Abstract
Single-molecule localization techniques are restricted by long acquisition and computational times, or the need of special fluorophores or biologically toxic photochemical environments. Here we propose a statistical super-resolution technique of wide-field fluorescence microscopy we call the multiple signal classification algorithm which has several advantages. It provides resolution down to at least 50 nm, requires fewer frames and lower excitation power and works even at high fluorophore concentrations. Further, it works with any fluorophore that exhibits blinking on the timescale of the recording. The multiple signal classification algorithm shows comparable or better performance in comparison with single-molecule localization techniques and four contemporary statistical super-resolution methods for experiments of in vitro actin filaments and other independently acquired experimental data sets. We also demonstrate super-resolution at timescales of 245 ms (using 49 frames acquired at 200 frames per second) in samples of live-cell microtubules and live-cell actin filaments imaged without imaging buffers.
Highlights
Single-molecule localization techniques are restricted by long acquisition and computational times, or the need of special fluorophores or biologically toxic photochemical environments
We demonstrate that MUltiple SIgnal Classification ALgorithm (MUSICAL) performs well in situations where stochastic optical reconstruction microscopy (STORM) fails due to high density of fluorophores
We show that MUSICAL can be used for live-cell fast imaging (B49 frames amounting to a total acquisition time of less than 250 ms) of live cells expressing standard green fluorescent protein (GFP) imaged in physiologically conducive buffer devoid of chemicals that influence blinking
Summary
Single-molecule localization techniques are restricted by long acquisition and computational times, or the need of special fluorophores or biologically toxic photochemical environments. Single-molecule localization microscopy (SMLM) techniques such as stochastic optical reconstruction microscopy (STORM) or photo-activated localization microscopy (PALM) are popular super-resolution techniques owing to their simplicity, few (if any) special requirements on instrumentation, and impressive resolution of B20 nm (refs 1–3) They require that the fluorophores exhibit long dark states, so that only a small subset of optically separable fluorophores are in the bright state in each frame of the image stack. The limitations of SMLM have motivated development of techniques that rely on statistical independence of blinking of individual fluorophores rather than on long dark states[5] Such techniques include super-resolution optical fluctuations imaging (SOFI6), Bayesian analysis of blinking and bleaching We show that MUSICAL can be used for live-cell fast imaging (B49 frames amounting to a total acquisition time of less than 250 ms) of live cells expressing standard green fluorescent protein (GFP) imaged in physiologically conducive buffer devoid of chemicals that influence blinking
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