How to define and optimize axial resolution in light-sheet microscopy: a simulation-based approach.

Elena Remacha,Lars Friedrich,Florian O Fahrbach,Julien Vermot

doi:10.1364/boe.11.000008

Abstract

"How thick is your light sheet?" is a question that has been asked frequently after talks showing impressive renderings of 3D data acquired by a light-sheet microscope. This question is motivated by the fact that most of the time the thickness of the light-sheet is uniquely associated to the axial resolution of the microscope. However, the link between light-sheet thickness and axial resolution has never been systematically assessed and it is still unclear how both are connected. The question is not trivial because commonly employed measures cannot readily be applied or do not lead to easily interpretable results for the many different types of light sheet. Here, we introduce a set of intuitive measures that helps to define the relationship between light sheet thickness and axial resolution by using simulation data. Unexpectedly, our analysis revealed a trade-off between better axial resolution and thinner light-sheet thickness. Our results are surprising because thicker light-sheets that provide lower image contrast have previously not been associated with better axial resolution. We conclude that classical Gaussian illumination beams should be used when image contrast is most important, and more advanced types of illumination represent a way to optimize axial resolution at the expense of image contrast.

Highlights

Light sheet microscopy [1] has proven to be an enabling technology for a number of research fields including developmental biology [2], cell biology [3], neuroscience [4] and many more [5]
Some light sheet with thin main lobes may provide higher axial resolution: For example, for equal depth of field and light sheet thickness, the resolution is increased by 30% but only by 10% for a light sheet that is twice as thick than the detection objectives depth of field, something that routinely happens for detection NAdet=1.0 and a light sheet created at NAill=0.1 which results in a depth of focus of dyLS = 2n /NA2 ≈130μm
In conclusion, we highlight the difference between the optical sectioning and the axial resolution that different light sheets provide

Summary

Introduction

Light sheet microscopy [1] has proven to be an enabling technology for a number of research fields including developmental biology [2], cell biology [3], neuroscience [4] and many more [5]. The purpose of the light sheet is to optically slice the sample to generate images with high contrast of thickly fluorescent samples. The thickness of the slice depends on the thickness of the light sheet. The light sheet fully determines optical sectioning. Illumination by a thicker light sheet results in the projection of a thicker section of the sample into a single image. In thickly fluorescent three-dimensional samples, optical sectioning (OS) is required to generate image contrast. Because resolution depends on contrast, i.e. the separation of individual features is only possible for a sufficient drop in signal in between two features, optical sectioning should be seen as one of the fundamental parameters of a light sheet microscope

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Discussion

Conclusion