Article pubs.acs.org/Langmuir
Solvation Dynamics under a Microscope: Single Giant Lipid Vesicle Supratik Sen Mojumdar, Shirsendu Ghosh, Tridib Mondal, and Kankan Bhattacharyya* Physical Chemistry Department, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India ABSTRACT: Picosecond spectroscopy under a confocal microscope is employed to study solvation dynamics of coumarin 153 (C153) inside a single giant lipid vesicle (1,2dilauroyl-sn-glycero-3-phosphocholine, DLPC) of diameter 20 μm. Fluorescence correlation spectroscopy (FCS) indicates that the diffusion coefficient (Dt) of the probe (coumarin153, C153) in the immobilized vesicle displays a wide distribution from ∼3 to 21 μm2 s−1. The distribution of Dt suggests that the microenvironment of the probe (C153) is highly heterogeneous and the local friction is different for probe molecules in different regions. The values of Dt is significantly smaller than that for the same dye in bulk water (550 μm2 s−1). This suggests that the probe is located in the interface or membrane region rather than in the water pool of the vesicle. The solvation time of C153 in different regions of the lipid vesicle varies between 750 to 1200 ps. This result clearly shows that a confocal microscope is able to resolve the spatial heterogeneity in local friction (i.e., Dt) and solvation dynamics within a lipid vesicle.
1. INTRODUCTION Recent advances in single molecule spectroscopy have demonstrated that properties of an individual molecule are different from those of the ensemble average. In bulk, the chemical properties are averaged over the Avogadro number of molecules. The variation in individual systems in an ensemble is revolutionizing our understanding of chemistry. It is observed that kinetics of enzyme-catalyzed reactions1,2 and electrontransfer processes in proteins3−5 are essentially heterogeneous and varies from one protein to another. Similar differences or fluctuations are observed in single molecule fluorescence resonance energy transfer (smFRET) between an enzyme and a substrate labeled with fluorophores and during protein folding−unfolding dynamics.6−8 We have previously shown that the diffusion coefficient in lipid9 and catanionic10 vesicles varies from one vesicle to another and attributed this to shape9 and size10 fluctuations. We have further shown that even in macroscopically homogeneous ionic liquid, diffusion coefficients exhibit a large distribution which may arise from structural heterogeneity.11 In a lipid vesicle, a highly hydrophobic membrane bilayer surrounds a very polar water pool. Thus, a lipid vesicle is a relatively simple model of a biological cell. Lipids are studied using fluorescence spectroscopy,12−24 vibrational spectroscopy coupled with quantum chemical calculations,25−27 computer simulations,28−35 NMR,36−39 X-ray and neutron scattering.40,41 There is a longstanding interest to understand the properties of the lipid vesicle (hence, in a cell) as a function of distance from the membrane.12−22 Previously, many groups studied solvation dynamics in a lipid vesicle in bulk water.23,24,42 In the case of a lipid vesicle in bulk water, one gets an average of many vesicles of various sizes, freely moving in bulk water. © 2012 American Chemical Society
In the present work, we attempt to study solvation dynamics within different regions of a single giant lipid vesicle using single molecule spectroscopy. For this, we focused on different positions of an individual lipid vesicle of 20 μm size immobilized on a microscopic slide using a confocal microscope. We will demonstrate that diffusion and solvation dynamics within a giant lipid vesicle are not the same everywhere and depend on the position and microenvironment around the probe. This observation is completely different from that observed in bulk (ensemble average). To the best of our knowledge, solvation dynamics at the single vesicle level has not been studied previously using a confocal microscope.
2. EXPERIMENTAL SECTION 2.1. Materials. Laser-grade dye, coumarin 153 (C153, Scheme 1A), was purchased from Exciton Inc. and used without further purification. The lipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC, Scheme 1B) was purchased from Avanti Polar Lipids and used without further purification. The phase transition temperature of DLPC is −1 °C. These lipids carry zero net charge at pH 7.4. The lipid vesicles were prepared following a reported protocol.43 Briefly, giant multilamellar vesicles were prepared by depositing a thin film of the phospholipid (typically 0.25 mL of 8 mg/mL of DLPC in chloroform solution) through the evaporation method. The completely dried lipid film was then prehydrated with water-saturated N2 for 15−20 min. The film was subsequently hydrated in 5 mL of 0.1 M sucrose solution, which had been N2 purged, and then sealed under argon and incubated at 37 °C overnight. As reported by Akashi et al.,43 the vesicles prepared following this procedure consist of giant multilamellar vesicles (≥20 μm) with a few giant unilamellar vesicles (≥20 μm) as well as a few small vesicles ( position 3 > (position 1, 2). 3.3. Solvation Dynamics in a Single Vesicle Studied by Confocal Microscope. Solvation dynamics in a single vesicle is studied by confocal microscope by focusing the exciting laser beam on six different positions (as indicated in Figure 1A) within a giant single immobilized lipid vesicle of 20 μm size. Figure 3A shows the picosecond transients for C153 in position 4 within a single lipid vesicle at different emission wavelengths. As shown in Figure 3A, there is a rise at long wavelength and decay at short wavelength. This is a clear signature of solvation dynamics. Figure 3B shows the difference in rise times between position 6 and position 1 within a giant lipid vesicle of 20 μm size. The average rise time gradually increases from 800 ps at position 6 to 1000 ps at position 1 of the 20 μm vesicle. The longer rise time for position 1 indicates slower solvation relative to position 6. The difference in rise times suggests that solvation dynamics varies significantly at different regions within the single giant lipid vesicle of 20 μm size.
Figure 4. Time-resolved emission spectra (TRES) of C153 (λex = 405 nm) at (A) position 1 at 0 ps (blue), 250 ps (green), 500 ps (orange), 1000 ps (olive), 2000 ps (wine), 5000 ps (violet), and 10000 ps (red). (B) Position 6 at 0 ps (blue), 250 ps (green), 500 ps (orange), 1000 ps (olive), 2000 ps (wine), and 4000 ps (red).
giant single vesicle. Figure 6 displays the ν vs t plots at different positions within the vesicle. The decay of C(t) can be fitted with a biexponential decay curve. The decay parameters are summar-
Figure 3. (A) Picosecond transients of C153 (λex = 405 nm) in DLPC vesicle (at position 4) at λem (i) 450 (blue), (ii) 510 nm (green), and (ii) 660 nm (red) obtained using confocal microscope. (B) Picosecond transients of C153 (at λex = 405 nm) at (i) position 6 (red) and (ii) position 1 (blue) at λem = 660 nm obtained using a confocal microscope. 10233
dx.doi.org/10.1021/la3014859 | Langmuir 2012, 28, 10230−10237
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larger than that at position 6 (Δν ∼1250 cm−1) (Figure 4). This suggests a faster solvation dynamics at position 6. Further, ν(0) displays a 500 cm−1 red shift at position 6 (ν(0) ∼19900 cm−1) compared to that at position 1 (ν(0) ∼20400 cm−1) confirming a more polar environment around the probe in the region around position 6 compared to that around position 1. The amount of solvation missed may, in principle, be calculated using the Fee−Maroncelli formula.45 This formula involves the absorption maximum. In the case of a single molecule, it is not possible to determine the absorption spectrum. Therefore, we could not quantitatively determine the amount of solvation missed and the true ν(0). Nevertheless, it is evident at positions 4−6 that the average solvation time is faster (Table 1) and the observed Stokes shift (Δν) smaller than those at positions 1−3. 3.4. Fluorescence Anisotropy Decay under a Microscope. The fluorescence anisotropy decay curves are shown in Figure 7A and the decay parameters are summarized in Table 2. It has been observed that the rotational relaxation of the probe (C153) near the water pool (position 4, 5, and 6) is faster (⟨τr⟩ ∼2000−2350 ps) while that in the less polar hydrophobic region (position 1, 2, and 3) is slower (⟨τr⟩ ∼2700−3050 ps).
Figure 5. Decay of the solvent response function, C(t) of C153 at (i) position 1 (blue), (ii) position 3 (olive), and (iii) position 6 (red) within the giant lipid vesicle (diameter ∼20 μm). The points denote the actual values of C(t) and the solid line denotes the best fit.
ized in Table 1. The decay components are found to be 500 and 2000 ps at position 1 within the vesicle while they are 500 and 1000 ps at position 6. The parameters of solvation dynamics and the dynamic Stokes shift (Δν) values for the six different positions within the lipid vesicle is summarized in Table 1. The magnitude of dynamic Stokes shift (Δν) agrees well with average solvation time. The total dynamic Stokes shift at position 1 (Δν ∼2000 cm−1) is
4. DISCUSSIONS The main aim of this work is to demonstrate that by using a confocal microscope one can study dynamics in different regions of a vesicle. Before summarizing the results, it may be pointed out that the spatial resolution of our setup is 0.61λ/N.A. ∼ 200 nm (since λex = 405 nm and N.A. = 1.2). This is much (100 times)
Figure 6. The total decay of emission energy, ν(t), of C153 at (A) position 1 (blue), (B) position 3 (olive), (C) position 4 (orange), and (D) position 6 (red) within the lipid vesicle. 10234
dx.doi.org/10.1021/la3014859 | Langmuir 2012, 28, 10230−10237
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Figure 7. (A) Fluorescence anisotropy decay of C153 (at λex = 405 nm) along with a fitted curve (black) at position 1 (blue) and position 6 (red) within the vesicle (diameter ∼20 μm) at λem = 510 nm. (B) Plot of fwhm (Γ) of the time-resolved emission spectra with time (t) at (i) position 1 (blue) and (ii) position 6 (red) within a giant lipid vesicle (diameter ∼20 μm).
Table 2. Anisotropy Decay Parameters of C153 (at λem = 510 nm) at Different Positions of a DLPC Vesicle (diameter ∼20 μm) at λex = 405 nm positions
r0
τ1 (a1) (ps)
τ2 (a2) (ps)
⟨τr⟩ (ps)
position 1 position 2 position 3 position 4 position 5 position 6
0.36 0.35 0.34 0.29 0.3 0.3
450 (0.3) 500 (0.3) 400 (0.3) 270 (0.38) 200 (0.48) 200 (0.5)
4150 (0.7) 4250 (0.7) 3750 (0.7) 3650 (0.62) 4100 (0.52) 3850 (0.5)
3050 3100 2700 2350 2250 2000
dynamics in 750−1200 ps indicates that the hydrophobic probe, C153, is located at the membrane (not inside the water pool) and reports dynamics of the water confined at the membrane and interfaces. Evidently, there is a clear variation of solvation time in different regions within the same single lipid vesicle of 20 μm diameter. The main polar entities present are the water molecules and the polar head groups of the lipids. All of them may contribute to the solvation dynamics. However, the water molecules seem to play a major role because of their very large numbers and also in view of recent studies on deuterium isotope effect on solvation dynamics.52 The ultraslow (∼1000 ps) dynamics in a lipid vesicle arises from hydrogen bonding (“bound water”) to polar head groups, and this is discussed in many recent works.50,51 The average solvation time measured at position 6 within the giant lipid vesicle (⟨τs⟩ ∼750 ps) is ∼1.6 times faster compared to that measured at position 1 (⟨τs⟩ ∼1220 ps) (each measurement was carried out on at least three vesicles of similar size, and similar trends have been observed). Thus, it may be concluded that the motion of the polar species responsible for solvation (water molecules and/or polar head groups) is more restricted at position 1 than at position 6. Let us now compare local friction (i.e., diffusion coefficients obtained from FCS) and solvation dynamics. FCS involves molecular motion over a distance of ∼200 nm while solvation involves motion of water and other polar groups around the probe (∼1 nm). Thus, these two measurements (FCS and solvation dynamics) involve a different length scale. The anisotropy decay also gives an estimate of the local friction. The local friction estimated from anisotropy decay correlates well with observed solvation time. Thus, the local viscosity (friction) in the polar region (4, 5, and 6) is lower than that in the regions (1, 2, and 3) inside the membranes away from the water pool. Figure 7B displays the variation of the full width at halfmaximum (Γ, fwhm) of the time-resolved emission spectra with time. It is readily seen that the fwhm exhibits an initial rise followed by decay. The width (Γ) is a measure of the heterogeneity or fluctuation of the local microenvironment (solvation energy) around the hydrophobic probe, C153. It may be noted that such nonmonotonic behavior of fwhm (initial increase followed by decrease) has been observed for C153 even in homogeneous liquid by Maroncelli and co-workers53,54 and also by Hof and co-workers23 for other probes in lipid vesicles in
smaller than the diameter of the vesicle (20 000 nm), hence, one can reliably resolve dynamics in different regions. From the FCS data, we can compare the diffusion coefficient (Dt) of the dye molecules (C153) with those in bulk water. From the Stokes−Einstein equation, Dt is related to viscosity, η (friction) as, Dt =
kBT 6πηrh
(5)
where rh denotes the hydrodynamic radius. As discussed in section 3.1, the Dt (3−21 μm2 s−1) in the vesicles is 25−180 times higher than that in bulk water (550 μm2 s−1) and hence, the friction (η) inside the vesicle is 25−180 times higher. This suggests that the dye molecules are located in the interface or membrane region and are not in the water pool where the friction is same as that in bulk water. The distribution in Dt suggests multiple values of friction in different locations within the vesicle. We have observed such spatial heterogeneity of friction (and hence distribution in Dt) previously in triblock copolymer mixed micelle/gel,11,49 catanionic vesicles,10 and in neat ionic liquid.11 The position of emission maxima, magnitude of Δν and average solvation time (⟨τs⟩) display the following trend. At hydrophobic positions (1, 2, and 3), λmax em is blue-shifted, Δν is larger, and ⟨τs⟩ is higher (∼1050−1200 ps). At polar positions (4, 5, and 6) λmax em is red-shifted, Δν is smaller and solvation dynamics is relatively faster (⟨τs⟩ ∼750−950 ps). In the water pool of size (diameter) ∼5 μm (5000 nm) the solvation dynamics (dielectric response) should resemble that of bulk water (both dynamics and static dielectric constant). Thus, in the water pool, solvation dynamics should be completed within
