Sciences

Approved: ______Douglas J. Doren, Ph.D. Interim Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS

A huge thank you to Dr. Lars Gundlach, whose mentorship and guidance made this work and my professional development possible.

My colleagues Samantha Doble, Joseph Avenoso, Han Yan, Mercury Li, Meng Jia, who are always willing to share their knowledge and expertise, and make lab awesome.

The support and love of my friends and family

iii TABLE OF CONTENTS

LIST OF TABLES ...... v LIST OF FIGURES ...... vi ABSTRACT ...... vii

Chapter

1 INTRODUCTION ...... 1

2 EXPERIMENTAL ...... 6

2.1 Lamellar Films ...... 6 2.2 Steady State Spectra ...... 6 2.3 Transient Absorption Spectroscopy ...... 7 2.4 Time Correlated Single Photon Counting ...... 9

3 RESULTS AND DISCUSSION ...... 11

4 CONCLUSION ...... 22

REFERENCES ...... 23

iv LIST OF TABLES

Table 1: Fluorescence lifetimes of PRODAN in various solvents ...... 14

v LIST OF FIGURES

Figure 1: Moiety of PRODAN derivatives. For PRODAN, R = CH3. For laurdan, R=C9H19...... 2

Figure 2: Mechanism of intramolecular charge transfer in PRODAN upon excitation...... 3

Figure 3: Diagram of TA measurement set up. The pump pulse, black, crosses with the probe pulse, gray, at the sample, followed by a pinhole to allow only the probe pulse to reach detection. [10] ...... 8

Figure 4: Measurement apparatus used for TA experiments with lamellar films. In the center of the cuvette lies the film on a thin glass medium. The pipette tip water reservoir humidifies the cuvette...... 9

Figure 5: emission spectra of various PRODAN solutions excited with 350 nm light 12

Figure 6: Emission spectra of PRODAN solutions with 410 nm excitation...... 13

Figure 7: Normalized absorbance spectra of PRODAN solutions ...... 14

Figure 8: TA maps of PRODAN in various solvents. A:methanol, B: ethanol, C: dimethyl sulfoxide, D: acetonitrile, E: acetone. The color scale, in units of OD, is common for all graphs in its row. For A and B Sapphire white light generation was used, and for C, D and E Calcium fluoride was used to generate white light...... 16

Figure 9: PRODAN emission maximum over time with sapphire white light generation for ethanol and methanol, and calcium fluoride white light generation for all others...... 18

Figure 10: TA maps of PRODAN loaded lamellar film (A) and a blank film (B) ...... 20

Figure 11: IR region of PRODAN emission spectra, presumably phosphorescence. .. 21

vi ABSTRACT

2-propionyl-6-dimethylamino naphthalene (PRODAN) is used in biomedical research due to the sensitivity of its emission spectrum to changes in the environment.

Typically, the fluorescence intensity at two wavelengths is analyzed to draw conclusions about water content, for example, of the PRODAN’s microenvironment. However, both polarity and hydrogen bonding ability of the environment play roles in PRODAN’s emission behavior. PRODAN was studied in a variety of solvents and mixtures, using steady state spectroscopy and time resolved techniques to bring a new perspective to the discussion of the nature of the excited states of PRODAN and the factors which give rise to them.

vii Chapter 1

INTRODUCTION

Both PRODAN and laurdan (figure 1) were first synthesized by Gregorio

Weber in 1979 for the study of dipolar relaxation1, and have since remained a hot topic in the scientific community. Since, the molecule and many functionalized derivatives have been studied by physical chemists and spectroscopists, and used as a probe by biologists for decades2, 3. PRODAN is a model system for the study of the phenomenon of solvatochromism, a property where a dye changes its color depending upon its surroundings (i.e. the solvent). This makes PRODAN and its derivatives very useful as probes in many biophysical studies. However, we must first understand the mechanism of solvatochromism and why PRODAN displays such a dramatic shift in fluorescence maxima- about 125 nm, or 10 eV difference from cyclohexane to water as the solvent4. Upon excitation, the dipole moment of the molecule increases with charge localizing to the amino and carbonyl residues. This causes reorientation of the solvent dipoles, which lowers the energy of the excited state1. This is the cause of the observed red shift of emission of PRODAN as solvent polarity increases.

1

Figure 1: Moiety of PRODAN derivatives. For PRODAN, R = CH3. For laurdan, R=C9H19

A dye in two situations, first surrounded by hydrocarbons, and second surrounded by water, may or may not enter an identical state upon excitation, and are acted upon by different forces as the solvation sphere re-orients. Different forces are being exerted on these identical dye molecules by different solvents, so distinct emission bands should be observed and follow a trend based on the polarity and H- bonding ability of the solvent. Experimentally this is the case1, 4, and the trend is understood.

For PRODAN, there is at least a locally excited (LE) state and an intramolecular charge transfer (ICT) state2. In polar solvent, PRODAN emits from the ICT state shown in figure 2. The primary goal of this study is to determine the dynamics of PRODAN in polar solvents leading up the ICT state. The current model assumes an initial transition to the locally excited state for all solvents2, as absorption changes very little with the solvent. Upon excitation to the locally excited state,

2 relatively little rearrangement of non-polar solvent occurs, but a polar solvent will rearrange its dipoles considerably to stabilize the excited state and allow a transition to the ICT state, where further rearrangement can occur. The greater the stabilization, the lower energy of the resulting transition and thus the degree to which emission is redshifted. However, there has been, and still is, considerable debate as to the nature of the excited state(s) of PRODAN4, 5. Let us first discuss the nature of the ICT state. The charge transfer occurs between the amino and carbonyl groups of PRODAN on either side of the naphthalene ring structure, which acts as a bridge as shown in figure 2. Charge is transferred from the amine to the carbonyl, resulting in a zwitterionic iminium enolate.

Figure 2: Mechanism of intramolecular charge transfer in PRODAN upon excitation.

Previous literature suggested that a twisting of donor and acceptor CT groups of the molecule was involved in excitation, and that the degree of twisting allowed played a role in the environmental sensitivity of PRODAN6. Experiments with PRODAN derivatized with ring structures to immobilize the donor and acceptor proved to not significantly change the molecules spectroscopic properties5. Thus, it is now understood that the excited state of PRODAN under non-extreme temperatures is

3 planar in polar and non-polar solvents. However it remains unclear whether PRODAN enters the same locally excited state in polar and nonpolar solvents, and if so in polar solvents, how the transition to the ICT state proceeds. The utility of PRODAN, and more commonly laurdan, as a probe is based on the molecules` solvatochromism. The emission of the dye is clearly dependant upon its surroundings, but not enough is understood for truly quantitative methods to be developed based on this property. Still, qualitative or relative information about the CT dyes` local environment has proved extremely useful. Laurdan has been widely used in cell biology to determine the nature of lipid bilayer microenvironment7. The lauryl tail residue associates with the lipid residues of the bilayer or vesicles, making laurdan the dye of choice for this application. Specifically, this method can be used to determine whether the lipids are in an ordered or disordered (sometimes referred to as liquid crystal or gel) phase. Typically the fluorescence of laurdan in membranes in measured at 440 and 490 nm. These two values are used to calculate a generalized polarization function (GP). This technique is of great importance to our understanding of membrane dynamics: laurdan GP was used to support the lipid raft hypothesis7, which is essential to our understanding of cellular processes. Laurdan GP has been involved in medicinal advancement as well, being used to study factors influencing exosome release by malignant tumor cells8, as well as being a dye often used in two photon fluorescence microscopy7. Because infrared light can penetrate tissue, the phenomenon of two photon absorbance is utilized to image tissue to a depth of up to a few mm. Consider laurdan absorbing a photon of wavelength 350 nm. By absorbing two photons of wavelength 700 nm, the same excited state can be achieved.

4 Laurdan associates within the hydrophobic zone of the phospholipid structures7, so changes in the makeup of the polar head have little to no effect on laurdan emission. The GP value is therefore determined by molecules within the lipid vesicles or bilayers, typically water. The most common use of this method is to use the GP value as a measure of water permeation into the lipid bilayer or vesicle, which is in turn a measure of how ordered the membrane is: a tightly packed highly ordered membrane will not be as permeable to water as less ordered and tightly packed membranes. These biomedical applications will undoubtedly benefit from a more sophisticated understanding of the solvatochromism dynamics of PRODAN moiety fluorescent probes. Through spectroscopic and computational methods, ultrafast PRODAN dynamics have been elucidated. Not only does this further our understanding of quantum processes and light matter interaction, but also has potential to have a positive impact on human health.

5 Chapter 2

EXPERIMENTAL

2.1 Lamellar Films PRODAN and laurdan were obtained from Invitrogen, and all solutions prepared for TA measurements were saturated. Spectroscopic grade solvents obtained from Fisher Chemicals were dried over sodium sulfate before use. Lamellar films were prepared on glass microscope cover slides according to “rock and roll method9,” however an upside down beaker on the bench top was used to dry the film in place of a glove box. Dipalmitoylphosphatidylcholine (DOPC) obtained from Fisher

Chemicals was the lipid used.

2.2 Steady State Spectra 1.25 micromolar solutions of PRODAN were prepared from a stock solution of PRODAN in acetone: After allocation, the acetone was allowed to dry off in open air (about 30 seconds, aliquot was 50 microliters) leaving PRODAN in the vial, to which the desired solvent was added and shaken well. This was done immediately before measurement for each solution. A scanning fluorometer, the Fluoromax-4 by Horiba (Edison, NJ), was used. Excitation was set to 350 and 410 nm with excitation entry slit width of 5 nm. Emission spectra was taken from 10 nm red of the excitation to 1000 nm. Exit slit width was 1 nm, with a data point taken every .5 nm. To ensure the system was working properly and consistently, a water blank was measured before and

6 after experimental measurements, and the emission peak at 397 nm was observed with consistent wavelength and intensity.

2.3 Transient Absorption Spectroscopy Transient absorption (TA) spectroscopy was performed with femtosecond time resolution. TA spectroscopy is a time resolved method involving pulsed laser excitation, called the "pump," and a pulse of broad-spectrum white light, called the "probe." The beams are crossed as shown in figure 10 such that the sample lie at their intersection and only the probe may pass through to detection. A TA measurement is an absorbance spectrum of a molecule’s excited state, that is, the transmitted probe after passing through the sample, which has been excited by the pump. By sampling a different pump probe delay time each pulse, this method is able to produce time resolved spectra. The TA signal contains 3 possible contributions10. The aforementioned absorbance by the excited state is the contribution typically of interest. Also present, however, is ground state bleach and stimulated emission. Both are a result of exciting the sample with the pump pulse. In ground state bleach, the signal of the unexcited sample is subtracted from the signal upon excitation. This would lead to a negative signal where the ground state absorbs, since the pump pulse excited some fraction of molecules. Once the probe arrives, there are fewer ground state molecules available to absorb, hence a negative signal upon subtraction of the ground state spectra. The stimulated emission signal is the emission of photons by excited sample. The addition of these photons to the probe pulse results in a negative signal in the absorption spectrum. Also a possibility is absorption by any states that the excited molecule decays to (other than the ground state, because this would only decrease the

7 negative signal of ground state bleach.). As an absorption process, this would result in a positive signal.

Figure 3: Diagram of TA measurement set up. The pump pulse, black, crosses with the probe pulse, gray, at the sample, followed by a pinhole to allow only the probe pulse to reach detection. [10]

Solutions for TA experiments were prepared and measured as follows: Solid dye was added to spectroscopic grade solvent until saturated. After vigorous mixing, an aliquot was transferred to a 1mm quartz cuvette, having been filtered if necessary.

The TA setup used begins with a Coherent Mantis Oscillator, Ti:Sapphire laser, and Coherent Legend Elite regenerative amplifier to generates 10 kHz pulsed light. SHG is achieved by BBO, and either a sapphire or calcium fluoride crystal was used for white light generation. The pump pulse is centered on 440 nm with a FWHM of less than 50 nm.

8 Lamellar films were prepared as previously stated, and placed in the cuvette apparatus shown in figure 4. A section of foam fabric sliced lengthwise down the center is placed in a glass 20x50x100 mm cuvette to hold the film media and the water reservoir. The water reservoir humidifies the air and allows hydration of the lamella to more closely model biological membranes. This reservoir was a 1000 microliter pipette tip with the tip melted shut. The rubber band serves two purposes. First, it ensures the cuvette is tightly sealed and sufficiently humidified. Second, it acts as a buffer to prevent damage to the cuvette caused by over tightening of the clamp.

Figure 4: Measurement apparatus used for TA experiments with lamellar films. In the center of the cuvette lies the film on a thin glass medium. The pipette tip water reservoir humidifies the cuvette.

2.4 Time Correlated Single Photon Counting Time correlated single photon counting (TCSPC) experiments were performed with an MPD SPD-050-CTC APD photon counting module. 400 nm light first passes

9 through a Thor labs colored glass low pass filter to remove the second harmonic of the excitation. A second colored glass high pass filter allows passing of emission, but not scattered excitation light, to detection. An oscilloscope receives a signal from the 10 kHz pulsed laser and the APD. The laser signal sets time zero, and the time until the signal from the APD is received is recorded. Each pulse cycle, no more than a single photon is counted. The result of many thousand cycles amounts to a decay curve of the excited state of the molecule of interest. A very low concentration is required for this method to work properly, as a concentrated sample may show an artificially short lifetime due to saturation at early times, i.e. the probability of an early time photon not being counted is so low that nearly no later time photons are counted. Solutions of PRODAN were prepared from a stock solution as done for steady state measurements. Immediately after preparation, the solutions were measured until ~40,000 counts were reached. The lifetime was determined by fitting the data to an exponential equation. Due to the nanosecond timescale of the lifetimes, the instrument response, which was measured to be 30-40 ps, is of a magnitude such that it does not need to be considered in calculation of the fluorescence lifetimes.

10 Chapter 3

RESULTS AND DISCUSSION

The emission spectra of PRODAN in various solvents are shown in figure 5.

Note the dramatic redshift, which increases with solvent polarity. Quantum yield comparison in different solvents cannot be considered quantitative, however, a clear trend is observed: the solutions with the highest quantum yield are near the center of the wavelength maxima distribution. Presumably, the same property determines both the quantum yield and the emission maxima of PRODAN solutions: the degree to which the solvent stabilizes the excited state. This could initially make the transition more accessible or kinetically favorable and thus increase the quantum yield. However, if at a point, the solvent stabilization is enough to allow transition to lower energy states non-radiatively, fluorescence will be quenched, lowering the quantum yield. Interestingly, a similar trend is observed with PRODAN derivatives dissolved in mixtures of toluene and alcohols5. As concentration of alcohol increases, the emission red shifts and quantum yield initially increases, to a point. As alcohol concentration increases further, and the emission redshifts past about 480-490 nm, quantum yield decreases. Clearly some relationship exists between emission maxima and quantum yield for PRODAN, and further study of this may yield interesting results. It must be addressed that quenching could also be caused by increased formation of PRODAN dimer, which for cyclohexane and water in particular could be driven by poor solubility. The concentration was sufficiently low to avoid this issue,

11 for example, the solubility of PRODAN in water11 is reported as roughly triple the concentration used.

Figure 5: emission spectra of various PRODAN solutions excited with 350 nm light

Figure 6 shows emission of PRODAN with excitation at 410 nm. Acetone and acetonitrile solutions show quenching by a factor of 10 where methanol and water are quenched by a factor of 2-3. Emission maxima is unaffected by this change in excitation.

12

Figure 6: Emission spectra of PRODAN solutions with 410 nm excitation.

This is consistent with an absorbance experiment performed with solutions of PRODAN in mixtures of acetone and methanol, shown in figure 7. The shoulder broadening on the red side of the absorbance peak with increasing methanol concentration is an observation of solvent stabilization allowing absorption of lower energy light.

13

Figure 7: Normalized absorbance spectra of PRODAN solutions

Fluorescence lifetimes (table 1) have been determined through TCSPC. More trials and solvent conditions would allow for a more rigorous and quantitative comparison, but a clear qualitative trend is apparent in these limited data: in non-polar solvents PRODAN fluoresces longer than in polar solvents. Therefore, it can be said that the emissive transition from the ICT state to the ground state is more efficient than that of the LE state to the ground state.

Table 1: Fluorescence lifetimes of PRODAN in various solvents

Solvent Acetone Methanol Toluene Dimethyl Sulfoxide picoseconds 5870 3570 7820 2810

TA results (figures 8, 9 and 10) bring a plethora of information about the dynamics of PRODAN and laurdan to the discussion. Note the positive signal, most

14 apparent for dye in protic solvents, located in the 650-675 nm region. This signal begins quite broad at time zero, and decays symmetrically to be much narrower over the course of a few hundred picoseconds. This is an absorption signal presumably by the excited state, but also possibly by dimerized dye. Interestingly, this signal is not observed with non-polar, aprotic solvents, although a similar broad positive signal is observed for PRODAN in cyclohexane and toluene at 500-650 nm. One might conclude from this that hydrogen bond stabilization of the excited state is prerequisite for the absorption of near IR photons, and that the excited state in non-polar solvents absorbs more energetic photons to achieve a transition to the same or a different state. It also appears that in methanol, the signal decays more quickly than in ethanol. Perhaps because methanol interacts more strongly due to being the more polar and less bulky of the two alcohols12.

15

Figure 8: TA maps of PRODAN in various solvents. A: methanol, B: ethanol, C: dimethyl sulfoxide, D: acetonitrile, E: acetone. The color scale, in units of OD, is common for all graphs in its row. For A and B Sapphire white light generation was used, and for C, D and E Calcium fluoride was used to generate white light.

Next, let us discuss the broad negative signal observed for polar solvents at approximately 500 nm. This signal is stimulated emission: the negative signal is at the same wavelength at which fluorescence is observed in steady state measurements.

Notice the absence of this signal for solutions of PRODAN in nonpolar solvents, which fluoresce further blue. The fluorescence signal for these solutions is below the detection range of the instrument. For all solvents, the signal is bluer at early times, and shifts to the expected wavelength over the course of a few to a few hundred picoseconds. This is a direct observation of excited state stabilization by solvent rearrangement. Initially upon excitation, the dye emits a bluer photon, in the 440-460

16 nm range for all tested solvents. But as time passes, the solvatosphere rearranges and lowers the energy of the excited state, resulting in emission of lower energy photons. PRODAN in acetone has a wavelength shift of about 15 nm that occurs over the course of 2 ps, from 440 to 455. In methanol and ethanol, the emission maxima begins at the same wavelength, but shifts much further over the course of hundreds of picoseconds, while in ethanol emission begins at the same wavelength as in methanol, but shifts about 10 nm less and takes nearly 150 ps to rearrange. Acetone and methanol have identical polarity12, so the difference in emission over time is due to hydrogen bonding alone. Clearly, the process of solvation sphere rearrangement is taking longer and having a greater effect for protic solvents. Some rearrangement is observed of mildly polar aprotic solvents as expected, as these solvents do indeed red shift the emission of PRODAN in steady state measurements.

The rearrangement of these aprotic solvents occurs more quickly than the more stabilizing rearrangement of protic solvents. Ethanol's shift relative to methanol is consistent with the model: ethanol is less protic, and should stabilize PRODAN to a lesser degree than methanol, as well as being bulkier and as a result taking more time to rearrange. Acetone, acetonitrile, and dimethyl sulfoxide are all very similar in shape, but with all time points red shifted according to polarity. It appears as though aprotic polar solvents act on PRODAN in a polarity dependent manner in the first few dozen or so femtoseconds upon excitation (resolution is too poor at these extremely early times to meaningfully interpret), and after this rapid initial action these solvents act upon PRODAN nearly identically, all shifting emission maximum about 18 nm.

17

Figure 9: PRODAN emission maximum over time with sapphire white light generation for ethanol and methanol, and calcium fluoride white light generation for all others.

Clearly both polarity and hydrogen bonding ability play a role in PRODAN emission dynamics, and this time resolved emission data suggest that hydrogen bonding ability acts on PRODAN in a long timescale solvation sphere rearrangement, while aprotic polarity acts much more quickly. It will require further study to determine whether the polarity of aprotic solvents causes red shift in the first few dozen femtoseconds, or allows emission to begin at red shifted wavelength. The current model of solvatochromism would predict that at time zero, all solutions emit at

18 the same wavelength, and upon rearrangement emission will shift to the wavelength observed in steady state measurements. This data supports this model for some solvents, but for the polar aprotic solvents the data only somewhat supports the model. TA experiments with better early time resolution will be paramount to the support or modification of the model. Also intriguing is a negative signal at about 700 nm observed for PRODAN in cyclohexane, toluene, and laurdan in DOPC lamellar films. This signal is very long lived: its presence before time zero of each cycle is evidence that it lasts at least 100 microseconds. This time scale suggests phosphorescence as the cause of the signal. However, one might reasonably think SHG is responsible for this signal, as it is about double the excitation wavelength of 340 nm. This cannot be the case, as with all variables constant the signal is only observed in non-polar solvents, and lamellar films. Furthermore, a blank film prepared identically to dye sensitized film but without laurdan, did not show this 700 nm signal.

19

Figure 10: TA maps of PRODAN loaded lamellar film (A) and a blank film (B)

Possibly related to the 700 nm phosphorescence is a novel feature observed in steady state spectra. In the 750- 1000 nm region a second set of peaks is observed for PRODAN in all solvents except water (figure 11). The difference between maxima for these low energy peaks is roughly equal to that of the high energy peaks, but the relative intensities are quite different. For example, toluene gives the most intense signal at the low energy region, while both acetone and acetonitrile fluoresce with a higher quantum yield in the high energy region. Note that the intensity of the peaks in this low energy region are ~ 20 times less intense than their blue counterparts. These peaks are 20-30 nm blue of where a SHG of the high energy signal should be observed, so it is reasonable to conclude that this signal is not a SHG artifact. It is rather curious that this easily observed phenomenon has never been reported in the

20 literature, as PRODAN phosphorescence could reveal triplet states and possibly interesting molecular dynamics.

Figure 11: IR region of PRODAN emission spectra, presumably phosphorescence.

Also interesting is the broad positive signal observed at 500-600 nm for PRODAN in toluene, and to a much lesser degree in cyclohexane. This absorption is only observed in these non-polar solvents, and the much stronger signal present in toluene suggests aromatic pi interactions are likely at play. This signal could correspond to the same transition as the more red absorption signal observed in protic solvents.

21 Chapter 4

CONCLUSION

In polar solvents, the partial charges present in the environment stabilizes the

ICT excited state sufficiently to make it accessible and a relatively large stokes shift is observed. In non-polar solvents, however, this is not the case, and PRODAN emits from a locally excited state. The stokes shift in non-polar solvents is considerably less than in polar, especially protic, solvents. Time resolved and steady state experiments have brought a great deal of new information to the PRODAN discussion. TA data shows emission redshift with femtosecond resolution, and offers further support of the locally excited and ICT excited state, as well as exciting new phenomena such as phosphorescence and excited state absorbance.

2 2 REFERENCES

1. G. Weber, and F. J. Farris. Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene. Biochemistry, 18: 3075–3078, 1979

2. Tiziana Parasassi, E. K. Krasnowska, L. Bagatolli, and E. Gratton. Laurdan and Prodan as Polarity-Sensitive Fluorescent Membrane Probes. Journal of Fluorescence. 8:367-373, 1998.

3. A. Marini, A. Munoz-Losa, A. Biancardi, and B. Mennucci. What is Solvatochromism?. J. Phys. Chem. B. 114: 17128–17135, 2010.

4. V.Y. Artukhov, O. M. Zharkova, J. P. Morozova. Features of absorption and fluorescence spectra of prodan. Spectrochimica Acta Part A ,68: 36–42, 2007.

5. M. Daneri, and C. J. Abelt. A higher-order preferential solvation model for the fluorescence of two PRODAN derivatives in toluene-alcohol mixtures. Journal of Photochemistry and Photobiology A: Chemistry, 310: 106–112., 2015.

6. P. Ilich, and F. G. Prendergast. Singlet Adiabatic States of Solvated PRODAN: A Semiempirical Molecular Orbital Study. J. Phys. Chem., 93: 4441-4447, 1989.

7. S. A. Sanchez, M.A.Tricerri, G. Gunther, and E. Gratton. Laurdan Generalized Polarization: from cuvette to microscope. Modern Research and Educational Topics in Microscopy, 1007-1014, 2007.

8. I. Parolini, C. Federici, C. Raggi, L. Lugini, S. Palleschi, A. De Milito, C. Coscia, E. Iessi, M. Logozzi, A. Molinari, M. Colone, M. Tatti, M. Sargiacomo, and S. Fais. Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells. JOURNAL OF BIOLOGICAL CHEMISTRY. 284: 34211–34222, 2009.

9. S. A. Tristram-Nagle. Preparation of Oriented, Fully Hydrated Lipid Samples for Structure Determination Using X-Ray Scattering. Methods Mol Biol, 400: 63–75, 2007.

10. R. Berera, R. van Grondelle, and J. T. M. Kennis. Ultrafast transient absorption spectroscopy: principles and application to photosynthetic systems. Photosynth Res. 101: 105–118, 2009.

23 11. H. M. Kim, H. J. Choo, S. Y. Jung, Y. G. Ko, W. H. Park, S.J. Jeon, C. H. Kim, T. Joo, and B. R. Cho. Supporting Information for: A Two-Photon Fluorescent Probe for Lipid Raft Imaging: C-Laurdan. Chembiochem, 8:553-9, 2007.

12. L. R. Snyder, J. J. Kirkland and J. L. Glajch. Practical HPLC Method Development, Second Edition. John Wiley & Sons, Inc. 722-723, 1997.

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