Abstract
A method is proposed for assessing the temporal resolution of structured illumination microscopy (SIM), by tracking the amplitude of different spatial frequency components over time, and comparing them to a temporally-oscillating ground-truth. This method is used to gain insight into the performance limits of SIM, along with alternative reconstruction techniques (termed ‘rolling SIM’) that claim to improve temporal resolution. Results show that the temporal resolution of SIM varies considerably between low and high spatial frequencies, and that, despite being used in several high profile papers and commercial microscope software, rolling SIM provides no increase in temporal resolution over conventional SIM.
Highlights
In wide-field microscopy, the sample is illuminated with uniform intensity, and the spatial resolution of the observed fluorescence distribution is limited by the microscope’s pass band – the microscope functions as a lowpass filter, attenuating higher spatial frequencies until they cannot be observed and cannot contribute to the image
The effective spatial resolution and information content achievable by structured illumination microscopy (SIM) and other super-resolution techniques have been subject to intense scrutiny in the last decade [23,24,25]
Though super-resolution methods open the door to the investigation of biological processes with unprecedented detail, they significantly increase the technical complexity of microscopes and image reconstruction techniques
Summary
The experiment is performed in silico by modulating the intensity of each frame with a known sine wave and taking the normalized root-mean-squared-error between this known sine wave and the temporal variation of each spatial frequency component in the reconstructed superresolution images By taking this error over a number of oscillation periods, it is possible to assess the temporal resolution for different spatial frequency components independently (see Fig. 2). Is an implementation of the original Gustafsson reconstruction method [3], so results are applicable to many, if not all other SIM reconstruction codes
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