Abstract
Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable.
Highlights
The last decade has seen a variety of microscopy methods which enable circumventing the classical diffraction limit of light microscopy [1]
We presented a new algorithm to pre-process image sequences using Fourier analysis of temporal
We presented newinalgorithm to blinking pre-process image sequences using analysis of fluctuations, which ahelps identifying single molecule signals overFourier background noise
Summary
The last decade has seen a variety of microscopy methods which enable circumventing the classical diffraction limit of light microscopy [1]. One prominent set of techniques utilizes the stochastic switching of single molecules between a fluorescent “on-state” and a non-fluorescent “off-state”. In these single molecule localization microscopy (SMLM) approaches, the problem of resolving two or more point emitters was shifted to the problem of obtaining their correct position [2]. The single molecule localization precision is essentially determined by the brightness of the signal over background noise, but hardly depends on the width of the signal, it allows for imaging structures at a resolution which is not limited by diffraction [3,4,5,6]. A related technique has been termed points accumulation for imaging in nanoscale topography (PAINT), and makes use of on/off signals upon binding of a fluorescent molecule to the biomolecule of interest [7,8].
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