Two-photon excitation action cross-sections of the autofluorescent proteins

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Abstract

We report on the values of the two-photon excitation action cross-sections of commercially available enhanced cyan, green, yellow, and red fluorescent proteins. The two-photon absorption spectra are very similar in shape to those measured for one-photon absorption. However, they exhibit a significant blueshift, which is attributed to the participation of a vibrational mode in the two-photon absorption process. The two-photon spectra are compared to that of flavine mononucleotide, which constitutes the main source of autofluorescence in mammalian cells. The definition of a relative detection yield between the autofluorescent proteins and flavine allows us to quantify the applicability of autofluorescent proteins in two-photon single-molecule studies in living cells.

Introduction

Major innovations in recent years have largely revolutionized the fluorescence imaging of biological samples. Two-photon excitation using near-infrared wavelength short laser pulses increased the achievable penetration depth in thick samples, which for the first time renders imaging of thick tissues possible [1], [2]. In addition, the out-of-focus photodamage of the samples is essentially avoided which further facilitates reliable volume imaging of fluorescent samples. Another advancement came from the discovery of the autofluorescent proteins originating from the jellyfish Aequorea victoria[3] ([4] and references therein) and the coral Discosoma sp. [5]. By fusing a specific protein with an autofluorescent protein, the functional imaging of complex processes in cells became possible for the first time: the role of microtubules in mitosis [6], functional oligomerization of cellular proteins [7], or changes in the intracellular Ca2+ level [8], [9]. One advantage of the fluorescence labelling technique in comparison to bioconjugation is its extremely high sensitivity which makes experiments feasible down to the level of a single molecule, opening a new avenue for cell biological and biophysical research.

While the applicability of the autofluorescent proteins for single-molecule research in vivo has recently been demonstrated [7], [10], [11], its general application is still in early development. One major issue is the detectability of a single fluorophore within the autofluorescence background inside a living cell. The main component of this autofluorescent background is flavine molecules which are present at particularly high abundance of 100–1000 molecules per focal volume element [12]. Due to the broad absorption of flavine in the blue/green part of the visible spectrum the excitation spectra of the cellular autofluorescence strongly overlaps with the excitation spectra of the fluorescent proteins. For this reason, only the two most redshifted varieties, the enhanced yellow fluorescent protein (eYFP) and the red fluorescent protein (DsRed), provide a significantly stronger wavelength discrimination compared to the flavines in wide-field single-molecule applications [10].

Here we report on the prospects of using two-photon excitation (TPE) in order to largely increase the signal-to-background ratio in our quest for general single-molecule detection of the autofluorescent proteins in live cells. The basic idea for two-photon excitation rests on the earlier observation that the two-photon absorption cross-section of a variety of fluorescent molecules scales super-linearly with the one-photon absorption cross-section [13]. Hence, the ratio of the effective excitation rate of a fluorophore with high one-photon absorption cross-section, like the fluorescent proteins, and a fluorophore with a low one-photon absorption cross-section, like flavines, will be largely increased for two-photon excitation. Indeed our data show a super-linear scaling behaviour which has been previously described only for small organic molecules. For exploitation and optimization of the two-photon excitation scheme we have fully characterized and compared the two-photon spectroscopic properties of commercially available autofluorescent proteins and that of flavine mononucleotide. Our findings might lead to a novel strategy of two-photon imaging of single molecules using the autofluorescent proteins as markers.

Section snippets

Sample preparation

The autofluorescent proteins were purified as described previously [10]. In brief, plasmids containing the coding sequences of the autofluorescent proteins with a C-terminal his6 tag (peXFP, Clontech) were transformed into E. coli and cultured at 37 °C. The cells were harvested, lyzed and the fluorescent protein was extracted using a column of chelating sepharose (Pharmacia Biotech). Concentrations of the fluorescent proteins were determined by measuring their absorption spectra. SDS-PAGE

Results and discussion

The foremost characteristic of two-photon induced fluorescence is the dependence of the signal on the square of the excitation intensity [16]. For each wavelength measured we have recorded a power series of the detected fluorescence signal for peak intensities between 1 and 424 MW/cm2 (Fig. 1). For all 84 experiments we found that the fluorescence signal, F, followed a power-law dependence on the excitation power, FIα with an average exponent of α=1.92±0.17 (mean ± SD). At the respective

Conclusions

We have shown that the use of two-photon excitation does largely enhance the detection ratio of all autofluorescent proteins against the autofluorescence background expected from flavines in living cells. The higher detectability is the result of the super-linear scaling between the one- and two-photon absorption cross-sections which has been previously reported for organic fluorophores. The presentation of the two-photon spectra allows one to optimize the excitation wavelength for any

Acknowledgements

This work was supported by funds from the Dutch ALW/FOM/NWO program for Physical Biology (99FBK03). L.C. acknowledges support from DGA/DSP (France) and the European Marie-Curie fellowship program (IHP-MCFI-1999-00736).

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    1

    Present address: CPMOH–UMR 5798 CNRS et Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France.

    2

    Present address: Pacific Northwest National Laboratory, MSIN: K8-88, Richland, WA 99352, USA.

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