Optimal Background Estimators in Single-Molecule FRET Microscopy.

Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Förster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique.

[1]  V. Birkedal,et al.  Single molecule FRET data analysis procedures for FRET efficiency determination: probing the conformations of nucleic acid structures. , 2013, Methods.

[2]  K. Weninger,et al.  Optimizing methods to recover absolute FRET efficiency from immobilized single molecules. , 2010, Biophysical journal.

[3]  David Yadin,et al.  Defining the limits of single-molecule FRET resolution in TIRF microscopy. , 2010, Biophysical journal.

[4]  Lasse L. Hildebrandt,et al.  iSMS: single-molecule FRET microscopy software , 2015, Nature Methods.

[5]  D. P. Fromm,et al.  Methods of single-molecule fluorescence spectroscopy and microscopy , 2003 .

[6]  D. Lamb,et al.  NC2 mobilizes TBP on core promoter TATA boxes , 2007, Nature Structural &Molecular Biology.

[7]  Rahul Roy,et al.  A practical guide to single-molecule FRET , 2008, Nature Methods.

[8]  V. Vandelinder,et al.  Continuous throughput and long-term observation of single-molecule FRET without immobilization , 2014, Nature Methods.

[9]  Michael D. Mason,et al.  Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy. , 2009, Methods in molecular biology.

[10]  S. Hess,et al.  Precisely and accurately localizing single emitters in fluorescence microscopy , 2014, Nature Methods.

[11]  Kees Jalink,et al.  The fidelity of stochastic single-molecule super-resolution reconstructions critically depends upon robust background estimation , 2014, Scientific Reports.

[12]  Daniel Herschlag,et al.  Multiple Native States Reveal Persistent Ruggedness of an RNA Folding Landscape , 2010, Nature.

[13]  B. Hadwen,et al.  The noise performance of electron multiplying charge-coupled devices , 2003 .

[14]  Steve B. Howell,et al.  TWO-DIMENSIONAL APERTURE PHOTOMETRY: SIGNAL-TO-NOISE RATIO OF POINT-SOURCE OBSERVATIONS AND OPTIMAL DATA-EXTRACTION TECHNIQUES , 1989 .

[15]  Lasse L. Hildebrandt,et al.  Structural dynamics of nucleic acids by single-molecule FRET. , 2013, Methods in cell biology.

[16]  A. Small,et al.  Fluorophore localization algorithms for super-resolution microscopy , 2014, Nature Methods.

[17]  D. F. Ogletree,et al.  Probing the interaction between single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor , 1996, Summaries of Papers Presented at the Quantum Electronics and Laser Science Conference.

[18]  S. Ram,et al.  Ultrahigh accuracy imaging modality for super-localization microscopy , 2013, Nature Methods.

[19]  Dmitri S. Pavlichin,et al.  Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data , 2012, PloS one.

[20]  Justin N. M. Pinkney,et al.  Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence , 2012, Proceedings of the National Academy of Sciences.

[21]  Jerry Chao,et al.  Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices , 2012, Multidimens. Syst. Signal Process..

[22]  Timothy D Craggs,et al.  Alternating-laser excitation: single-molecule FRET and beyond. , 2014, Chemical Society reviews.

[23]  C. Joo,et al.  Single-molecule FRET with total internal reflection microscopy. , 2012, Cold Spring Harbor protocols.

[24]  Lasse L. Hildebrandt,et al.  Single molecule FRET analysis of the 11 discrete steps of a DNA actuator. , 2014, Journal of the American Chemical Society.

[25]  H. Flyvbjerg,et al.  Optimized localization-analysis for single-molecule tracking and super-resolution microscopy , 2010, Nature Methods.

[26]  C. Joo,et al.  Advances in single-molecule fluorescence methods for molecular biology. , 2008, Annual review of biochemistry.

[27]  Nam Ki Lee,et al.  Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. , 2005, Biophysical journal.

[28]  R. Ebright,et al.  Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes. , 2006, Biophysical journal.

[29]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.