Camera-based single-molecule FRET detection with improved time resolution

Here the authors report on significant improvements in time-resolution and throughput in camera-based single-molecule detection by combining stroboscopic alternating-laser excitation with dynamic probability distribution analysis.

SOLVING SINGLE BIOMOLECULES BY ADVANCED FRET-BASED SINGLE-MOLECULE FLUORESCENCE TECHNIQUES

The use of Förster resonance energy transfer (FRET) has undergone a renaissance in the last two decades, especially in the study of structure of biomolecules, biomolecular interactions, and dynamics. Thanks to powerful advances in single-molecule fluorescence (SMF) techniques, seeing molecules at work is a reality, which has helped to build up the mindset of molecular machines. In the last few years, many technical developments have broadened the applications of SMF-FRET, expanding the amount of information that can be recovered from individual molecules. Here, we focus on the non-standard SMF-FRET techniques, such as two-color coincidence detection (TCCD), alternating laser excitation (ALEX), multiparameter fluorescence detection (MFD); the addition of fluorescence lifetime as an orthogonal dimension in single-molecule experiments; or the development of novel and improved methods of analysis constituting to a set of advanced methodologies that may become routine tools in a close future. [Formula: see text]Special Issue Comment: This review about advanced single-molecule FRET techniques is specially related to the review by Jørgensen and Hatzakis,6 who detail experimetal strategies to solve the activity of single enzymes. The advanced techniques described in our paper may serve as interesting alternatives when applied to enzyme studies. Our manuscript is also related to the reviews in this Special Issue that deal with model solving.22,130

Four-Color Alternating-Laser Excitation Single-Molecule Fluorescence Spectroscopy for Next-Generation Biodetection Assays

BACKGROUND Single-molecule detection (SMD) technologies are well suited for clinical diagnostic applications by offering the prospect of minimizing precious patient sample requirements while maximizing clinical information content. Not yet available, however, is a universal SMD-based platform technology that permits multiplexed detection of both nucleic acid and protein targets and that is suitable for automation and integration into the clinical laboratory work flow. METHODS We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescence-aided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ≥2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously. RESULTS We show detection, differentiation, and quantification—in a single well—of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug–resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood. CONCLUSIONS The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly (“sample in, answer out”) point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample.

Red light, green light: probing single molecules using alternating-laser excitation

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein–nucleic acid interactions.