Solvation Dynamics of Biological Water in a Single Live Cell under a

Jan 21, 2013 - Time-resolved confocal microscopy has been applied to study the cytoplasm and nucleus region of a single live Chinese hamster ovary (CH...
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Solvation Dynamics of Biological Water in a Single Live Cell under a Confocal Microscope Dibyendu Kumar Sasmal, Shirsendu Ghosh, Atanu Kumar Das, and Kankan Bhattacharyya* Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India S Supporting Information *

ABSTRACT: Time-resolved confocal microscopy has been applied to study the cytoplasm and nucleus region of a single live Chinese hamster ovary (CHO) cell. To select the cytoplasm and the nucleus region, two different fluorescent probes are used. A hydrophobic fluorescent dye, DCM, localizes preferentially in the cytoplasm region of a CHO cell. A DNA binding dye, DAPI, is found to be inside the nucleus of the cell. The locations of the probes are clearly seen in the image. Emission maxima of the dyes (DCM in cytoplasm and DAPI in the nucleus) are compared to those of the same dyes in different solvents. From this, it is concluded that the polarity (dielectric constant, ε) of the microenvironment of DCM in the cytoplasm is ∼15. The nucleus is found to be much more polar with ε ≈ 60 (as reported by DAPI). The diffusion coefficient (and hence viscosity) in the cytoplasm and the nucleus was determined using fluorescence correlation spectroscopy (FCS). The diffusion coefficient (Dt) of the dye (DCM) in the cytoplasm is 2 μm2 s−1 and is ∼150 times slower than that in bulk water (buffer). Dt of DAPI in the nucleus (15 μm2 s−1) is 30 times slower than that in bulk water (buffer). The extremely slow diffusion inside the cell has been ascribed to higher viscosity and also to the binding of the probes (DCM and DAPI) to large biological macromolecules. The solvation dynamics of water in the cytoplasm (monitored by DCM) exhibits an average relaxation time ⟨τsol⟩ of 1250 ± 50 ps, which is about 1000 times slower than in bulk water (1 ps). The solvation dynamics inside the nucleus (studied using DAPI) is about 2-fold faster, ⟨τsol⟩ ≈ 775 ps. The higher polarity, faster diffusion, and faster solvation dynamics in the nucleus indicates that it is less crowded and less restricted than the cytoplasm. membrane lipids in a live cell.11 Lu and co-workers applied AFM-SERS to study redox proteins.12 In this work, we attempt to determine and compare the polarity, viscosity, and solvation dynamics in the cytoplasm and the nucleus of a cell. For this study, we have chosen a Chinese hamster ovary (CHO) cell. We used two different fluorescent probes to study the cytoplasm and the nucleus of a live CHO cell. It will be demonstrated that a hydrophobic probe, DCM, is preferentially localized in the cytoplasm region whereas a DNA binding dye, DAPI, resides inside the nucleus of the cell. We compare the emission maximum, diffusion, and solvation dynamics of the two dyes to highlight the differences in the two regions inside the cell. The emission maxima of the solvation probes are polarity-sensitive because of the difference in dipole moments in the ground and excited states and the differential stabilization of the ground and excited states by solvation.14 It may be mentioned previously that Chattopadhyay and his group16−18 used solvation probes (laurdan, prodan, and di-8-ANEPPS) to study the depth-dependent polarity of membranes in a lipid vesicle from the steady-state

1. INTRODUCTION The biological cell is one of the most interesting and most complicated machines designed by nature. The most important architectural parts of a eukaryotic cell are the cell wall (membranes), the cytoplasm, and the nucleus. Each of these three parts contains many complex constituents. The cell wall surrounds the entire cell and protects its content. The cytoplasm fills up the entire region between the nucleus and the cell wall. Recent advances in single-molecule spectroscopy (SMS) have opened up many new avenues to study a live cell.1−13 This technique has been used to capture the fluorescence lifetime trajectory during transcription,1 lipid rafts in live cells,2 time-resolved and two-photon emission imaging microscopy in a live cell,3 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 Using time-resolved confocal microscopy, one can easily record the fluorescence decay under a microscope and thus study ultrafast chemical processes under a microscope. So far, timeresolved microscopy has been used to study FRET and anisotropy in a biological system.1−15 Recently, Philip et al. studied protein-binding dynamics9 and Sakan and Weninger studied the conformation of a protein inside a cell using smFRET.10 Eggeling et al. studied the nanoscale dynamics of © 2013 American Chemical Society

Received: November 2, 2012 Revised: January 21, 2013 Published: January 21, 2013 2289

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emission maxima. Chattopadhyay et al., however, did not study the solvation dynamics or FCS as a function of depth using time-resolved spectroscopy. They also did not study the nucleus region of a cell. Hoff and co-workers studied the solvation dynamics in phospholipid bilayers using ABA-C15.19 In view of the crucial role of water in biology, many groups have previously studied solvation dynamics in biological systems using ultrafast laser spectroscopy,20−29 large-scale computer simulation,30−33 and theory.34,35 The relaxation time of water near a protein is 10−100 times slower than that in bulk water (1 ps).20,21 Such relaxations have been observed in proteins,20,33−35 DNA,27−32 and lipids.36,37 The solvation dynamics studies discussed above are all in an ensemble, which reports an average over an exceedingly large number of systems. To obtain regionwise precise information in a single cell, it is necessary to carry out time-resolved studies by focusing light on different regions using a confocal microscope. Most recently, we have reported for the first time the study of solvation dynamics inside a single lipid vesicle by combining confocal microscopy and time-resolved spectroscopy.36 Polarity, diffusion, and solvation control the chemical reactivity. A knowledge of these properties inside a cell is fundamental to understand chemical and biological activity. Very recently, some groups have applied NMR relxation13 to study the dynamics in a cell. However, they studied an ensemble containing an extremely large number of cells. In this work, we report for the first time the solvation dynamics inside a single live Chinese hamster ovary (CHO) cell using timeresolved confocal microscopy. We used laser dyes DCM and DAPI as probes. The laser dyes were chosen because of their convenient wavelengths of excitation (∼400 nm for one photon and 800 nm for two photons) and robust photostability for studying dynamics (FCS and solvation).

Scheme 1. Structures of (A) DCM, (B) DAPI, and (C) HPTS

studies, we kept the laser power at or below ∼0.27 μW. At this power, prolonged exposure does not cause cell damage, as is observed from the images. Fluorescence from the dye in the cells was separated with a dichroic mirror (HQ490DCXR, Chroma). To block the exciting laser light, a suitable long-pass filter (510LP, Chroma, for excitation at 470 nm) 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 detected by two single-photon-counting avalanche photodiodes (SPAD1 and SPAD2). Appropriate narrow-band-pass filters were used (e.g., XBPA510, 540, etc., Asahi Spectra) to collect the TCSPC decay at specific emission wavelengths. The use of two detectors eliminates the artifacts in FCS measurements arising from the after pulse. The origin of the after pulse in FCS measurements and its elimination using two detectors has been discussed by Enderlein et al.38 The signal was subsequently processed by the PicoHarp-300 timecorrelated, single-photon-counting module (PicoQuant) to generate a TCSPC histogram. 2.2.2.2. 2PE. For the two-photon experiment, a mode-locked Tisapphire laser (Tsunami, Spectra Physics) with a repetition rate of 80 MHz pumped by a 5 W Millennia laser (Spectra Physics) was used. The laser power was kept below ∼4 mW to collect TCSPC and FCS data for a live cell. To reduce the intensity of the laser, a combination filter wheel (Thorlab) was placed before the beam expander. The beam expander (Thorlab) was used to fill the back aperture of the objective. To separate the fluorescence from the exciting laser (along the same path), we used a dichroic mirror (725DCSP-XR, Chroma) and appropriate short-pass filters (FF01-750/SP-25). The fluorescence was focused through a pinhole (30 μm). The emitted light with different polarizations was separated using a polarizer cube (Chroma) and detected by two single-photon-counting avalanche photodiodes (SPAD1 and SPAD2). Appropriate narrow-band-pass filters were used (e.g., XBPA510, 540, etc., Asahi Spectra) to collect the TCSPC decay at specific emission wavelengths. The signal was subsequently processed by the PicoHarp-300 time-correlated, single-photoncounting module (PicoQuant) to generate a TCSPC histogram. Two-photon excitation has a number of advantages over onephoton excitation such as lower photodamage (bleaching), improved cell survival, and tissue penetration.39 For this reason, all of the data

2. EXPERIMENTAL SECTION 2.1. Materials. Laser-grade dye 4-(dicyanomethylene)-2-methyl-6(p-dimethyl-aminostyryl)-4H-pyran (DCM, Scheme 1A) was used as received from Exciton Inc. 4′,6-Diamidino-2-phenylindole (DAPI, Scheme 1B) was purchased from Sigma-Aldrich and used as received. For the cell culture, we used a 35 mm coverslip-bottom dish (BD BioCoat). 2.2. Methods. 2.2.1. Cell Preparation. Chinese hamster ovary (CHO) cells were grown in a phenol red-free DMEM medium with 10% fetal bovine serum and 1% pen strep glutamine (From Gibco) in an atmosphere of 5% (v/v) CO2-enriched air at 37 °C. For microscopy, cells were cultured overnight in a 35 mm glass-bottomed Petri dish. The cells were rinsed at least three times with phosphatebuffered saline (PBS) buffer solution before being stained with fluorescent dye. For the solvation dynamics studies, 2 mL of a 500 nM dye solution in ethanol (DCM)/water (DAPI) was prepared. For the TCSPC experiment, from the stock dye solution 50 μL was added to the Petri dish and then incubated for about 10 min. For the FCS measurement, 10 μL (50 nM) 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 being stained, cells were used for the microscopy study within 10 min. 2.2.2. Experimental Setup for One-Photon and Two-Photon (1PE and 2PE) Microscopy. We used a combination of a confocal microscope (Olympus IX-71) and a TCSPC setup (PicoQuant, MicroTime 200). A water-immersion objective (magnification 60× and numerical aperture (NA) ∼1.2) was used to focus the excitation light (405 and 470 nm for 1PE and 810 nm for 2PE) onto the glassbottomed dish. 2.2.2.1. 1PE. For the one-photon excitation experiment, we used a pulsed diode laser with a repetition rate of ∼40 MHz. For live cell 2290

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Figure 1. Two-photon excitation (2PE) confocal image of CHO cells stained with (A) R6G, (B) DCM, (C) DAPI, and (D) DAPI and DCM. 2.2.3. Picosecond Time-Resolved Fluorescence. The parallel (I∥, parallel to the polarization of the exciting light) and perpendicular (I⊥) components 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:

reported in this work (except the anisotropy decay) are collected using 2PE. Other results with 1PE are given as Supporting Information. 2.2.2.3. Emission Spectra. The emission spectra of DCM in a CHO cell and in ethanol were recorded using an electron-multiplying charge-coupled device (EMCCD, ANDOR Technology) attached to a spectrograph (ANDOR Technology, Shamrock series) for both onephoton and two-photon excitation experiments. 2.2.2.4. Fitting of FCS Curves. The autocorrelation function G(τ) is defined as14

G(τ ) =

δF(0)δF(τ ) F 2

Imagic(t ) = I (t ) cos2(54.75°) + I⊥(t ) sin 2(54.75°) =

(1)

−1/2 −1 1⎛ τ ⎞ ⎛ τ ⎞ ⎟ ⎜1 + ⎟ ⎜1 + N⎝ τD ⎠ ⎝ τDS2 ⎠

(2)

r(t ) =

In this equation, τD denotes the diffusion time in the confocal volume, τ is the delay time, and N represents the average number of molecules in the confocal volume. The diffusion constant of the dyes can be determined using the following equation for two-photon excitation14

Dt =

I − GI⊥ I + 2GI⊥

(5)

The G factor for this microscope setup was measured by tail fitting of fluorescein.4 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.40,41 The solvation dynamics is described by the decay of the solvent correlation function, C(t), defined as

ωxy 2 8τD

(4)

2.2.3.1. Fluorescence Lifetime Measurement. To record IRF, we used a bare slide and collected the scattered laser light. The fwhm of the IRF for excitation at 810 nm (femtosecond laser) is ∼50 ps. 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. 2.2.3.2. Fluorescence Anisotropy Measurement. The anisotropy function, r(t), was obtained under 1PE using the equation

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 model,14

G(τ ) =

⎛1⎞ ⎛2⎞ ⎜ ⎟I (t ) + ⎜ ⎟I (t ) ⎝3⎠ ⎝3⎠ ⊥

(3)

where ωxy is the transverse radius (∼526 nm) of the confocal volume. 2291

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Figure 2. Normalized autocorrelation function of (A) DCM dye in water (green) and the cytoplasm of a single CHO cell (red) (λex = 810 nm), (B) DAPI dye in water (green) and the nucleus of a single CHO cell (red) (λex = 810 nm), (C) DCM dye in the cytoplasm of a single CHO cell (red) (λex = 810 nm) and DAPI dye in the nucleus of a single CHO cell (red) (λex = 810 nm), and (D) DCM dye in HSA protein (green) and HPTS dye in HSA protein (red) (λex = 405 nm).

C(t ) =

ν(t ) − ν(∞) ν(0) − ν(∞)

region. We did not make any measurements on the bright spots that presumably arise from the lipid droplets as discussed previously by many groups.42 3.2. FCS Measurement: Diffusion in the Cytoplasm and Nucleus. To determine the diffusion coefficient and hence the friction (viscosity), we used fluorescence correlation spectroscopy (FCS). Figure 2 describe the FCS curves in the nucleus (recorded using DAPI) and in the cytoplasm (reported by DCM). The number of dye molecules (DCM and DAPI) present in the confocal volume in each case may be obtained from the value of G(0).14,15 In the present case, from the FCS data the number of dye molecules within the confocal volume is estimated to be ∼20. The FCS data was also recorded under two-photon excitation (2PE). In bulk water, the Dt of DCM is 300 μm2 s−1 (Table 1).43 The FCS data (Figure 2) indicates that the diffusion coefficient

(6)

where ν(0), ν(t), and ν(∞) are the emission maxima (frequencies) at times 0, t, and ∞, respectively. The solvent correlation function, C(t), was fitted to a multiexponential: C(t ) =

∑ aie−t/ τi i

(7)

3. RESULTS 3.1. Confocal Image. Figure 1 shows the confocal image of a single Chinese hamster ovary (CHO) cell obtained under two-photon excitation. To minimize autofluorescence, we used a phenol red-free culture (DMEM) medium. To see different regions, we stained the cell with different dyes. Because of the phosphate groups of the phospholipid membranes, a cell wall carries a net negative charge. As a result, a positively charged dye, rhodamine 6G (R6G), preferentially binds to the membrane region as shown in Figure 1A. From Figure 1B, it is clearly seen that the hydrophobic dye, DCM, is located inside the cytoplasm of the cell (indicated by brown, i.e., a high fluorescence intensity). Very few of the DCM dye molecules accumulate in the membrane and the nucleus (Figure 1B). From the images, it is clear that different parts of the cytoplasm have a wide variation in intensity presumably because of the different dye concentrations in the endoplasm, lipid droplets, ribosomes, and other organelles in the cytoplasm. In the present work, we focus on the region of uniform intensity for recording the emission spectrum, FCS, and solvation dynamics using DCM as a probe in the cytoplasm. In contrast to DCM, DNA binding dye DAPI is located in the nucleus of the cell (Figure 1C), which contains the genetic information (and DNA) of the cell. Figure 1D shows a confocal image of CHO cells stained with both DAPI (blue) and DCM (red), which clearly reveals both the cytoplasm and the nucleus

Table 1. Diffusion Constants of the Dyes in Different Systems dye

cell (μm2/s)

water (μm2/s)

DCM DAPI

2 (in cytoplasm) 15 (in nucleus)

300 430

(Dt) of DCM in cytoplasm is 2 μm2 s−1 (i.e., 150 times slower than that in bulk water). The diffusion coefficient of DCM in the cytoplasm measured by us is close to the value (∼1 μm2 s−1) of the diffusion coefficient of an albumin protein inside the cytoplasm reported by Lang et al.44 Thus it seems that DCM moves within the cytoplasm not in the free state but being bound to a biological macromolecule. The diffusion of DCM or HPTS (Scheme 1) bound to human serum albumin protein in bulk water is ∼55 μm2 s−1 (Figure 2D). The diffusion coefficient of bound DCM is about 25 times smaller in the cytoplasm (2 μm2 s−1) compared to that of DCM bound to 2292

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Figure 3. Emission spectra: (A) DCM in ethanol (red), cytoplasm of CHO cells (black), 2-propanol (green), and 2-methyl propan-2-ol (blue). (B) DAPI in water (red), nucleus of CHO cells (black), 50/50 methanol/water (green), and methanol (blue).

Figure 4. (A) Fluorescence decays of DCM under two-photon excitation (2PE) at 810 nm in the cytoplasm of a single live CHO cell. (B, C) Comparison between the decay in autofluorescence (green, CHO cell without DCM) and DCM in the CHO cell (red) under 2PE. (D) Decay of the solvent response function, C(t), of DCM under 2PE (λex = 810 nm). Insets show time-resolved emission spectra (TRES): 0 ps (black), 500 ps (red), 1500 ps (green), and 5000 ps (blue).

HSA in bulk water (55 μm2 s−1). This suggests that the local viscosity inside the cytoplasm is 27 times larger than that in bulk water. The diffusion coefficient of DAPI in the nucleus is 15 μm2 −1 s . This is 30 times slower than that of DAPI in buffer (430 μm2 s−1). In this case, DAPI may also move inside the nucleus while being bound to DNA. The diffusion of bound DAPI inside the nucleus is ∼7 times faster than that of bound DCM (2 μm2 s−1) in the cytoplasm. Thus, the nucleus seems to be less crowded than the cytoplasm. 3.3. Emission Maxima and Polarity of the Cytoplasm and Nucleus. The emission maximum of both DCM45−48 and DAPI49−52 depends strongly on the polarity of the medium and hence is routinely used to measure the polarity of biological systems.45−52

Figure 3A,B describes the emission spectra of DCM and DAPI, respectively, inside the CHO cell along with those in several solvents (all recorded under 2PE in the microscope). In the case of DCM, the emission maximum of the dye in the cytoplasm (at 600 nm) is found to be blue-shifted compared to that in bulk ethanol (615 nm). For DAPI, the emission maxima is blue-shifted by 5 nm compared to that of bulk buffered water (465 nm). This suggests that the cell is less polar than bulk water. A closer comparison of the emission maxima reveals further differences in the polarity of the cytoplasm and nucleus region. For DCM, the emission maxima in cytoplasm (∼600 nm) is almost identical to that of DCM in isopropanol (602 nm), is blue-shifted from that in ethanol (615 nm), and is red-shifted compared to that (595 nm) in 2-methyl propan-2-ol. Thus, the 2293

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Figure 5. (A) Fluorescence decays of DAPI under two-photon excitation (2PE) at 810 nm in a nucleus of a single live CHO cell. (B) Time-resolved emission spectra (TRES): 0 ps (black), 500 ps (red), 1500 ps (green), and 4000 ps (blue). (C) Comparison of the decay of the C(t) of DAPI in the nucleus (red) and DCM in the cytoplasm (blue) under 2PE.

Table 2. Parameters of the Solvation Correlation Function C(t) in Different Systems Obtained under Two-Photon Excitation position

excitation

emission maxima (nm)

Δν (cm−1)

τ1 (a1) (ps)

τ2 (a2) (ps)

⟨τsol⟩ (ps)

cytoplasm (DCM) nucleus (DAPI)

2 PE 2 PE

600 460

1350 650

200 (0.4) 190 (0.54)

1950 (0.6) 1460 (0.46)

1250 775

hydrogen bond energies are not uniform, and this is the source of the broadening O−H stretch in the IR spectrum and is an area of intense recent activity.56,57 The broader emission spectrum in water (compared to that in methanol) may originate from this (i.e., hydrogen bonding). 3.4. Solvation Dynamics of DCM in Cytoplasm. To record the fluorescence decays, we had to use a much higher concentration of dye molecules (∼50 times higher than that used for FCS studies). Thus, the number of dye molecules present in the confocal volume is nearly 1000. Figure 4 shows the wavelength-dependent fluorescence decays of DCM in a single live cell recorded under 2PE. As discussed previously by many workers,14,40,41 in the case of solvation dynamics one observes a decay at a short wavelength (blue end) and a rise at a long wavelength (red end). In the present case for both probes, we observed a distinct rise at the red end of emission (240 ps at λem ≈ 720 nm for DCM and 140 ps at λem ≈ 600 nm for DAPI). This clearly indicates solvation dynamics occurring inside the cell. The time constants of decay and rise for 2PE and 1PE (Supporting Information) are identical, but their amplitudes vary. It is clearly seen that under the conditions of our experiment (1PE and 2PE) the contributions of autofluorescence are negligible (Figures 4B,C and S3 in Supporting Information). The time-resolved emission spectra (TRES, Figure 5) were constructed from the steady-state emission and decays. The TRES suggest that the total dynamic stokes shift (DSS) is 1350 ± 100 cm−1 (Table 2). Figure 4D shows the decay of C(t) and the time-resolved emission spectra (TRES) under 2PE (λex =

polarity of cytoplasm is in between that of isopropanol (ε = 18)53 and 2-methyl propan-2-ol (ε = 11).54 The polarity or dielectric constant (ε) of cytoplasm is estimated to be ∼15.53,54 This is about 5-fold smaller than that of water, and hence the microenvironment of DCM in the cytoplasm is much less polar than that of bulk water. For DAPI, the emission maximum in the nucleus is at 460 nm and is identical to that in a 50% water/methanol mixture (v/v). This is red-shifted from that in methanol (455 nm) and blue-shifted from that in water (465 nm). The position of the emission maximum of DAPI in the CHO nucleus (460 nm) observed in this work is very close to that reported for DAPI bound to the minor groove of DNA27,28,49,50 and DAPI in the nucleus of various biological cells.51,52 Thus, the polarity of the nucleus of a CHO cell (reported by DAPI) is similar to that of a 50% water/methanol mixture. Thus dielectric constant (ε) of the nucleus is ∼60.55 This is slightly lower than that in bulk water (∼78) and is much higher than that reported by DCM (∼15) in the cytoplasm. It may be noted that the width of the emission spectra (or, more precisely, the fwhm) of both probes in ethanol and methanol is smaller than that in the cell (cytoplasm or nucleus) and in water. The fwhm is a measure of the fluctuations (inhomogeneity) of the energies of interaction of the solute with the medium. In the case of the cell, the main source of fluctuation is the inherent inhomogeneity of the complex media with many components. It is interesting to note that even in water the fwhm is quite broad. It may be noted that water forms a stronger hydrogen bond than alcohols. In water, the 2294

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810 nm). The decay suggests two time components: 200 ps (40%) and 1950 ps (60%) with an average solvation time ⟨τsol⟩ of ∼1250 ps (Table 2) under 2PE. The total dynamic Stokes shift of DCM in cytoplasm is nearly 3, 1.4, and 1.3 times greater than those of DCM in anionic micelle SDS (450 cm−1),47 neutral TX-100 (950 cm−1),47 and HSA protein (1030 cm−1)48, respectively. 3.5. Solvation Dynamics of DAPI in the Nucleus. Figure 5A shows the wavelength-dependent fluorescence decays of DAPI in a single live cell under two-photon excitation (2PE). Figure 5B shows time-resolved emission spectra (TRES): 0 ps (black), 500 ps (red), 1500 ps (green), and 4000 ps (blue). Figure 5C shows a comparison of the decay of the C(t) of DAPI in the nucleus and DCM in the cytoplasm under 2PE. The decay C(t) of DAPI has two components: 190 ps (54%) and 1460 ps (46%). The parameters of C(t) under 2PE are also summarized in Table 2. The observed dynamic Stokes shift of DAPI in the nucleus is found to be ∼650 cm−1. It may be noted that recently Sen and co-workers reported a total DSS of ∼700 cm−1 for DAPI bound to the minor groove of DNA.27,28 3.6. Anisotropy Decay in the Cytoplasm and Nucleus. The anisotropy decay of DCM in the cytoplasm and DAPI in the nucleus (Table 3) is found to be much slower than that in buffer and may be fitted to a biexponential function: ⎡ ⎛ t ⎞ ⎛ t ⎞⎤ r(t ) = r0⎢β exp⎜ − ⎟ + (1 − β)exp⎜ − ⎟⎥ ⎢⎣ ⎝ τslow ⎠ ⎝ τfast ⎠⎥⎦

Figure 6A,B shows the anisotropic decay of DCM and DAPI, respectively, recorded under 1PE. Note that under 1PE, in the anisotropic decay, r0 = 0.4. The average rotational time of DCM is 270 ps in water and 1000 ps in the cytoplasm of the cell, so the molecular rotation in the cytoplasm is ∼4 times slower than that in bulk water. Figure 6B shows the anisotropic decay of DAPI in buffer and in the nucleus of a CHO cell. It shows that the average rotational time of DAPI in water is ∼175 ps, which slows down to 600 ps for DAPI bound to the nucleus. Thus, the rotational dynamics of DAPI in the nucleus is ∼3 times slower than that in bulk water.

4. DISCUSSION The most important findings of this work are the following. First, DCM and DAPI localize in different regions of the CHO cell (cytoplasm and nucleus, respectively). Second, the polarity of the nucleus (ε ≈ 60) is much higher than that of cytoplasm (ε ≈ 15). Third, the viscosity (friction) of the nucleus is much less than that of the cytoplasm, both of which are more viscous than bulk water. Fourth, water molecules in the nucleus exhibit 2-fold-faster solvation dynamics than the cytoplasm. These observations may be rationalized in terms of the general architecture of the cell. The cytoplasm of a cell is a gellike material consisting of a 3D network (microtrabecular lattice) of protein-rich strands. There are a large number of units such as ribosome, mitochondria, and endosomes for different kinds of cellular functions. The nucleus, however, is a very polar region because of the presence of a large number of ions (DNA and counterions). The mode and site of binding of DAPI in DNA has been studied by many groups.27−29,49−52 DAPI binds to the minor groove of DNA, making hydrogen bonds with AT base pairs. DAPI does not intercalate in DNA.27−29,49−52 Because of its strong binding to DNA, DAPI preferentially stays in the nucleus and was used earlier to record the microscopic image of the cell.51,52 Hydrophobic dye DCM is much more soluble in a hydrophobic cytoplasmic region than the nucleus.45−48

(8)

Table 3. Anisotropic Decay Parameters of the Dyes in Different Systemsa

a

system

r0

τr1 (ar1), ps

DCM in water DCM in cytoplasm DAPI in water DAPI in nucleus

0.36 0.36 0.38 0.35

270 130 (0.76) 175 100 (0.67)

τr2 (ar2), ps 3850 (0.24) 1600 (0.33)

⟨τrot⟩, ps 270 1000 175 600

All data were recorded using confocal microscopy.

Figure 6. Fluorescence anisotropic decay of (A) DCM (at λex = 470 nm) in water (A1) and in cytoplasm (A2) at λem = 570 nm and (B) DAPI (at λex = 405 nm) in water (B1) and in the nucleus (B2) at λem = 450 nm. 2295

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reactions (proton/electron transfer) and also the differences in dynamics in different regions of a cytoplasm remain to be elucidated. However, it may be emphasized that inside the cytoplasm and nucleus the probe remains bound to a biological macromolecule that has not been clearly identified in this work. The future challenge is to obtain more precise information using specific solvation probes covalently attached to functional biological molecules on specific locations of a cell (mitochondria, endosome, etc).

One of the striking observations is that the dielectric constant (ε) of the nucleus (∼60) is about 4 times higher than that (∼15) in the cytoplasm. The markedly higher polarity of the nucleus may be attributed to the presence of ions in the nucleus. Also, this indicates that the DAPI probe does not intercalate inside the DNA and remains vastly exposed. This is consistent with the binding of DAPI to DNA in bulk water.27,28,49−52 The diffusion constants or the viscosity (translational friction) values obtained from FCS also indicate that the gellike cytoplasm is about 5 times more viscous than the nucleus. Because the observed diffusion coefficient of DCM in the cytoplasm (2 μm2 s−1) is very close to that reported (1 μm2 s−1) for a protein (albumin)44 in the cytoplasm, it seems that DCM remains bound to a protein or other biological molecule during diffusion within the cytoplasm. Because the diffusion coefficient of DCM bound to human serum albumin in bulk water is 55 μm2 s−1, we estimate that the cytoplasm is ∼27 times more viscous than bulk water. Finally, the solvation dynamics is 2-fold faster in the nucleus than in the cytoplasm. The faster dynamics in the nucleus is consistent with the higher polarity and viscosity in the cytoplasm. It may, however, be pointed out that though the static dielectric constant of the nucleus is close to that of bulk water the solvation dynamics in the nucleus is more than 700 times slower. It seems that the bound water molecules in the nucleus, though slow, are two times faster than in the cytoplasm. The polarity or dielectric constant depends first on the ability of the water molecules to orient in response to an external electric field.58 Second, the dipole moment of one water molecule increases if it is hydrogen bonded to another because of mutual dynamic polarization, and this increases the dielectric constant.59 The lower polarity and slower solvation dynamics in the cytoplasm suggest that there is a large-scale destruction of the water−water hydrogen bond and most of the water molecules remain bound to the macromolecules. The lower solvation dynamics in the cytoplasm and nucleus may be attributed to bound-free interconversion as envisaged by Nandi and Bagchi.34,35 The point to note is that the effect of bound water and water immobilization is more prominent in the cytoplasm than in the nucleus. It is also apparent that compared to translational diffusion (150-fold) the rotational diffusion is retarded to a smaller extent (3- to 4-fold) inside the cell relative to bulk water. The large magnitude of translational diffusion may be attributed to the presence of many organelles in the cytoplasm, which hinders the translational diffusion of DCM. Translational diffusion occurs over a large distance (λ/2NA ≈ 800/2 × 1.2 ≈ 333 nm). The rotational motion, however, reports friction within a small region around the molecule (DCM) and is hence faster.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Decay parameters of the fluorescence decay and C(t) of DCM in the cytoplasm of a CHO cell observed by 1PE and 2PE. Confocal images of CHO cell auto-fluorescence. Emission spectra and fluorescence decay, TRES. Decay of the solvent response function. This material is available free of charge via the Internet at http://pubs.acs.org. 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 the Department of Science and Technology, India, for IRHPA project “Center for Ultrafast Spectroscopy and Microscopy” (project no. IR/S1/CU 02/2009), the Council for Scientific and Industrial Research (CSIR), and the J. C. Bose Fellowship for generous research support. D.K.S., S.G., and A.K.D. thank CSIR for awarding fellowships. We are grateful to Sankha Pattanayek (Department of Organic Chemistry, IACS) for his help and support regarding cell culturing.

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REFERENCES

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5. CONCLUSIONS This work demonstrates for the first time the differences in polarity, viscosity, and solvation dynamics in the nucleus compared to those in the cytoplasm of a single live cell. It is shown that the dielectric constant of the nucleus is 4-fold higher compared to that in the cytoplasm whereas the diffusion and solvation in the nucleus are 7-fold and 2-fold faster, respectively. This suggests that the nucleus is less crowded and more labile and experiences a lower screening of the Coulomb force field. The implications of these processes in polar 2296

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