A practical guide to single-molecule FRET

Abstract

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

Figure 1

Single molecule FRET. (a) FRET Efficiency, E as a function of inter-dye distance (R) for a R₀ = 50 Å. Donor dye directly excited with incident laser either fluoresces or transfers energy to acceptor dye depending upon its proximity. At R = R₀, E = 0.5 while at smaller distances, it is > 0.5 and vice versa according to the function shown by the blue line. Notice the linearity of the E values adjacent to R₀. (b) Example of a two color smFRET data. Data is acquired in the form of intensities of donor and acceptor (top panel) from which apparent FRET efficiency (bottom panel) is calculated. A mutant hairpin ribozyme which carries the donor and acceptor on different arms of the same molecule undergoes transitions between three FRET states (E1, E2 and E3). The anti-correlated nature of the donor and acceptor signal indicates that these intensity changes are due to energy transfer. Dye molecules also show transitions to dark states e.g. acceptor intensity transiently drops to zero (∼9 s) or completely photobleaches (∼18.5 s).

Figure 2

Schematic for smFRET spectroscopy. Labels - M, mirror; DM, dichroic mirror; L, lens; CCD, charge coupled device camera; BE, beam expander; PBS, polarizing beam splitter; λ/2, half waveplate (a) TIR excitation and single FRET pair emission detection. Tethered single molecules are either excited by PTIR or OTIR. Fluorescence is collected using the objective and the slit generates a final imaging area that is half of the CCD imaging area. The collimated image is split into the donor and acceptor emissions and imaged side by side on the CCD camera (see camera image in inset). (b) TIR excitation schemes (enlarged view of the box in (a)). In PTIR, laser beam focused at a large incident angle (θ_c > 68°) on the prism placed on the top of the sample creates an evanescent field (EF) at the quartz/water interface on the slide. Alternately in OTIR excitation, the focused laser beam strikes at the periphery of the objective back focal plane causing TIR at the glass/water interface on the coverslip close to the objective. (c) Emission detection for three color scheme. Image is framed using a slit such that final image size covers one-third of the CCD chip. A set of dichroics allow separation of the individual emissions from the three fluorophores and imaging them simultaneously.

Figure 3

Surface immobilization strategies for smFRET experiments. (a) Biotin-BSA proteins bound non-specifically to the surface tether biotinylated molecules with the aid of multivalent avidin proteins (e.g. neutravidin). (b) Mixture of biotin-PEG and PEG is covalently attached to amino silanized slide surface. Biotins on the PEG can bind DNA (or protein) engineered with a biotin moiety with the help of a sandwiched neutravidin protein. PEG coating prevents the non-specific binding. (c) PEG coated surfaces can be engineered to carry Ni²⁺ or Cu²⁺ chelated on iminodiacetic acid (IDA) (or nitrilotriacetic acid (NTA)) groups that bind efficiently to 6× His-tagged proteins while retaining functional activity. (d) Using dimyristoyl phospatidylcholine (DMPC) at room temperature, vesicles selectively permeable to small molecules can be used to trap biomolecules. A fraction of biotinylated lipid allows specific tethering of the vesicles to the biotin-PEG surface. D and A labels stand for donor and acceptor dyes.

Figure 4

Sample Chamber. (a) A sample chamber is made by sandwiching a microscope slide and a coverslip with double-sided tape and by sealing with epoxy. The holes on the slide are used for the inlet and outlet of solution exchange. (b) A syringe is connected to the chamber through tubing and a pipette tip that contains a solution is snugly plugged into an inlet hole. When the syringe is pulled, the solution is introduced into a chamber. Reproduced from ref. 7 with permission from Cold Spring Harbor Laboratory Press.

Box Figure 1

Single molecule FRET schemes.