Abstract
Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary ‘blinking’ without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation.
Highlights
Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers
One of the major branches in the field is referred to as single molecule localization microscopy (SMLM) and includes methods such as photo-activated localization microscopy[1] (PALM), Stochastic optical reconstruction microscopy[2] (STORM), point accumulation in nanoscale topology[3] (PAINT), and their descendants[4]
Due to the non-fluorogenic nature of imagers, DNA-PAINT experiments are typically performed using some sort of selective plane illumination and/or detection, such as total internal reflection fluorescence (TIRF) microscopy[6], oblique illumination[7], or spinning disk confocal microscopy[8]
Summary
Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary ‘blinking’ without the use of rather complex optical or chemical activation schemes It still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Besides offering spectrally-unlimited multiplexing capabilities (Exchange-PAINT)[9] and quantitative imaging (qPAINT)[10], DNA-PAINT can achieve spatial resolutions down to ~5 nm using standard TIRF microscopy[5] As it is the case for all SMLM methods, reconstructed images have to be carefully interpreted, as they can be prone to artifacts arising e.g., from inhomogeneous illumination caused by the Gaussian laser profile[11,12]. We identify imaging and quantification artifacts introduced by inhomogeneous sample illumination in DNA-
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