Ratiometric measurement and identification of single diffusing molecules

doi:10.1016/S0301-0104(99)00132-9

Chemical Physics

Volume 247, Issue 1, 1 August 1999, Pages 85-106

https://doi.org/10.1016/S0301-0104(99)00132-9 Get rights and content

Abstract

The concept of ratiometric identification and separation of sub-populations of single biomolecules which are labeled with individual fluorophores and are freely diffusing (or flowing) through the laser excitation volume is introduced. The efficacy of the scheme versus the signal and noise level is considered and a general theoretical framework and data analysis schemes are introduced. Experimental results that demonstrate distributions of single molecule fluorescence polarization anisotropy (smFPA) and single pair fluorescence resonance energy transfer (spFRET) in solution are presented. Ultimate resolution for identifying molecular observables such as conformational states, degree of freedom of motion and spectral distribution are discussed.

Introduction

Recent advances in single molecule detection (SMD) and spectroscopy (SMS) by laser induced fluorescence have opened up new possibilities for the study of biomolecules 1, 2, 3. Significant progress in optical microscopy, advances in detector technology and sophisticated biochemistry have made feasible the observation of individual biomolecules and their conformational dynamics, both under equilibrium and non-equilibrium conditions. This extreme detection sensitivity allows one to study biological processes on a truly molecular basis and therefore bridge the gap between microscopic theoretical descriptions and conventional, macroscopic ensemble measurements. It is expected that single molecule methods will provide new insights, at the molecular level, on important biological phenomena such as protein folding, bio-polymer dynamics and enzymatic reactions.

It has been previously speculated [4] and recently demonstrated [5] that extrinsic (and possibly intrinsic) fluorescent probes can report on macromolecules' conformational states and their fluctuations. Naturally, a question arises as to the role of fluctuations in the functional properties of biomolecules. To this end, a variety of ensemble steady state and time resolved spectroscopic tools were developed with various degrees of success. Time resolved measurements have been especially successful in sorting out different fluctuation time scales and/or conformational distributions [6].

One can generally distinguish between two different types of single molecule experiments according to the freedom of motion of the biomolecules under study. In one approach, molecules are immobilized on a solid-liquid interface or embedded in gels. Confocal, wide-field or total internal reflection geometries are then used to excite fluorescence and record it in time, until the photodestruction of the fluorophore (on the order of few seconds for molecules in aqueous solution). From such measurements, single molecule time trajectories of various molecular properties and their dynamical fluctuations can be extracted. Such measurements also allow the observation of stochastic events that would otherwise be hidden in an ensemble measurement due to the lack of synchronization. Two recent experiments have illustrated the potential of this approach and have already provided information on enzymatic reactions 5, 7.

An alternative way, which is introduced in this paper, consists of ratiometric single molecule measurements of biomolecules which are flowing or freely diffusing in a liquid. In this case, a laser beam is focused into a dilute aqueous sample. when a fluorescently labeled biomolecule traverses the focal volume, a burst of photons is generated. The observation time, limited by the transit time of the molecules in the laser beam, is short (about 1 ms). Consequently, long time trajectories cannot be acquired in this manner. However, compared to the case of immobilized molecules, this approach has the following advantages: (i) dye-surface interactions, which can significantly affect the photophysical properties of the fluorophores [2], are non-existing; (ii) data from a large number of molecules can be collected in a relatively short period of time, providing more meaningful statistics; (iii) the molecular observables extracted from the burst data can be tabulated in a histogram and therefore inhomogeneities and sub-populations can be identified.

Analytical chemistry applications of SMD/SMS (originating from cell flow cytometry technology developed at Los Alamos in the early eighties) have been proposed and demonstrated 8, 9. So far, photon bursts from molecules confined to a focused sheath flow have been analyzed for their brightness and fluorescence lifetime. Using these observables, identification of sub-populations in a heterogeneous solution has recently been demonstrated 10, 11, 12. SMD/SMS measurements of diffusing molecules by two photon excitation have also been demonstrated [13], but recent observation suggest accelerated photodestruction under these conditions 14, 15. Diffusion detection techniques offer one of the most fascinating ways for rapid DNA sequencing. This very challenging effort, pioneered by Keller and coworkers at Los Alamos [16] is now pursued by many other groups [17].

Rigler et al. [18] further developed a related technique for diffusing molecules in solutions, fluorescence correlation spectroscopy (FCS), which was first introduced in the early seventies [19]. It should be noted, however, that FCS is usually not employed in the single molecule regime. For best results, it requires a few molecules to be present in the laser excitation volume for any given time. The technique consists in recording the fluorescence emission fluctuations from these molecules and computing the intensity autocorrelation function. Any process that leads to changes in the light intensity such as diffusion or changes in photophysical properties can in principle be detected and analyzed by FCS. For instance, measurements of diffusion coefficients, triplet state lifetimes, photobleaching characteristics and conformational changes have been demonstrated with FCS 20, 21, 22, 23, 24, 25.

In this paper, we extend the applications of single molecule diffusion experiments to ratiometric measurements for which a molecular observable is calculated as a ratio of two quantities. Two different molecular properties are first considered: fluorescence polarization anisotropy (FPA) and fluorescence resonance energy transfer (FRET). These two observables provide valuable information on the conformational states and freedom of motion of the molecules. Ratiometric detection of emission spectral peak position is also discussed.

In an ensemble FPA experiment, molecules are excited with polarized light and their fluorescence is collected according to its polarization 26, 27. The polarization anisotropy r is defined by: $r= I_{∥} −I_{⊥} I_{∥} +2I_{⊥}$ where I_∥ and I_⊥ are the fluorescence intensities with polarization parallel and perpendicular to the excitation light. The observable r provides valuable information about the freedom of orientational motion of the construct dye/macromolecule.

In an ensemble FRET experiment, a fluorophore (the donor) is excited by a laser and transfers its excitation energy to a second molecule (the acceptor) through a non-radiative dipole-dipole coupling. This process, described by Förster [28], has a strong dependence on the distance and orientation between the two dyes and can be used to report on biomolecule conformations [26]. The FRET efficiency E, which represents the fraction of energy transferred, can be calculated according to: $E= I_{a} I_{a} +I_{d}$ where I_d and I_a are the respective fluorescence intensities of the donor and acceptor molecules.

The ratiometric coincident single molecule approach opens up new ways for single molecule diffusion experiments. In particular, it suppresses intensity fluctuations, characteristic to single molecule measurement. Using this method, sub-populations of molecules in heterogeneous solutions can be identified. This paper is organized as follows: in Section 2we describe the experimental set-up and the main characteristics of the data acquisition scheme. In Section 3we develop the data analysis and lay out the theoretical framework for ratiometric measurements in the single molecule limit. It is shown that it is possible to reconstruct the complete distribution of a ratiometric molecular property and the achievable resolution of the technique for sub-population separation is discussed. The methodology is then demonstrated for single molecule (sm)FPA in Section 4and single pair (sp)FRET in Section 5and experimental data is compared with simple models. In Section 6the utility of the ratiometric approach for spectral indicators is discussed. Finally, a comparison between the ratiometric single molecule method and other techniques and possible future developments are discussed in Section 7.

Section snippets

Experimental set-up

Our experimental set-up consists of a confocal microscope coupled to a high sensitivity detection scheme. A sample is prepared by placing a small volume (about 15 μl) of the solution between two HF cleaned glass coverslips (cf. Fig. 1). An epi-illumination excitation (dichroic mirror D₁) is used and the laser light (488 nm or 514 nm in the experiments reported here) is focused onto this sample through a high numerical aperture objective L (Zeiss, 100×, NA 1.4). In order to detect freely

Ratiometric measurements in the single molecule limit

In this section we lay the theoretical framework and develop a general analysis scheme applicable to ratiometric observables. As described above, a pair of data points (I_aⁱ,I_bⁱ) is recorded for each time bin i, corresponding to the number of detected photons in each channel. To identify the time bins containing photon bursts from single molecules, a standard threshold algorithm is employed. This algorithm is described first. Next, the characteristics of the phenomenological ratio d=I_a/(I_a+I_b)

Single molecule Fluorescence Polarization Anisotropy

Fluorescence polarization of ensemble of molecules have long been used in biochemistry to extract information about the size, shape and flexibility of macromolecules 26, 27. In particular, steady-state and time-resolved fluorescence polarization anisotropy (FPA) have been used to study rotational dynamics of macromolecules. When linearly polarized light is used for excitation, only a subset of molecules, with dipoles aligned mostly along the excitation polarization, is selected from the

Single molecule Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) has proven to be a useful spectroscopic technique for measuring distances in the 10-75 Å range 26, 27, 41. To measure the distance between two points, the first is labeled with a donor fluorescent molecule, and the second is labeled with an acceptor molecule (which is usually, but not necessarily fluorescent). The acceptor is chosen such that its absorption is in resonance with the donor emission. Energy is transferred from the donor to the acceptor

Spectral indicators

The FPA and FRET observables discussed in the previous sections mostly relate to conformational properties and freedom of motion of the molecules. The dual-channel coincident burst approach can be applied to other observables as well. In this section we discuss spectral indicators. These are fluorescent dyes which change their optical properties in response to ion binding, local potential or other chemical/electrostatic changes in their local environment. The change in optical response can be

Discussion

Single molecule spectroscopy of immobilized molecules (on surfaces or in gels) can provide detailed information on working macromolecules 2, 5. However, the immobilization process itself can introduce additional broadening to the distributions of the measured properties. The development of the ratiometric coincident burst approach for freely diffusing (or flowing) single molecules should provide us with a machinery to study macromolecules' structure and function in equilibrium and

Conclusion

The ratiometric coincident burst approach of freely diffusing/flowing single molecules offers many exciting possibilities for in-vitro biophysical and biochemical characterization of macromolecules; the study of their function and structure; ultimate sensitivity and high information content in analytical chemistry applications and screening of target molecules for drug discovery. Rather straightforward improvements in hardware, software and experimental methodologies should facilitate

Acknowledgements

We thank J. Grunwell, J. Glass, A.E. Faulhaber, T. Lacoste and T. Laurence for their valuable help. Financial support for this work was provided by the National Science Foundation under Grant No.CHE-971-4390, by the Laboratory Directed Research and Development Program of Lawrence Berkeley National Laboratory under U.S. Department of Energy, contract No. DE-AC03-76SF00098, by the National Institutes of Health (Grant No. GM49220) and by the Office of Naval Research Contract N0001498F0402. P. G.

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¹: Contributed equally to this work.

²: Present address: Department of Physics, Stanford University, Stanford, CA 94305, USA.

³: To whom reprint requests should be addressed. E-mail: [email protected]

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Ratiometric measurement and identification of single diffusing molecules

Abstract

Introduction

Section snippets

Experimental set-up

Ratiometric measurements in the single molecule limit

Single molecule Fluorescence Polarization Anisotropy

Single molecule Fluorescence Resonance Energy Transfer

Spectral indicators

Discussion

Conclusion

Acknowledgements

Biophys. J

Chem. Phys. Lett

Biophys. J

Biophys. J

Biophys. J

Ann. Rev. Biophysics and Biomolecular Chem

Ann. Rev. Phys. Chem

Science

Proc. Natl. Acad. Sci. USA

Proc. Natl. Acad. Sci. USA

Science

Applied Spectroscopy

Proc. Natl. Acad. Sci. USA

Bioimaging

Optics Lett

Bioimaging

J. of Phys. Chem. A

Chem