Quantitative sectioning and noise analysis for structured illumination microscopy

Liang Gao,Nathan Hagen,Tomasz S Tkaczyk

doi:10.1364/oe.20.000403

Abstract

Structured illumination (SI) has long been regarded as a nonquantitative technique for obtaining sectioned microscopic images. Its lack of quantitative results has restricted the use of SI sectioning to qualitative imaging experiments, and has also limited researchers’ ability to compare SI against competing sectioning methods such as confocal microscopy. We show how to modify the standard SI sectioning algorithm to make the technique quantitative, and provide formulas for calculating the noise in the sectioned images. The results indicate that, for an illumination source providing the same spatially-integrated photon flux at the object plane, and for the same effective slice thicknesses, SI sectioning can provide higher SNR images than confocal microscopy for an equivalent setup when the modulation contrast exceeds about 0.09.

Highlights

Structured illumination (SI) is an optical sectioning technique compatible with widefield imaging microscopy, and has been shown to provide a depth resolution comparable to confocal microscopy [1]
Whereas the measured noise is obtained by taking the standard deviation of a sequence of 1000 measurements, the photon shot noise standard deviation σp is estimated by taking the square root of the mean number of photons collected
It has often been argued that structure illumination sectioning microscopy is a nonquantitative technique, we have shown that a quantitative version of the algorithm can be obtained by adding a proper scaling factor

Summary

Introduction

Structured illumination (SI) is an optical sectioning technique compatible with widefield imaging microscopy, and has been shown to provide a depth resolution comparable to confocal microscopy [1]. While the resulting photon number estimate will be noisier than would be the case for imaging the slice without out-of-focus layers present, the mean value of the correctly scaled algorithm will equal to the mean photon count one would obtain with a standard widefield microscope. This permits researchers to use standard methods [16] of correcting for the objective lens’ numerical aperture, optical transmission, and detector quantum efficiency to determine photon counts at the sectioned plane relative to the number of photoelectrons detected at the sensor plane. For any contrast below this value, confocal imaging will out-perform SI

Sectioning algorithm

Widefield image algorithm

Variance and SNR of sectioned images

Estimating the modulation contrast

Experimental results

Sectioned imaging of 2 μm fluorescent beads without out-of-focus light

Conclusion