Physics Laboratory
Optical Technology Division
Laser Applications Group

Single Molecule Fluorescence Detection

Introduction

New techniques, discoveries and uses of single molecule fluorescence detection are becoming increasingly abundant in scientific literature. This new field has great potential in science and technology. For example, the use of single molecule emission spectra, lifetime, and intensity fluctuation behavior may be used as a sensitive probe of the local environment in which the molecule resides. The physical properties of materials have also been probed using the lateral and rotational diffusion of single molecules. Conformational dynamics including protein folding can be studied using single molecule fluorescence intensity and spectra fluctuation analysis, fluorescence resonance energy transfer methods (FRET) and orientation studies. and l Applications in which single molecules are used to study lateral and rotational motion, and protein folding are also in progress. However, significant work in understanding the photophysics and dynamic properties of dye molecules in various environments is still needed. Recently, we have developed a confocal microscope capable of mapping both the location and orientation of single molecules, observing orientation and intensity changes over time, and obtaining single molecule emission spectroscopy. We are in the process of applying single molecule detection techniques to fluorophores embedded in a wide range of organic thin films from common polymers to biomimetic Langmuir-Blodgett films.

Confocal Microscope For Single Molecule Fluorescence Imaging, Orientation Analysis and Spectroscopy

The microscope used for this work is built around a standard inverted optical microscope. The beam from a diode pumped, frequency doubled CW YAG laser, after treatment with polarization and beam expansion optics, is sent into the back of this microscope and is focussed to a 400 nm spot on the sample surface with an oil immersion objective. The active area of an avalanche photodiode is positioned in the image plane to detect laser induced fluorescence. The sample is scanned or positioned with respect to the stationary excitation spot using a motion amplifying flexure stage driven by piezo stack transducers. The electo-optic modulator (EOM) and l/4 retarder are oriented to generate linearly polarized light with an angle proportional to the voltage applied to the EOM.

Orientation Imaging

For orientation imaging, the polarization angle of linearly polarized laser excitation is rotated through several cycles at each pixel in an image. The EOM is driven with a 125 Hz ramp waveform and the fluorescence signal is observed for 32 ms (1 ms bin times). Induced fluorescence has a sinusoidal dependence and the phase of the signal indicates the orientation of the absorption dipole. For a sample with DiIC1(3) molecules in a PS film, the fluorescence intensity modulation for a line in an image in a grayscale format is shown. The shift in phase due to randomly oriented molecules is easily noticed. The modulation phase and amplitude for each pixel is obtained using Fourier Transform methods. Note that the phase scale "wraps around," i.e. 90 degrees = - 90 degrees.

Below are full images (a single line of which is shown above) of the polarization averaged intensity and absorption dipole orientation (upper 2 images). For stationary single molecules, the phase is well defined and blocks of color indicating orientation result. However, for pixels of low or non-modulated fluorescence, the modulation phase is undefined and the result is full-scale noise in those regions. To facilitate visualization of the single molecule orientations, the intensity image is used as a reference to threshold out pixels with insufficient signal to measure a phase (lower images).

In addition to imaging the orientation of a field of single molecules, the technique described above is easily adapted to monitor the orientation of single molecules as a function of time. The fluorescence intensity modulation, polarization-averaged intensity, and calculated phase for four different DiIC1 molecules embedded in a thin film of polystyrene are shown below. The first data set (top, right) shows the expected result: a molecule has a stationary orientation until an irreversible photobleach. However, the other data sets show a surprising result: large shifts in the absorption dipole orientation are observed. The progression shows increasing rotational activity. More than half of the molecules observed showed at least one rotational jump. On a qualitative level, the rate of rotational jumps does not appear to be power dependent, and thus is not likely to be laser induced.

As mentioned, one of the potential applications of single molecule detection techniques will be use of characteristic fluorescence properties of single fluorophores embedded in a matrix to understand the local nanoscale chemistry and physics of that matrix. For example, one might imagine probing the local ionic strength or rigidity at specific locations around an organelle inside a living cell by monitoring the fluorescence characteristics of single molecules attached to those sites. We have sought to identify the relationships between local properties and fluorescence characteristics by monitoring emission spectra and intensity fluctuation behavior of fluorophores embedded in various materials. While this has proven to be a difficult task due to the wide distribution of fluorescence properties for different molecules in most samples studied to date, this work is continuing with optimism. The figures below demonstrate the wide range of emission spectra and intensity fluctuations for DiI molecules on a glass surface.

Relevant Publications
K. D. Weston, P. J. Carson, J.A. Dearo, and S. K. Buratto, "Single-Molecule Fluorescence Detection of Surface-Bound Species in Vacuum," Chem. Phys. Lett. 308, 58 (1999).
K. D. Weston, P. J. Carson, H. Metiu, and S. K. Buratto, "Room Temperature Fluorescence Characteristics of Single Molecules Adsorbed on a Glass Surface," J. Chem. Phys. 109, 7474 (1998).
M. D. Mason, G. M. Credo, K. D. Weston, and S. K. Buratto, "Luminescence of Individual Porous Si Chromophores," Phys. Rev. Lett. 80, 5405 (1998).
K. D. Weston and S. K. Buratto, "Millisecond Intensity Fluctuations of Single Molecules at Room Temperature," J. Phys. Chem. A. 102, 3635 (1998).

For technical information or questions, please contact:

Lori S. Goldner
Phone: (301) 975-3792
Fax: (301) 840-8551
Email: lori.goldner@nist.gov

Any comments or questions regarding this web page may be directed to arvella.kuehl@nist.gov.

Program Highlights
Optical Technology Division
Last update: October 2001