Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell

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Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell: Time-Resolved Confocal Microscopy Shirsendu Ghosh, Shyamtanu Chattoraj, Tridib Mondal, and Kankan Bhattacharyya* Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India S Supporting Information *

ABSTRACT: Different regions of a single live Chinese hamster ovary (CHO) cell are probed by time-resolved confocal microscopy. We used coumarin 153 (C153) as a probe. The dye localizes in the cytoplasm, nucleus, and lipid droplets, as is clearly revealed by the image. The fluorescence correlation spectroscopy (FCS) data shows that the microviscosity of lipid droplets is ∼34 ± 3 cP. The microviscosities of nucleus and cytoplasm are found to be 13 ± 1 and 14.5 ± 1 cP, respectively. The average solvation time (⟨τs⟩) in the lipid droplets (3600 ± 50 ps) is slower than that in the nucleus (⟨τs⟩ = 750 ± 50 ps) and cytoplasm (⟨τs⟩ = 1100 ± 50 ps). From the position of emission maxima of C153, the polarity of the nucleus is estimated to be similar to that of a mixture containing 26% DMSO in triacetin (η ∼ 11.2 cP, ε ∼ 26.2). The cytoplasm resembles a mixture of 18% DMSO in triacetin (η ∼ 12.6 cP, ε ∼ 21.9). The polarity of lipid droplets is less than that of pure triacetin (η ∼ 21.7 cP, ε ∼ 7.11).

1. INTRODUCTION Recent advances in confocal microscopy and single molecule spectroscopy have provided new insight into the dynamics in different regions of a live cell.1−7 For instance, fluorescence lifetime trajectory during transcription1 and lipid rafts in a live cell2 have been identified using single molecule spectroscopy (SMS). SMS has also been used to capture time-resolved and two-photon emission imaging microscopy in a live cell3 and coherence at the active site of an enzyme.7 Apart from imaging, several groups have used SMS to study diffusion in different regions of a cell.5,6 Time-resolved confocal microscopy may be used to record fluorescence decay under a microscope and thus to unravel ultrafast chemical processes under a microscope. So far, time-resolved microscopy has been employed to study diffusion, conformational dynamics, Förster resonance energy transfer (FRET), and anisotropy in a biological system.1−18 Recently, Philip et al. studied protein binding dynamics,9 while conformational dynamics of a protein inside a cell was studied by Sakan and Weninger using single molecule fluorescence resonance energy transfer (smFRET).10 Lu and co-workers combined atomic force microscopy (AFM) and confocal surface enhanced Raman scattering (SERS) spectroscopy to study redox proteins.12 Usually spectra and dynamics of a fluorescent probe in a nucleus are obtained by extracting the nucleus using nonionic detergents.19 However, in a confocal microscope, such procedures are not needed. One can directly record spectra and dynamics under in vivo conditions without the need of in vitro measurements. Recently, we have applied time-resolved confocal microscopy to study spectroscopy, diffusion, and solvation dynamics in the © XXXX American Chemical Society

cytoplasm and the nucleus of a single live chinese hamster ovary (CHO) cell.20 In a previous work, we used different flourophores, DCM (4-(dicyanomethylene)-2-methyl-6-(p-dimethyl-aminostyryl)-4H-pyran) and DAPI (4′,6-diamidino-2phenylindole), to probe the cytoplasm and nucleus, respectively. For the hydrophobic dye DCM, the emission maxima in cytoplasm (∼600 nm) is almost identical to that of DCM in isopropanol (602 nm) and is in between that in ethanol (615 nm, ε =18) and in 2-methyl propan-2-ol (595 nm, ε = 11). Thus, the polarity or dielectric constant (ε) of cytoplasm is estimated to be ∼15. DAPI is a well-known DNA-binding dye and binds to the minor groove of DNA, and hence, DAPI is located inside the nucleus. The emission maximum of DAPI in the nucleus (∼460 nm) is identical to that in a 50% water/ methanol mixture (v/v) (ε ∼ 60). Thus, from the position of emission maxima, we concluded that cytoplasm (ε ∼ 15) is less polar than the nucleus (ε ∼ 60).20 We also studied solvation dynamics of water in the cytoplasm and the nucleus, respectively, using DCM and DAPI as probes. It is observed that the solvation dynamics in the nucleus (average solvation time ⟨τs⟩ ∼ 775 ± 50 ps) is 2-fold faster than that in the cytoplasm (⟨τs⟩ ∼ 1250 ± 50 ps).20 Note, in bulk water, solvation dynamics occurs in ∼1 ps time scale.21−23 From fluorescence correlation spectroscopy (FCS), we studied the diffusion coefficient (Dt) of DCM in the cytoplasm and of DAPI in the nucleus.20 DCM exhibits very slow diffusion Received: March 6, 2013 Revised: May 24, 2013

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in cytoplasm (Dt ∼ 2 μm2 s−1),20 which is 150 times slower than that of DCM in bulk water (300 μm2 s−1).24 If we apply the Stokes−Einstein equation (eq 1) Rh =

kBT 6πηDt

bulk water (1 ps).21−23 The solvation dynamics studies are carried out in bulk and give an ensemble average over a very large number of systems. In contrast, time-resolved microscopy monitors to a small region (focal spot of size (0.6λ/N.A.)), which is of the order of 200−300 nm.

(1)

2. EXPERIMENTAL SECTION 2.1. Materials. Laser-grade dye, coumarin 153 (C153, Scheme 1), was purchased from Exciton Inc. and used without further purification. Dimethyl sulfoxide (DMSO, Sigma) and triacetin (LobaChemie) were used as received.

and assume the hydrodynamic radius (Rh) is the same as that of the dye (DCM) in water (i.e., 0.7 nm),24 Dt ∼ 2 μm2 s−1 corresponds to η ∼156 cP. This is very much higher than the reported25−27 values of viscosity (∼8 cP) in the cytoplasm. The abnormally low value of Dt (high viscosity) may arise from binding of DCM to a macromolecule (or vesicle) inside the cytoplasm. If we assume that the viscosity in the cytoplasm is ∼8 cP, the Rh of the macromolecular host containing DCM is estimated to be 15.6 nm. The Dt value of DAPI in the nucleus (∼15 μm2 s−1) is about 30 times smaller than that of DAPI in bulk water (∼430 μm2 s−1).20 This may also be ascribed to binding of DAPI to a macromolecule of Rh ∼ 2.1 nm. In summary, the FCS data indicates that both DCM and DAPI bind to a macromolecule and hence reports the microenvironment of the binding site of the macromolecule (of course, under in vivo conditions inside a cell). In the present work, we employ a new fluorescent probe coumarin 153 (C153) which does not bind to any macromolecules inside the cell (as will be revealed by the FCS data to be discussed later). Thus, C153 truly reports the microenvironment inside the cell. C153 offers another special advantage that it penetrates many different cellular structures, namely, cytoplasm, nucleus, and lipid droplets. Thus, using the same probe, we could study different regions of a cell. It may be mentioned that Kucherak et al. previously reported fluorescence microscopic images of different regions of a cell using nile red derivative.11 They, however, did not measure the diffusion coefficient and dynamics in different regions of a cell.11 Photophysics of C153 in many solvents has been discussed in great detail by Maroncelli et al.23 Briefly, the dipole moment of C153 increases in the excited state and this results in solvent reorganizations.23 In the present work, we use C153 to compare the dynamics in the nucleus, cytoplasm, and lipid droplet regions of a single live CHO cell. Lipid droplets, also known as lipid bodies, oil bodies, or adiposomes, are lipid-rich cellular organelles, which are used to regulate the storage and hydrolysis of neutral lipids.28−33 The lipid droplets are characterized as a few bright spots in the cytoplasm region of the cell.28−31,33 Their size varies from 20 to 40 nm to 100 μm.32 The structure and composition of the lipid droplets are not well characterized. It is generally believed that, in a lipid droplet, there is a neutral lipid core consisting mainly of triacylglycerols (TAGs) and cholesteryl esters which is surrounded by a phospholipid monolayer.33 In this work, we attempted to get additional information on the lipid droplets by confocal microscopy using the fluorescent probe C153. We have estimated the polarity of lipid droplets from emission maxima obtained under microscope, viscosity from FCS, and solvent dynamics by time-resolved confocal microscopy. Many groups have applied ultrafast laser spectroscopy to study the dynamics of water in biological systems. A large body of experimental results21−23,34−43 as well as computer simulation44−47 and theoretical studies48,49 are carried out which suggest that the relaxation times of water in different biological assemblies are 10−1000 times slower compared to

Scheme 1. Structure of Coumarin 153 (C153)

2.2. Methods. 2.2.1. Cell Preparation. Methods for cell preparation were discussed in our previous publication.20 Briefly, Chinese hamster ovary (CHO) cells were grown in phenol-red-free DMEM medium with 10% fetal bovine serum, 1% Pen Strep Glutamine (from Gibco) in an atmosphere of 5% (v/v) CO2 enriched air at 37 °C. For microscopy, cells are cultured overnight in a 35 mm glass bottom Petri dish. For the solvation dynamics studies, 2 mL of a 1 μM solution of the dye (C153) in DMSO was prepared. For TCSPC experiment, after rinsing the cells three times with phosphate buffered saline (PBS) buffer solution, 50 μL of the stock dye solution was added to the Petri dish and then incubated for about 10 min. For the FCS measurement, 10 μL of the dye solution was added to the Petri dish and then incubated for about 10 min. The stained cell was rinsed with PBS solution five to six times. After staining, cells were used for microscopic study within 10 min. All experiments are carried out at ∼25 °C. 2.2.2. Experimental Setup for One-Photon and Two-Photon (1PE and 2PE) Microscopy. The experimental setup for one-photon and two-photon (1PE and 2PE) microscopy has been described in our previous publication.20 Briefly, a combination of confocal microscope (Olympus IX-71) and TCSPC setup (PicoQuant, MicroTime 200) has been used in this study. A water immersion objective (magnification 60× and numerical aperture (NA) ≈ 1.2) was used. Thus, the diffraction limited spot size is 0.6λ/1.2 ∼ λ/2. The excitation sources are a pulsed picosecond diode laser (PDL 828-S “SEPIA II”, PicoQuant for 1PE at 405 nm) and a mode-locked Ti-sapphire laser (Tsunami, Spectra Physics, for 2PE at 810 nm). For live cell studies, we kept the laser power at or below ∼0.27 μW for 1PE and 4 mW for 2PE. Fluorescence from the dye in the cells was separated by a dichroic mirror (Z405RDC, Chroma for 1PE and 725DCSP-XR, Chroma for 2PE). To block the exciting laser light, a suitable filter (430LP, Chroma, for 1PE and FF01-750/SP-25 for 2PE) was used before the detectors. The fluorescence was focused through a pinhole (30 μm). The emitted light with different polarizations was separated using a polarizer cube (Chroma) and was detected by two singlephoton-counting avalanche photodiodes (SPAD1 and SPAD2). Appropriate narrow band-pass filters are used (e.g., XBPA510, 540, etc., Asahi Spectra) to collect TCSPC decay at specific emission wavelengths. The signal was subsequently processed by the PicoHarp300 time-correlated single-photon-counting module (PicoQuant) to generate a TCSPC histogram. Emission Spectra. The emission spectra of C153 in a CHO cell were recorded using an electron multiplying charge-coupled device (EMCCD, ANDOR Technology) attached to a spectrograph (ANDOR Technology, Shamrock series). B

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Fitting of FCS Curves. The autocorrelation function G(τ) is defined as13

G(τ ) =

⟨δF(0)δF(τ )⟩ ⟨F ⟩2

(2)

where ⟨F⟩ is the average intensity and δF(τ) is the fluctuation in the intensity at a delay τ around the mean value, i.e., δF(τ) = ⟨F⟩ − F(τ). We fitted G(τ) to a 3D-diffusion model13

G(τ ) =

1 (1 + τ /τD)−1(1 + τ /τDS2)−1/2 N

(3)

In this equation, τD denotes the diffusion time in the confocal volume, τ is the delay time, N represents the average number of molecules in the confocal volume, and S is the structure parameter. The diffusion constant of the dyes can be determined using the following equation for two-photon excitation.13

Dt =

ωxy 2 8τD

(4)

where ωxy is the transverse radius (∼526 nm) of the confocal volume. 2.2.3. Picosecond Time-Resolved Fluorescence. The parallel (I∥, parallel to the polarization of the exciting light) and perpendicular (I⊥) components of the fluorescence were recorded by using the two detectors (SPADs). I∥ and I⊥ were combined to generate the fluorescence lifetime decays at magic angle conditions as follows:

Figure 1. One-photon (1PE) confocal image of CHO cell stained by C153.

cytoplasm region, and a few bright spots. This clearly indicates that C153 stays in three different regions. The bright spots are ascribed to lipid droplets following previous literature reports28−31,33 and by co-localization study (Figure S1, Supporting Information). It is clearly seen that the size of the bright spot is larger than the diffraction limited spot size (λ/2 ∼ 200 nm). From the image, the size of the lipid droplets is calculated to be ∼0.84 μm (840 nm). This is consistent with previous reports on the size of the lipid droplets in a cell.32 3.2. FCS Measurement: Diffusion in Nucleus, Cytoplasm, and Lipid Droplets. In order to study diffusion in different regions of a cell, we used fluorescence correlation spectroscopy (FCS). The number of dye molecules (C153) present in the confocal volume in each case may be obtained from the value of G(0).13 In the present case, from the FCS data, the number of dye molecules within the confocal volume is estimated to be ∼30. The FCS data was recorded under twophoton excitation (2PE). 3.2.1. FCS in Nucleus and Cytoplasm: Size of Diffusing Particle. Figure 2 describes the FCS curves for the dye C153 in the nucleus, cytoplasm, and lipid droplets (bright spots) and their comparisons. In bulk water, Dt of C153 is 550 μm2 s−1.52 The FCS data (Figure 2 and Table 1) indicates the diffusion coefficient (Dt) of C153 is 45 μm2 s−1 in the nucleus, 39 μm2 s−1 in the cytoplasm, and 17 μm2 s−1 in lipid droplets. Using these values of Dt and the hydrodynamic radius of C153 in water (Rh = 0.38 nm),52 we have estimated the viscosities of the nucleus and cytoplasm using the Stokes−Einstein relationship (eq 1). Previously, many groups have reported the values of viscosity of cytoplasm of ∼8 cP.25−27 The viscosity of the nucleus is reported to be ∼8 cP.27 In this present study, the viscosity of nucleus and cytoplasm are found to be 13 ± 1 and 14.5 ± 1 cP, respectively. From this, it may be concluded that the dye molecule moves in the cytoplasm and nucleus either in the free state or bound to very small macromolecules. 3.2.2. FCS in Lipid Droplets. The Dt values in lipid droplets are more than 2-fold smaller than that of C153 in nucleus and cytoplasm. Note the size of lipid droplet (Rh = 840/2 = 420 nm) is about 1000 times that of C153. Thus, diffusion of the lipid droplets, as a whole, is too slow to be observed. In this case, the FCS data corresponds to motion of C153 within the

Imagic(t ) = I∥(t ) cos2(54.75°) + I⊥(t ) sin 2(54.75°) = (1/3)I∥(t ) + (2/3)I⊥(t )

(5)

Fluorescence Lifetime Measurement. For recording IRF, we used a bare slide and collected the scattered laser light. The FWHM of the IRF for excitation at 470 nm is ∼100 ps. The fluorescence decay is deconvoluted using the IRF and DAS6 v6.3 software. Fluorescence Anisotropy Measurement. The anisotropy function, r(t), was obtained under 1PE using the formula r(t ) =

I∥ − GI⊥ I∥ + 2GI⊥

(6)

The G factor for this microscope setup was measured by tail fitting of fluorescein4 and was found to be ∼2.86. 2.2.4. Solvation Dynamics under a Microscope. The time-resolved emission spectra (TRES) were constructed using the parameters of best fit to the fluorescence decays and the steady-state emission spectrum following the procedure described by Maroncelli and Fleming.50,51 The solvation dynamics is described by a decrease in emission energy (frequency, ν) with increase in time. If ν(0), ν(t), and ν(∞) are the emission frequencies at time 0, t, and ∞, respectively, the decreases in emission energy with time are described by v(t ) = v(∞) + [v(0) − v(∞)] ∑ aie−t/ τi i

(7)

The time constants for solvation (τi) are obtained from the fitting of ν(t) vs t curves using eq 7. The other parameters, ν(0) and ν(∞), are also obtained from this fitting. For recording the fluorescence decays, we had to use a much higher concentration of the dye molecules (∼50 times higher than that used for FCS studies). Thus, the number of dye molecules present in the confocal volume is nearly 1500.

3. RESULTS 3.1. Confocal Image. Figure 1 shows the confocal image of a single Chinese hamster ovary (CHO) cell stained by C153. In order to minimize autofluorescence, we used a phenol-red-free culture (DMEM) medium. From the image, three different regions of a cell can be clearly observednucleus region, C

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Figure 2. Normalized autocorrelation function of coumarin 153 (C153) dye in a single CHO cell (λex = 810 nm).

Table 1. Diffusion Coefficients of C153 in CHO Cell region

Dt (μm2 s−1)

η (cP)

nucleus cytoplasm lipid droplets

45 ± 3 39 ± 3 17 ± 2

13 ± 1 14.5 ± 1 34 ± 3

lipid droplets. We used the observed Dt value of C153 in lipid droplets to estimate the microviscosity inside the lipid droplets using eq 1. The microviscosity sensed by C153 molecules in lipid droplets is ∼34 ± 3 cP. Previously, Bisbya et al.53 reported the viscosity in lipid emulsion droplets (not inside cells) and estimated a local viscosity of 50−100 cP. The microviscosity estimated by our FCS data for lipid droplets inside the cell is close to this. To the best of our knowledge, this is the first report on the viscosity inside the lipid droplets in a cell. 3.3. Emission Maxima and Polarity of Nucleus, Cytoplasm, and Lipid Droplets. The emission maximum of C153 is extremely sensitive to the polarity of the medium and shifts from 549 nm in water to 501 nm in ethyl acetate.54 Figure 3 shows the emission spectra of C153 in nucleus, cytoplasm, and lipid droplets. The observed emission maximum of C153 in lipid droplets, cytoplasm, and nucleus are 502, 521, and 525 nm, respectively (Table 2). To compare the emission maxima with solvent of known polarity, we have chosen viscous solvents (Figure S2, Supporting Information). The emission maximum of C153 in the nucleus is identical to that of C153 in 26% (v/v) DMSO in triacetin (η ∼ 11.2 cP, ε ∼ 26.2). The emission maximum of C153 in cytoplasm resembles that in 18% (v/v) DMSO in triacetin (η ∼ 12.6 cP, ε ∼ 21.9). Finally, the emission maximum of C153 in lipid droplets (502 nm) is much more blue-shifted compared to that in triacetin (λem ∼ 510 nm). Thus, the polarity of lipid droplet is much less than that of triacetin (η ∼ 21.7 cP, ε = 7.11).55 It may be mentioned

Figure 3. Emission spectra of C153 in a CHO cell.

Table 2. λmax em of C153 in Different Regions of a CHO Cell region

λmax em (nm)

similar to

dielectric constanta (εeff)

nucleus cytoplasm lipid droplets

525 521 502

26% DMSO in triacetin 18% DMSO in triacetin triacetin (λem ∼ 510 nm)

∼26.2 ∼21.9 cytoplasm ≈ nucleus. This is of the same order of the viscosity of different regions of the cell as obtained from FCS. However, one should remember that anisotropy decay (rotation) and FCS (translation) correspond to different kinds of diffusion of the probe (C153).

cP).25−27 One possible reason for this discrepancy might be because of refractive index mismatch and consequent error in single spot FCS and “crowding effect”.58 The other possibility could be an increase in the size of the probe because of binding to a small biomolecule and consequent decrease in Dt. We did not get a different value of Dt (η) in different regions of cytoplasm. One possible reason may be as follows. In an FCS experiment, translational diffusion occurs over a large distance (λ/2 ≈ 800/2 ≈ 400 nm for 2PE). Thus, the viscosity measured by FCS is an average over a region of radius ∼0.4 μ (400 nm). It is possible that the viscosity of cytoplasm near the membrane may differ from that near the nucleus of a cell. In fact, the η in the nucleus (13 ± 1 cP) measured by FCS is very close to that in the cytoplasm (14.5 ± 1 cP). The present FCS study does not reveal an appreciable difference in the viscosity of cytoplasm and nucleus and the difference is within experimental error. The viscosity of the lipid droplet is ∼2.5fold slower compared to that of the nucleus. The calculated microviscosity of the lipid droplet is 34 ± 3 cP. This may be ascribed to the restricted environment of the interior of lipid droplets created by the large lipid molecules.28−31,33 Fourth, solvation dynamics in the cytoplasm (⟨τs⟩ = 1100 ps) is ∼1.5 times slower than that in the nucleus (⟨τs⟩ = 750 ps). The faster dynamics in the nucleus is consistent with the slightly higher local polarity of the microenvironment of the nucleus than that in the cytoplasm. In contrast, the solvation dynamics in lipid droplets (⟨τs⟩ = 3600 ps) is 5-fold slower compared to that in the nucleus. This shows that the lipid environment inside lipid droplets is highly restricted.28−31,33 The polarity or dielectric constant of the medium is controlled by the ability of the water molecules to reorient in response to an external electric field.59 Further, hydrogen bonding between water molecules increases the individual dipole moment of one water molecule because of mutual dynamic polarization and this increases the dielectric constant.60 The lower polarity and slower solvation dynamics in the lipid droplets compared to those in the cytoplasm or in the nucleus suggest that in the lipid droplets there is a large scale destruction of water−water hydrogen bond and most of the water molecules remain bound to the macromolecules. It may be recalled that in bulk water the solvation dynamics in water occurs in a 1 ps time scale.21−23 In this work, we observed that the average solvation times are 3600, 1100, and 750 ps in lipid droplets, cytoplasm, and nucleus, respectively. This is substantially slower than that of bulk water. Similar time scales were reported by us earlier in a time-resolved microscopic study on lipid vesicles and in a CHO cell.20,43 The ultraslow response of water in the vicinity of biomolecules may arise from the polar residues of the protein and other biomolecules as well as the water molecules. The relative contribution of water or the macromolecular biological host is a matter of intense recent debate and has been analyzed in detail by large scale computer simulations and many experiments.34−51 The point to note is the solvation dynamics in the lipid droplets is much slower than that in the cytoplasm or nucleus. Fifth, the same trend is observed in anisotropy decay as ⟨τs⟩. The ⟨τrot⟩ values in the nucleus (800 ± 100 ps) and cytoplasm (850 ± 100 ps) are comparable, which is consistent with viscosity measured by FCS. The ∼2-fold slower ⟨τrot⟩ in lipid droplet (1750 ± 100 ps) compared to nucleus (or cytoplasm) is similar to that of the ∼2.5-fold higher viscosity of lipid droplets measured by FCS (Table 1). This may be ascribed to

4. DISCUSSION The most important findings of this work are the following. First, the same dye C153 is distributed over the entire cell, and the image and co-localization study using nile red clearly show three different regions of the cell (namely, nucleus, cytoplasm, and lipid droplets). Thus, the probe may be used to study spectra, diffusion, solvation, and anisotropy decay in three different regions. Second, local polarity of the three regions of cell as sensed by C153 probe is much less than that of water as the observed emission maxima are blue-shifted from that in water (λem ∼ 549 nm).54 We have compared the emission maxima of C153 in three different regions of cell with that of C153 in viscous (η > 10 cP) solvent mixtures to get an idea about the polarity (dielectric constant) of the regions of a living CHO cell (Figure S2, Supporting Information). The local polarity of cytoplasm (more precisely, λmax em of C153 = 521 nm) is comparable to that of 18% DMSO in triacetin (ε ∼ 21.9). The polarity of the nucleus (λmax em = 521 nm) is similar to that of 26% DMSO in triacetin (ε ∼ 26.2). The slightly higher polarity of the nucleus compared to that of the cytoplasm may be attributed to the presence of ions and polar residues in the nucleus. The local polarity of the microenvironment of lipid droplet is very much lower than that of nucleus or cytoplasm. Its polarity is less than that of pure triacetin (ε < 7.11) and is equal to that of ethyl acetate (η ∼ 0.4 cP, ε = 6.02, λmax em = 502 nm). The extremely low polarity of the lipid droplets may be due to its structure, as it has a neutral lipid core surrounded by a phospholipid membrane.28−31,33 Third, the microviscosity (i.e., diffusion) of the microenvironment in the nucleus and cytoplasm is comparable. From the FCS data, we have estimated the viscosity of the nucleus and cytoplasm to be 13 ± 1 and 14.5 ± 1 cP. It is concluded that the C153 molecule moves in the nucleus and cytoplasm almost freely and does not bind to large biological macromolecules. In our previous work, the FCS data indicates that DCM and DAPI bind to a macromolecule and hence shows the microenvironment of the binding site of the macromolecule (of course, under in vivo conditions inside a cell). The special advantage of the C153 dye is that it does not bind to any large biological macromolecule (as indicated by FCS data) and hence reports the actual environment of the cytoplasm and nucleus region of a cell. Previously, many groups have applied various techniques to measure the viscosity of the cytoplasm of a living cell. However, there is no consensus on the value of the viscosity of cytoplasm. The viscosity of the cytoplasm has been reported to be ∼8 cP.25−27 The local viscosity in the cytoplasm, measured in these works from the Dt value measured from FCS and using the Stokes−Einstein equation, is 14.5 ± 1 cP. This is ∼1.5-fold higher than the reported values of viscosity (∼8 F

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the restricted environment created by the giant neutral lipid molecules inside the lipid droplets.28−33 Note that translational diffusion occurs over a large distance (λ/2 ≈ 800/2 ≈ 400 nm). The rotational motion, on the other hand, reports friction within a small region around the molecule (C153) and hence may be faster. In spite of this, the agreement between our FCS data and the anisotropy decay are quite close.

5. CONCLUSION This work demonstrates that different regions of a cell may be probed by the same dye C153. Using time- and space-resolved confocal microscopy, we showed that the lipid droplets are more viscous, slower, and less polar than the nucleus or the cytoplasm, and exhibit 3-fold slower solvation dynamics compared to the cytoplasm. Compared with the nucleus (η ∼ 14.5 cP, ε ∼ 26.2), the cytoplasm shows a similar viscosity (η ∼ 13 cP) but a lower polarity (ε ∼ 21.9) and 1.5-fold slower solvation dynamics.

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ASSOCIATED CONTENT

S Supporting Information *

Co-localization study of C153 and nile red in a CHO cell, comparison of emission maxima of C153 in different regions of a CHO cell with viscous solvent, fluorescence decay of C153 in three different regions of a CHO cell and their comparisons observed by 1PE, and decay of the emission energy, νt, and rotational relaxation (anisotropy decay). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (91)-33-2473-2805. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to Department of Science and Technology, India, for IRHPA Project entitled “Center for Ultrafast Spectroscopy and Microscopy” (Project No. IR/S1/CU 02/ 2009), Council for Scientific and Industrial Research (CSIR), and J. C. Bose Fellowship for generous research support. S.G., S.C., and T.M. thank CSIR for awarding fellowships. We are grateful to Sankha Pattanayek (Dept. of Org. Chem., IACS) for his help and support for cell culture.

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REFERENCES

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