4.4 Article

Probability-based particle detection that enables threshold-free and robust in vivo single-molecule tracking

期刊

MOLECULAR BIOLOGY OF THE CELL
卷 26, 期 22, 页码 4057-4062

出版社

AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E15-06-0448

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资金

  1. National Science Foundation [0954836]
  2. Direct For Mathematical & Physical Scien
  3. Division Of Physics [0954836] Funding Source: National Science Foundation

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Single-molecule detection in fluorescence nanoscopy has become a powerful tool in cell biology but can present vexing issues in image analysis, such as limited signal, unspecific background, empirically set thresholds, image filtering, and false-positive detection limiting overall detection efficiency. Here we present a framework in which expert knowledge and parameter tweaking are replaced with a probability-based hypothesis test. Our method delivers robust and threshold-free signal detection with a defined error estimate and improved detection of weaker signals. The probability value has consequences for downstream data analysis, such as weighing a series of detections and corresponding probabilities, Bayesian propagation of probability, or defining metrics in tracking applications. We show that the method outperforms all current approaches, yielding a detection efficiency of >70% and a false-positive detection rate of <5% under conditions down to 17 photons/pixel background and 180 photons/molecule signal, which is beneficial for any kind of photon-limited application. Examples include limited brightness and photostability, phototoxicity in live-cell single-molecule imaging, and use of new labels for nanoscopy. We present simulations, experimental data, and tracking of low-signal mRNAs in yeast cells.

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