Total Internal Reflection Two-Dimensional Fluorescence Lifetime

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

pubs.acs.org/JPCB

Total Internal Reflection Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy Takuhiro Otosu and Shoichi Yamaguchi* Department of Applied Chemistry, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura, Saitama 338-8570, Japan S Supporting Information *

ABSTRACT: Fluorescence lifetime correlation analysis is becoming a powerful tool to understand the conformational heterogeneity of biomolecules and their dynamics with an unprecedented detection sensitivity and time resolution. However, its application to the study of biomembranes is very limited. Here, we report on two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) in combination with total internal reflection (TIR) microscopy (TIR 2D-FLCS). High depth resolution in TIR microscopy and speciesspecific correlation analysis in 2D FLCS give us the opportunity to selectively analyze molecules in or on a supported lipid bilayer, a model biomembrane formed on the glass surface. Feasibility experiments performed in this study clearly demonstrated that TIR 2D-FLCS has a potential to selectively analyze the diffusion and the conformational dynamics of proteins peripherally bound on the membrane in the presence of substantial amounts of unbound molecules in the bulk phase.

■

INTRODUCTION Fluorescence correlation spectroscopy (FCS) has been widely applied not only to the study of biomolecules in solution but also to the study of biomembranes.1−5 Those include the diffusion analysis of lipids in model biomembranes and the study of the association/dissociation kinetics of proteins on the membranes.2,6,7 The advantages of FCS over other singlemolecule spectroscopies are its detection sensitivity, time resolution and statistical reliability, which makes FCS a powerful tool in the field of biology.8 In conventional FCS, one analyzes the fluctuation of fluorescence intensity so that the origin of the fluctuation must be correctly identified for quantitative interpretation of the data.1,9,10 Actually, this is a challenging task, especially when more than two fluorescent species are involved in the fluorescence fluctuation. This drawback is especially problematic when studying cooperative dynamics of several constituents on biomembranes or analyzing the diffusion and the conformational dynamics of weakly bound peripheral membrane proteins in the presence of substantial amount of unbound molecules in the bulk phase. Because understanding the dynamics of peripheral membrane proteins upon binding to lipid membranes is crucial to elucidate various biological functions mediated by such proteins,11−14 further improvement of the FCS technique is highly required. To overcome this drawback, improved variants of FCS have been reported so far. Those include fluorescence crosscorrelation spectroscopy (FCCS), fluorescence lifetime correlation spectroscopy (FLCS) and two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS).15−23 Among them, the correlation analysis of fluorescence lifetime in 2D © XXXX American Chemical Society

FLCS (and FLCS) is advantageous due to its high sensitivity to the detection of multiple species.22−25 Indeed, several reports have shown the usefulness of these techniques to detect the conformational heterogeneity of biomolecules in solution and their conformational dynamics.23,25,26 However, the application to the study of biomembranes is still limited.27 One possible reason is the low depth resolution of a conventional confocal microscope that is usually used for 2D FLCS and FLCS, which makes it difficult to selectively analyze biomembranes and membrane proteins. Here, we performed 2D FLCS on a total-internal reflection microscope (TIR 2D-FLCS) with the aim of analyzing the diffusion and the conformational dynamics of lipids and membrane proteins in/on a supported lipid bilayer (SLB), a model biomembrane formed on a glass substrate. High depthresolution achieved by the evanescent-wave excitation of TIR and the fluorescence lifetime-correlation analysis in 2D FLCS enable the quantitative analysis of molecules on or in the vicinity of SLB. Very recently, a TIR-based FLCS instrument was reported by Weger and Hoffmann-Jacobsen.28 However, the actual application of the system to the fluorescence lifetime correlation analysis has not been done so far. In this study, the feasibility of TIR 2D-FLCS is demonstrated for the first time by performing simultaneous FCS analysis of two molecules, fluorescent lipids in SLB and dye-labeled DNA in bulk solution above the SLB, each of which has different fluorescence Received: February 2, 2018 Revised: May 15, 2018

A

DOI: 10.1021/acs.jpcb.8b01176 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B lifetime. Actually, this model system mimics the situation that peripheral membrane proteins such as cytochrome c occasionally bind to a lipid membrane and change the conformation (fluorescence lifetime) on the membrane. The results in this study showed that TIR 2D-FLCS can be a powerful tool to selectively analyze the diffusion, the conformational dynamics, and the association/dissociation kinetics of molecules weakly bound on biomembranes in the presence of substantial unbound molecules in the bulk phase.

■

MATERIALS AND METHODS Samples. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho- L -serine (DOPS) were purchased from Avanti Polar Lipids, and (tetramethylrhodamine-6-thiocarbamoyl)-1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine (TRITC-DHPE) was purchased from Biotium. Double-stranded DNA (15 bp) that is labeled with Cy3 at the 3′-terminus of one strand (hereafter denoted as Cy3-DNA) was purchased from Hokkaido SystemScience. Other reagents were of analytical grade and used without purification. Preparation of a Supported Lipid Bilayer. A supported lipid bilayer (SLB) was prepared by the vesicle-fusion method described in the literature.6 Briefly, a lipid mixture (DOPC:DOPS = 9:1 (w/w)) containing a small amount of fluorescent lipids (TRITC-DHPE, ∼3 × 10−5 wt %) in chloroform was dried on a glass vial in a low-pressure desiccator for ∼3 h. The resulting lipid film was suspended by using the buffer (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 1 mM CaCl2). Liposomes were prepared by the extrusion of the lipid suspension using a 100 nm polycarbonate filter and a miniextruder (Avanti Polar Lipids). Liposome solution was then added on a glass coverslip that was precleaned by piranha solution. After the incubation for 30 min, the glass surface was rigorously washed using the same buffer to remove unreacted liposomes, which gives a homogeneous SLB on the glass surface. Cy3-DNA (∼1 μM) dissolved in the same buffer was then added to the solution phase above the SLB. Final concentration of Cy3-DNA was approximately 15 nM. Measurements were done after 30 min incubation. TIR 2D-FLCS Instrument. Figure 1 shows a schematic of a TIR 2D-FLCS instrument. A picosecond diode laser (BDL510-SMN, Becker & Hickl GmbH) is used for the excitation light source. The repetition rate is set to 50 MHz. After expanding the beam by passing through a couple of focus lens with different focal lengths, the laser power is attenuated by using neutral density (ND) filters. The beam diameter after the expansion is ∼5 mm. The beam is then separated into two by a trapezoidal beamsplitter and two beams pass through the different optical paths after being reflected by a right-angle mirror. In this study, the performance of TIR-FCS was demonstrated by comparing the results with those of conventional confocal-based FCS. For that purpose, one of two beams is used for TIR-FCS (and TIR 2D-FLCS) and the other is used for confocal FCS. In a TIR geometry, the beam diameter is reduced to ∼1 mm and the beam enters the periphery of the objective (CFI Apo TIRF, 60×, NA 1.49, NIKON). The incident angle of the beam at the glass surface is set to ∼69°. (The incident angle is measured by using a hemisphere.6) This angle is enough for total internal-reflection at the glass−water interface. Thus, the beam is totally reflected, and the evanescent wave is generated on the glass surface. On the other hand, the beam enters the center of the objective in a

Figure 1. Schematic illustration of two-dimensional fluorescence lifetime correlation spectroscopy instrument utilizing total internalreflection microscope. Key: BE, beam expander; BS, beam splitter; DM, dichroic mirror; BF, bandpass filter; LF, long-pass filter; SPAD, single-photon avalanche photodiode; TCSPC, time-correlated singlephoton counting.

confocal geometry. One of two geometries can be selected by blocking one of the two laser beams in front of the second right-angle mirror. Therefore, this instrument enables us to easily switch the system either for TIR-FCS or confocal FCS without substantial changes in the optical system. As for the detection of fluorescence, the same optical configuration is used for both geometries. Fluorescence from the sample is collected by the same objective and separated from Rayleigh scattering by passing through a dichroic mirror, a long-pass filter (ET542lp, Chroma Technology) and a bandpass filter (ET575/50m, Chroma Technology). The fluorescence is then detected by a single-photon avalanche photodiode (SPD-050-CTE-FC, Micro Photon Devices) through a multimode fiber (the core diameter: 50 μm). A tube lens with f = 200 mm is placed in front of the multimode fiber to focus the fluorescence on the fiber end. The small core diameter of the fiber works as a pinhole. It is noted that the lateral position of the fiber end is slightly adjusted when switching the system either for TIR-FCS or confocal FCS. On the other hand, the position along the optical axis is kept the same. The electric signals from the avalanche photodiode are sent to a photon counting board (SPC-130EM, Becker & Hickl), and temporal information, that is, macrotime (T) and microtime (t), is collected for all detected photons. Macrotime is the absolute detection time of photon from the start of the experiment whereas microtime is the emission delay time of the photon detection with respect to the corresponding excitation pulse. The latter is calculated by referring synchronous signals from the diode laser. The microtime resolution is 4.1 ps in this study. Estimation of the Lateral and Depth Dimensions of the Detection Area. In a TIR configuration, the lateral dimension of the detection area is estimated from the magnification value of the objective, the reference and actual focal lengths of a tube lens (the reference focal length is 200 mm in our setup), and the size of a pinhole. In the TIR 2DFLCS instrument reported in this study, the lateral detection radius was then estimated to be 0.42 μm. On the other hand, the depth dimension of the detection area is predominantly determined by the penetration depth (d) of the evanescent B


Article

The Journal of Physical Chemistry B

noted that eq 4 assumes the Gaussian profile of the molecular detection function, which is not strictly correct in TIR-FCS.34 Therefore, this might also be the reason for the difference between the expected and the actual lateral dimension. On the basis of the measurement described above, the lateral detection radius and the detection depth in the TIR 2D-FLCS instrument were estimated to be 0.77 μm and 91.5 nm, respectively. Data Analysis. The analysis of photon data is composed of two parts. First, 2D FLCS analysis is performed on the photon data to identify the independent lifetime species and to determine their fluorescence lifetimes (or corresponding fluorescence decay curves).22,23,35 Second, FLCS analysis is applied to calculate the fluorescence correlation curve of each independent species by utilizing the fluorescence decay curves of those species that are obtained by 2D FLCS analysis.15,21,27,36 The details of 2D FLCS and FLCS analyses are described in the literatures and in the Supporting Information.15,22,23,35 Briefly, in 2D FLCS analysis, one searches photon pairs that have the macrotime interval of ΔT between two photons and builds a 2D histogram of the photon pairs according to the microtimes of two photons in the pairs.35 In the obtained 2D emission-delay correlation map (M(ΔT, t′, t″)), t′ and t″ correspond to the microtimes of first and second photons in photon pairs, respectively. After subtracting the uncorrelated signals from the map, 2D inverse Laplace transform (ILT) is performed on the resulting correlation map (Mcor (ΔT, t′, t″)) to convert the map to a 2D lifetime correlation map (M̃ (ΔT, τ′, τ″)) with the help of 2D maximum entropy method (MEM).22,23 Because Mcor (ΔT, t′, t″) is described with the sum of the single molecule correlation, which can be represented by the following equations,

wave and is estimated from the incident angle (θ) of the excitation beam as follows:29 d=

λ (n12 sin 2 θ − n2 2)−0.5 4π

(1)

In eq 1, λ is the excitation wavelength, and n1 and n2 are the reflective indices of glass and water, respectively. The depth dimension in the TIR 2D-FLCS instrument was then estimated to be 86 nm. However, these estimated values can be different from the actual ones due to several reasons such as the optical aberration and the uncertainty of the incident angle. Therefore, the actual dimensions were analyzed by measuring the diffusion of the standard samples with known diffusion coefficients. First, the depth dimension of the detection area was analyzed by measuring the diffusion of tetramethylrhodamine (TAMRA) in 10 mM Tris-HCl, pH 7.35 containing 150 mM NaCl. In this measurement, 150 mM NaCl was added to the solution to minimize the electrostatic interaction between TAMRA and glass.30 Fluorescence correlation curve of TAMRA in the vicinity of the glass surface measured with TIR 2D-FLCS setup is shown in Figure S1a. The data were fitted with the following theoretical equation:31 −1⎧ ⎛ ΔT ⎞ ΔT ⎞ ⎪⎛ ΔT ⎞ 1⎛ − G(ΔT ) = 1 + ⎜1 + 2 ⎟ ⎨ 1 exp ⎜ ⎟ ⎜ ⎟ ⎪ N⎝ 2τz ⎠ ω τz ⎠ ⎩ ⎝ ⎝ 4τz ⎠

⎛ ΔT ⎞ erfc⎜⎜ ⎟⎟ + ⎝ 4τz ⎠

⎫ ΔT ⎪ ⎬ ⎪ πτz ⎭

(2)

In eq 2, N is the average number of the sample molecules in the detection area, ω is the ratio between the lateral and depth dimensions of the detection area, and τz is the diffusion time of the sample along the depth direction defined by eq 3,

L

Mcor(ΔT ; ti′, t ″j ) =

(3)

exp( −t ″j /τl″)

where h corresponds to the detection depth and D is the diffusion coefficient of the sample. On the basis of the fitting result and the diffusion coefficient of rhodamine 6G in solution (4.1 × 10−10 m2 s−1),32 the depth dimension (h) was estimated to be 91.5 nm. (The fitting curve is also shown in Figure S1a.) This value is a good agreement with the value estimated from the incident angle (86 nm). Next, the lateral dimension of the detection area was analyzed by measuring the diffusion of fluorescent lipids in a supported DOPC bilayer (DOPC SLB). Fluorescence correlation curve of the fluorescent lipids in DOPC SLB is shown in Figure S1b. The data were fitted with the following theoretical equation:

τxy = w 2 /4D

∑ ∑ M̃ (ΔT ; τk′, τl″) exp(−ti′/τk′) k=1 l=1

τz = h2 /4D

G(ΔT ) = 1 +

L

−1 ΔT ⎞⎟ 1 ⎛⎜ + 1 τxy ⎟⎠ N ⎜⎝

(6)

n

M̃ (ΔT ; τk′, τl″) =

∑ ai(τk′)ai(τl″)

(7)

i=1

this conversion procedure tells us the number (n) and the lifetime distribution (a(τ)) of independent species resolved at ΔT. (In eq 6, L is the available number of τ points in the analysis.) After determining the number and the fluorescence lifetimes (fluorescence decay curves) of the independent species by 2D FLCS analysis, FLCS analysis is performed using that information.37 In FLCS analysis, one can calculate the filter function ( f(t)) of each species by using the decay curve of each species (that is determined by 2D FLCS analysis) and the ensemble-averaged fluorescence decay curve that is obtained by building a microtime histogram of all detected photons.15 Then, the autocorrelation of species i as well as the crosscorrelation between species i and j can be calculated using the filter function as follows:

(4) (5)

where w corresponds to the lateral radius of the detection area. On the basis of the fitting result and the diffusion coefficient of fluorescent lipids in DOPC SLB (4.0 × 10−12 m2 s−1) that was reported by Benda et al.,33 the lateral radius of the detection area was estimated to be 0.77 μm. This value is slightly larger than the value (0.42 μm) expected from the optical configuration. This difference might stem from the optical aberration or small misalignment of the optics. However, it is

gij(ΔT ) =

⟨Ii(T )Ij(T + ΔT )⟩ ⟨Ii(T )⟩⟨Ij(T )⟩ t max

=

t max

∑t = 0 ∑t ′= 0 fi (t )f j (t ′)⟨I(t , T )I(t ′, T + ΔT )⟩ t max

t max

∑t = 0 ⟨fi (t )I(t , T )⟩∑t ′= 0 ⟨f j (t ′)I(t ′, T )⟩ (8)

C


Article


Figure 2. Fluorescence decay curves (a) and the fluorescence correlation curves (b, c) of fluorescent lipids in a supported DOPC/DOPS (9/1 (w/ w)) bilayer and Cy3-labeled DNA in a solution phase. Data were measured with either total internal-reflection (TIR) geometry (red solid line) or confocal geometry (blue solid line). The fluorescence decay curves shown in part a were normalized by the counts at microtime of 0 ns. The correlation curves shown in part c were normalized by the correlation amplitude at a macrotime delay of 10 μs.

Point brackets represent T-averaging, I(T) is the fluorescence intensity at T and tmax is the maximum microtime (or the corresponding microtime channel) used for the analysis. If i = j, gij (ΔT) corresponds to the autocorrelation of species i whereas gij (ΔT) corresponds to the cross-correlation between species i and j when i ≠ j. Species-specific autocorrelation then gives us the information about the diffusion and the photophysics of the corresponding species. On the other hand, cross-correlation between two species represents the interconversion between these species and its time scale. The combination of 2D FLCS and FLCS analyses thus enables us to analyze the diffusion and the conformational dynamics of each independent species from their specific fluorescence correlation curves in a model-free manner.37

lipids, is a major component in the data of a TIR geometry. On the other hand, faster decay component corresponding to DNA is dominant in the data measured with a confocal geometry (Figure 2c). Thus, the result shown in Figure 2 clearly showed that the selective detection of molecules in the vicinity of the glass surface is possible with a TIR 2D-FLCS instrument. Next, 2D FLCS analysis was performed on photon data measured with a TIR geometry to identify the independent species and to determine their fluorescence lifetime distributions. In this analysis, 2D emission-delay correlation maps at ΔT = 50−300 and 400−700 μs were constructed and globally analyzed by a global 2D MEM analysis (see Supporting Information).23 Results are shown in Figure 3. (2D emissiondelay correlation maps at ΔT = 50−300 and 400−700 μs are shown in Figure S2.) On the basis of the analysis, two major components (red and blue solid lines) and one minor component (green solid line) were detected as shown in Figure 3a, which are denoted as sp1, sp2, and sp3. To identify the origin of these species, Laplace transform was performed on the independent lifetime distributions to convert the data to the fluorescence decay curves. Calculated fluorescence decay curve of each species is shown in Figure 3b. For comparison, the fluorescence decay curves of fluorescent lipids in 10% DOPS SLB and of Cy3-DNA, both of which were measured with a TIR geometry, are also shown. In the latter measurement, a coverslip was coated with 10% DOPS SLB without fluorescence lipids. As shown in Figure 3b, the fluorescence decay curve of sp1 matches with that of Cy3DNA, and the decay curve of sp2 is indistinguishable from that of fluorescent lipids in 10% DOPS SLB. On the basis of this result, one can safely assign sp1 to Cy3-DNA and sp2 to fluorescent lipids. Remaining species (sp3) was then assigned to a fluorescent contaminant contained in DOPC or DOPS lipids (Supporting Information and Figure S3). Therefore, the result shown in Figure 3 strongly suggests that the extraction of independent species and their fluorescence lifetimes (decay curves) is possible with TIR 2D-FLCS. The final goal of this study is to extract the species-specific correlation curves of fluorescent lipids and Cy3-DNA. For that purpose, FLCS analysis was performed on the photon data by using the fluorescence decay curves of sp1, sp2, and sp3 shown in Figure 3b. Filter functions of sp1, sp2, and sp3 were calculated based on the procedure described in the Supporting Information, and the species-specific correlation curves were calculated using eq 8. (Filter function of each species is shown in Figure S4.) Figure 4 shows the autocorrelation curves of sp1 and sp2, and the cross-correlation between sp1 and sp2. (Other auto- and cross-correlation curves (e.g., the autocorrelation of sp3) are shown in Figure S5.) For comparison, the correlation curves of fluorescent lipids in 10% DOPS SLB and of Cy3-

■

RESULTS AND DISCUSSION As a feasibility test, TIR 2D-FLCS was performed on a sample containing fluorescence lipids in a supported DOPC/DOPS (9/1 (w/w)) bilayer (10% DOPS SLB) and Cy3-labeled DNA (Cy3-DNA) in the bulk solution above the SLB. Fluorescence lifetime of TAMRA (a chromophore of TRITC-DHPE) is reported to be ∼2.3 ns whereas that of Cy3 is ∼0.3 ns by the manufacturer. Therefore, the fluorescence lifetime of Cy3-DNA is expected to be substantially shorter than that of fluorescent lipids even though the lifetime of Cy3 becomes longer upon conjugation with DNA.38,39 The electrostatic interaction between DNA and 10% DOPS SLB is repulsive, and thus, DNA resides predominantly in a solution phase. Therefore, the diffusion of DNA is anticipated to be much faster than that of fluorescent lipids. Thus, this sample is suitable to check whether the fluorescence lifetime and the diffusion time (or coefficient) of each fluorescent species (fluorescent lipids and DNA) are correctly retrieved by TIR 2D-FLCS. First, the performance of TIR 2D-FLCS instrument was checked by measuring ensemble-averaged fluorescence decay and fluorescence correlation curves of the sample with a TIR or confocal geometry. The focal plane is adjusted to SLB in the whole measurements. As observed in Figure 2a, the fluorescence decay curve measured with a confocal geometry decays rapidly with a lifetime of ∼1 ns. On the other hand, a substantial contribution from a longer lifetime component, which presumably corresponds to the signals from fluorescent lipids on SLB, is clearly seen in the data measured with a TIR geometry. The contribution of fluorescence lipids is also seen in the fluorescence correlation curve obtained with the same geometry (Figure 2b,c). The correlation curve measured with a TIR geometry decays with two decay times, one with ∼100 μs and the another with ∼1 s. Longer decay component, which is assignable to the translational diffusion time of fluorescent D


Article


Figure 3. Independent lifetime distributions (a) and the corresponding fluorescence decay curves (b) that are obtained from the photon data by 2D FLCS analysis. Photon data were measured on a sample containing fluorescent lipids in a supported DOPC/DOPS (9/1 (w/ w)) bilayer and Cy3-labeled DNA in a solution phase. In the analysis, two-dimensional emission-delay correlation maps at ΔT = 50−300 and 400−700 μs were constructed and globally analyzed by a global 2D MEM analysis. Laplace transform was performed on the lifetime distributions in part a, and the calculated fluorescence decay curves are shown in part b (broken line). In part b, the fluorescence decay curves of Cy3-labeled DNA on a supported DOPC/DOPS (9/1 (w/w)) bilayer without fluorescent lipids (DNA, red solid line) and of fluorescent lipids (∼0.1 wt %) in a supported DOPC/DOPS (9/1 (w/ w)) bilayer (TRITC, blue solid line) are also shown for comparison. The data shown in part b are normalized by the counts at a microtime of 0 ns.

Figure 4. Species-specific autocorrelations of sp1 (red broken line) and sp2 (blue broken line), and the cross-correlation between sp1 and sp2 (green broken line). For comparison, the correlation curve of Cy3DNA on a supported DOPC/DOPS (9/1 (w/w)) bilayer without fluorescent lipids (red solid line) and that of TRITC-DHPE in a supported DOPC/DOPS (9/1 (w/w)) bilayer (blue solid line) are also shown, both of which were normalized to match the curves to those of sp1 and sp2, respectively.

peripheral membrane proteins in the presence of substantial unbound molecules in the bulk phase. Understanding the association/dissociation kinetics of peripheral membrane proteins and their conformational dynamics upon binding to lipid membranes are essential to elucidate the sophisticated signal transductions and respiratory reactions mediated by these proteins.11,12,40 Thus, application of TIR 2D-FLCS will open up the opportunity to understand the molecular mechanism underlying such biological functions.

DNA in the vicinity of 10% DOPS SLB (without fluorescent lipids) are also shown. As is clearly observed in Figure 4, the autocorrelation curves of sp1 and sp2 perfectly match with those of Cy3-DNA and fluorescent lipids, respectively, confirming the result of 2D FLCS analysis. The crosscorrelation between sp1 and sp2 is unity in the whole ΔT regions. Other cross-correlations (e.g., between sp2 and sp3) also show gij (ΔT) = 1 for all ΔT regions (Figure S5). The absence of cross-correlation is reasonable because Cy3-DNA, a fluorescent lipid and a contaminant are independent and no interconversion among these species is expected to occur. Thus, the results of 2D FLCS and FLCS analyses strongly suggest that the diffusion and the conformational dynamics of each independent species are correctly and separately analyzed based on their species-specific auto- as well as cross-correlation curves.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b01176. Detailed descriptions of 2D FLCS and FLCS analyses, the assignment of sp3, and supporting figures (Figures S1−S5) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*(S.Y.) E-mail: [email protected]. Telephone: +8148-858-3521.

■

ORCID

CONCLUSIONS In this study, TIR 2D-FLCS instrument was constructed and applied to a sample containing fluorescence lipids in 10% DOPS SLB and Cy3-DNA in the bulk solution above the SLB. The results demonstrated that the fluorescence lifetime and correlation curve of each molecule are correctly retrieved by the combination of 2D FLCS and FLCS analyses. This also suggests that TIR 2D-FLCS is applicable to selectively analyze the diffusion and the conformational dynamics of weakly bound

Takuhiro Otosu: 0000-0001-8421-242X Shoichi Yamaguchi: 0000-0002-2710-5983 Author Contributions

T.O. performed the experiments. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. E


Article


■

(20) Olofsson, L.; Margeat, E. Pulsed Interleaved Excitation Fluorescence Spectroscopy with a Supercontinuum Source. Opt. Express 2013, 21, 3370−3378. (21) Kapusta, P.; Machan, R.; Benda, A.; Hof, M. Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook. Int. J. Mol. Sci. 2012, 13, 12890−12910. (22) Ishii, K.; Tahara, T. Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy. 1. Principle. J. Phys. Chem. B 2013, 117, 11414−11422. (23) Ishii, K.; Tahara, T. Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy. 2. Application. J. Phys. Chem. B 2013, 117, 11423−11432. (24) Otosu, T.; Ishii, K.; Oikawa, H.; Arai, M.; Takahashi, S.; Tahara, T. Highly Heterogeneous Nature of the Native and Unfolded States of the B Domain of Protein A Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy. J. Phys. Chem. B 2017, 121, 5463−5473. (25) Otosu, T.; Ishii, K.; Tahara, T. Microsecond Protein Dynamics Observed at the Single-Molecule Level. Nat. Commun. 2015, 6, 7685. (26) Felekyan, S.; Kalinin, S.; Sanabria, H.; Valeri, A.; Seidel, C. A. M. Filtered FCS: Species Auto- and Cross-Correlation Functions Highlight Binding and Dynamics in Biomolecules. ChemPhysChem 2012, 13, 1036−1053. (27) Benda, A.; Fagul’ova, V.; Deyneka, A.; Enderlein, J.; Hof, M. Fluorescence Lifetime Correlation Spectroscopy Combined with Lifetime Tuning: New Perspectives in Supported Phospholipid Bilayer Research. Langmuir 2006, 22, 9580−9585. (28) Weger, K.; Hoffmann-Jacobsen, K. A. Total Internal ReflectionFluorescence Correlation Spectroscopy Setup with Pulsed Diode Laser Excitation. Rev. Sci. Instrum. 2017, 88, 093102. (29) Hassler, K.; Leutenegger, M.; Rigler, P.; Rao, R.; Rigler, R.; Gö sch, M.; Lasser, T. Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS) with Low Background and High Count-Rate Per Molecule. Opt. Express 2005, 13, 7415−7423. (30) Blom, H.; Hassler, K.; Chmyrov, A.; Widengren, J. Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy. Int. J. Mol. Sci. 2010, 11, 386−406. (31) Hassler, K.; Anhut, T.; Rigler, R.; Gösch, M.; Lasser, T. High Count Rates with Total Internal Reflection Fluorescence Correlation Spectroscopy. Biophys. J. 2005, 88, L01−L03. (32) Müller, C. B.; Loman, A.; Pacheco, V.; Koberling, F.; Richtering, W.; Enderlein, J.; et al. Precise Measurement of Diffusion by MultiColor Dual-Focus Fluorescence Correlation Spectroscopy. EPL 2008, 83, 46001. (33) Benda, A.; Beneš, M.; Mareček, V.; Lhotsky, A.; Hermens, W. Th.; Hof, M. How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy. Langmuir 2003, 19, 4120−4126. (34) Ries, J.; Petrov, E. P.; Schwille, P. Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion and Surface-Generated Fluorescence. Biophys. J. 2008, 95, 390−399. (35) Ishii, K.; Tahara, T. Extracting Decay Curves of the Correlated Fluorescence Photons Measured in Fluorescence Correlation Spectroscopy. Chem. Phys. Lett. 2012, 519-520, 130−133. (36) Kapusta, P.; Wahl, M.; Benda, A.; Hof, M.; Enderlein, J. Fluorescence Lifetime Correlation Spectroscopy. J. Fluoresc. 2007, 17, 43−48. (37) Cheng, C.-H.; Ishii, K.; Tahara, T. RNA and DNA Hairpin Dynamics Studied by Temperature-Controlled 2D Fluorescence Lifetime Correlation Spectroscopy. Proc. Asian Spectrosc. Conf. 2017, 92. (38) Sanborn, M. E.; Connolly, B. K.; Gurunathan, K.; Levitus, M. Fluorescence Properties and Photophysics of the Sulfoindocyanine Cy3 Linked Covalently to DNA. J. Phys. Chem. B 2007, 111, 11064− 11074. (39) Spiriti, J.; Binder, J. K.; Levitus, M.; van der Vaart, A. Cy3-DNA Stacking Interactions Strongly Depend on the Identity of the Terminal Basepair. Biophys. J. 2011, 100, 1049−1057.

ACKNOWLEDGMENTS We thank Prof. Kunihiko Ishii and Prof. Tahei Tahara for the analytical codes of 2D FLCS and FLCS. This work is partly supported by the Tenure-track program in Saitama University (SUTT project) and JSPS KAKENHI Grant Number 17K19097.

■

REFERENCES

(1) Elson, E. L. Fluorescence Correlation Spectroscopy: Past, Present, Future. Biophys. J. 2011, 101, 2855−2870. (2) Machan, R.; Hof, M. Lipid Diffusion in Planar Membranes Investigated by Fluorescence Correlation Spectroscopy. Biochim. Biophys. Acta, Biomembr. 2010, 1798, 1377−1391. (3) Rose, M.; Hirmiz, N.; Moran-Mirabal, J. M.; Fradin, C. Lipid Diffusion in Supported Lipid Bilayers: A Comparison between LineScanning Fluorescence Correlation Spectroscopy and Single-Particle Tracking. Membranes 2015, 5, 702−721. (4) Sterling, S. M.; Allgeyer, E. S.; Fick, J.; Prudovsky, I.; Mason, M. D.; Neivandt, D. J. Phospholipid Diffusion Coefficients of Cushioned Model Membranes Determined via Z-Scan Fluorescence Correlation Spectroscopy. Langmuir 2013, 29, 7966−7974. (5) Schulze, A.; Beliu, G.; Helmerich, D. A.; Schubert, J.; Pearl, L. H.; Prodromou, C.; Neuweiler, H. Cooperation of Local Motions in the Hsp90 Molecular Chaperone ATPase Mechanism. Nat. Chem. Biol. 2016, 12, 628−635. (6) Otosu, T.; Yamaguchi, S. Communication: Development of Standing Evanescent-Wave Fluorescence Correlation Spectroscopy and its Application to the Lateral Diffusion of Lipids in a Supported Lipid Bilayer. J. Chem. Phys. 2017, 147, 041101. (7) Lieto, A. M.; Cush, R. C.; Thompson, N. L. Ligand-Receptor Kinetics Measured by Total Internal Reflection with Fluorescence Correlation Spectroscopy. Biophys. J. 2003, 85, 3294−3302. (8) Tian, Y.; Martinez, M. M.; Pappas, D. Fluorescence Correlation Spectroscopy: A Review of Biochemical and Microfluidic Applications. Appl. Spectrosc. 2011, 65, 115−124. (9) Bacia, K.; Haustein, E.; Schwille, P. Fluorescence Correlation Spectroscopy: Principles and Applications. Cold Spring Harb. Protoc 2014, 2014, 709−725. (10) Schwille, P.; Haustein, E. Fluorescence Correlation Spectroscopy, and Introduction to its Concerpts and Applications. Biophysics Textbook Online; 2004; pp 1−33; https://www.biophysics.org/ Portals/1/PDFs/Education/schwille.pdf. (11) Stahelin, R. V. Monitoring Peripheral Protein Oligomerization on Biological Membranes. Methods Cell Biol. 2013, 117, 359−371. (12) Whited, A. M.; Johs, A. The Interactions of Peripheral Membrane Proteins with Biological Membranes. Chem. Phys. Lipids 2015, 192, 51−59. (13) Corbalan-Garcia, S.; Gomez-Fernandez, J. C. Signaling through C2 Domains: More Than One Lipid Target. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 1536−1547. (14) Karp, G. Cell and Molecular Biology; Concepts and Experiments, 7th ed.; Wiley Global Education: 2013. (15) Enderlein, J.; Gregor, I. Using Fluorescence Lifetime for Discriminating Detector Afterpulsing in Fluorescence Correlation Spectroscopy. Rev. Sci. Instrum. 2005, 76, 033102. (16) Ishii, K.; Tahara, T. Resolving Inhomogeneity Using LifetimeWeighted Fluorescence Correlation Spectroscopy. J. Phys. Chem. B 2010, 114, 12383−12391. (17) Muller, B. K.; Zaychikov, E.; Brauchle, C.; Lamb, D. C. Pulsed Interleaved Excitation. Biophys. J. 2005, 89, 3508−3522. (18) Kettling, U.; Koltermann, A.; Schwille, P.; Eigen, M. Real-Time Enzyme Kinetics Monitored by Dual-Color Fluorescence CrossCorrelation Spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 1416−1420. (19) Krieger, J. W.; Singh, A. P.; Garbe, C. S.; Wohland, T.; Langowski, J. Dual-Color Fluorescence Cross-Correlation Spectroscopy on a Single Plane Illumination Microscope (SPIM-FCCS). Opt. Express 2014, 22, 2358−2375. F


Article

The Journal of Physical Chemistry B (40) Tuominen, E. K. J.; Wallace, C. J. A.; Kinnunen, P. K. J. Phospholipid-Cytochrome c Interaction - Evidence for the Extended Lipid Anchorage. J. Biol. Chem. 2002, 277, 8822−8826.

G


Total Internal Reflection Two-Dimensional Fluorescence Lifetime

Recommend Documents