(GFP) with fluorescence lifetime imaging - CiteSeerX

2 downloads 0 Views 76KB Size Report
resolved fluorescence anisotropy imaging (tr-FAIM). K. Suhling, D.M. Davis. Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College ...
Probing the local environment of green fluorescent protein (GFP) with fluorescence lifetime imaging (FLIM) and timeresolved fluorescence anisotropy imaging (tr-FAIM) K. Suhling, D.M. Davis Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College of Science, Technology & Medicine, London SW7 2AZ, UK.

[email protected]

D. Phillips Department of Chemistry, Imperial College of Science, Technology & Medicine, London SW7 2AY, UK.

J. Siegel, S. Lévêque-Fort1, S.E.D. Webb, P.M.W. French Photonics Group, Physics Department, Imperial College of Science, Technology & Medicine, London SW7 2BW, UK. 1 present address: Laboratoire de Photophysique Moleculaire, 91405-Orsay cedex, France.

Abstract: Wide-field time-domain fluorescence lifetime imaging and time-resolved fluorescence anisotropy imaging of green fluorescent protein can be used to probe the biophysical environment of specific proteins. 2001 Optical Society of America OCIS codes: (170.2520) Medical optics and biotechnology; (180.2520) Microscopy; (260.2510) Physical Optics; (300.2530,300.6280) Spectroscopy

1. Introduction The green fluorescent protein (GFP) of the jellyfish Aequorea victoria and its variants are widely used in cell imaging applications to reveal the location of proteins [1]. However, such imaging of fluorescence intensity does not generally provide information about the biophysical environment of the protein. Fluorescence lifetime imaging (FLIM) [2] is a technique that, in addition to position and intensity, also provides information concerning the average time a fluorophore remains in its excited state after excitation. FLIM of the fluorescence decay of GFP has, for example, been used to report on fluorescence resonance energy transfer (FRET) [3]. We demonstrate here that the GFP fluorescence lifetime depends on the biophysical environment of the fluorophore and so can be used to directly probe the GFP environment. In particular, it can report local variations in refractive index. We also report what is, to our knowledge, the first demonstration of wide-field time-resolved fluorescence anisotropy imaging (trFAIM) which may be used to report local variations in viscosity. Recently, we presented preliminary time-correlated single photon counting studies to identify the parameter that affects the fluorescence lifetime of GFP [4]. We found this to be the refractive index of the environment of GFP, and we showed that in mixtures of water and glycerol, the inverse GFP fluorescence lifetime scales with the square of the refractive index, as predicted by the Strickler Berg formula [5]: ~ ~ ε (~ v) ~ 1 ∫ I (v ) dv (1) = 2.88 × 10 − 9 n 2 ~ dv − 3 ~ ~ ~ v τ0 ∫ I ( v ) v dv



where τ0 is the natural fluorescence lifetime, n the refractive index, I the fluorescence emission, ε the extinction coefficient and ν~ the wavenumber. In this paper, we extend our study of the dependence of the GFP fluorescence lifetime on the refractive index to imaging with time-domain FLIM. We note that changes in viscosity have also been observed to perturb the fluorescence lifetime in FLIM maps, e.g. [6], but the GFP fluorescence lifetime does not depend on the local viscosity [7], since the GFP fluorophore is rigidly attached to a barrel-shaped cage which prevents internal twisting and protects it from collisional encounters with oxygen [1]. Thus, the GFP fluorescence lifetime cannot be used to probe the viscosity of its environment, although this parameter would be of interest in cell biology. The time-resolved fluorescence anisotropy r(t), however, does depend on the mobility of the protein, and this in turn is strongly influenced by the viscosity. The time-resolved fluorescence anisotropy r(t) is defined as [8]

r (t ) =

I || (t ) − I ⊥ (t )

(2)

I || (t ) + 2 ⋅ I ⊥ (t )

where I || (t ) and I ⊥ (t ) are the fluorescence intensity decays parallel and perpendicular to the polarization to the excitation, respectively. The time-resolved fluorescence anisotropy is related to the rotational correlation time θ according to (3) r (t ) = r0 e −t / θ where r0 is the initial anisotropy. In the case of a non-hindered spherically symmetric molecule, the rotational correlation time is directly proportional to the viscosity η ηV (4) θ= kT where V is the volume of the rotating molecule, k is the Boltzmann constant and T is the absolute temperature.

2. Results and Discussion To demonstrate that the variations in the local environment of GFP can be imaged, we deployed GFP in a series of solvent concentrations of glycerol and polyethylene glycol in a multiwell plate and interrogated this array of samples using wide-field time-domain FLIM [6]. By acquiring a series of time gated images and fitting them to a single exponential decay in each pixel, the fluorescence lifetime in each pixel of the image was determined. The FLIM map of 10 occupied wells of a multiwell plate (fig. 1a) displays the fluorescence lifetime of GFP in mixtures of aqueous buffer and polyethylene glycol (top row) and mixtures of aqueous buffer and glycerol (bottom row). A gradual decrease of the GFP fluorescence lifetime is evident as the glycerol and polyethylene glycol content, and thus the refractive index, is increased. Note that if the GFP fluorescence lifetime were dependent on the viscosity, the opposite trend, i.e. an increase of the fluorescence lifetime with viscosity, would be observed [4, 6]. A plot of the inverse fluorescence lifetime of GFP versus the square of the refractive index is linear, as predicted by equation 1 (fig. 1b). This shows that FLIM is sufficiently accurate to distinguish between different GFP lifetimes and thus image the refractive index of the GFP’s local environment.

buffer polyethylene glycol glycerol

0.42

-1

τ / ns

-1

0.45

0.39 1.8

1.9

2.0

n

a)

2.1

2

b)

Fig 1. Multiwell plate FLIM of GFP. a) The fluorescence lifetime image. Each square represents a 4 mm by 4 mm well with a capacity of 125 µl. Top row: GFP in mixtures of aqueous buffer and polyethylene glycol, from left to right: buffer, 10% polyethylene glycol , 30% polyethylene glycol , 50% polyethylene glycol , 70% polyethylene glycol. Bottom row: GFP in mixtures of aqueous buffer and glycerol, from left to right: 70% glycerol, 50% glycerol, 30% glycerol, 10% glycerol, buffer. The grayscale represents the fluorescence lifetime of GFP over a range from 2.1 ns (dark) to 2.7 ns (light). A gradual decrease of the GFP fluorescence lifetime is evident as the glycerol and polyethylene glycol content, and thus the refractive index, is increased. Therefore, our time-domain FLIM system is sufficiently accurate to detect the GFP lifetime differences due to the refractive index. b) To demonstrate the applicability of the Strickler Berg formula, equation 1, the corresponding inverse fluorescence lifetimes of GFP are plotted versus the square of the refractive index.

In order to obtain the viscosity of the environment of GFP, we have extended wide-field time-gated fluorescence lifetime imaging (FLIM) [6] to time-resolved fluorescence anisotropy imaging (tr-FAIM). This is done by using a

polarization difference imager which splits the image into two spatially identical components with polarizations that are perpendicular to each other. This enables the simultaneous acquisition, with a single wide-field detector, of images at polarizations parallel and perpendicular to that of the excitation. Acquiring a series of time-gated images and analyzing them according to equations 2 & 3, provides a map of the rotational correlation time and thus the viscosity in each pixel of the image. Preliminary experiments with rhodamine 6G in various solvents show a difference in the FLIM images between the parallel and perpendicular components (providing that the rotational correlation time is sufficiently long). By appropriate processing of the parallel and perpendicular polarized sets of time-gated fluorescence intensity images, wide-field images of the polarization anisotropy and the rotational depolarization time can be displayed. Having demonstrated the tr-FAIM technique with rhodamine 6G, we are currently working towards applying the technique to GFP.

3. Conclusion The application of FLIM to image the local refractive index of the environment of GFP, and of tr-FAIM to image the local viscosity of the environment of GFP appears to be a promising novel approach to report on the biophysical environment of specific GFP-tagged proteins in live cells. One application would be in live-cell imaging of receptors moving between membrane microdomains of different refractive index and different viscosity. We gratefully acknowledge support for this work from the UK Biotechnology and Biological Science Research Council (BBSRC) and the Engineering and Physics sciences Research Council (EPSRC). Stephen Webb acknowledges an EPSRC studentship.

References [1] [2] [3] [4] [5] [6] [7] [8]

K. F. Sullivan and S. A. Kay, Green Fluorescent Proteins, Methods in Cell Biology 58 (1999). J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, "Fluorescence lifetime imaging," Analytical Biochemistry 202, 316-330 (1992). A. G. Harpur, F. S. Wouters, and P. I. H. Bastiaens, "Imaging FRET between spectrally similar GFP molecules in cells," Nature Biotechnology 19, 167-169 (2001). K. Suhling, D. M. Davis, Z. Petrášek, J. Siegel, and D. Phillips, "The influence of the refractive index on EGFP fluorescence lifetimes in mixtures of water and glycerol," Proc. S.P.I.E. 4259, 92-101 (2001). S. J. Strickler and R. A. Berg, "Relationship between absorption intensity and fluorescence lifetime of molecules," Journal of Chemical Physics 37(4), 814-820 (1962). Dowling, K., M. J. Dayel, et al. (1998). “Fluorescence lifetime imaging with picosecond resolution for biomedical applications.” Optics Letters 23(10): 810-812. K. Suhling, D. M. Davis, and D. Phillips, "The influence of solvent viscosity on the fluorescence decay and time-resolved anisotropy of green fluorescent protein," Journal of Fluorescence in press. J. R. Lakowicz, Principles of Fluorescence Spectroscopy 2nd edition (1999).