Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme.

Maxime Woringer,Christophe Zimmer,Mustafa Mir,Xavier Darzacq

doi:10.1364/oe.25.013668

Abstract

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy.

Highlights

Imaging fluorescently labeled biological structures with high spatio-temporal resolution requires judicious compromises between the conflicting goals of achieving high signal-to-noise ratio (SNR) and temporal resolution while keeping the excitation power low to minimize photobleaching and phototoxicity
We present a new compressed sensing scheme for 3D fluorescence microscopy that can be applied to a wide range of microscopes
We demonstrate a widely applicable 3D compressed sensing scheme in which images are compressed along the z axis during acquisition

Summary

Introduction

Imaging fluorescently labeled biological structures with high spatio-temporal resolution requires judicious compromises between the conflicting goals of achieving high signal-to-noise ratio (SNR) and temporal resolution while keeping the excitation power low to minimize photobleaching and phototoxicity. To obtain a higher SNR, one can either increase the exposure time, thereby reducing imaging speed, or increase the illumination power, thereby increasing photodamage. These tradeoffs are further exacerbated in 3D imaging, which is often required in biological applications, such as calcium imaging in neurons or transient mitotic events in a developing embryo. Hundreds or thousands of planes are needed to image samples 10-1,000 μm thick, dramatically increasing acquisition time and light exposure. Light sheet illumination considerably reduces photodamage and allows prolonged imaging of living cells in 3D, the required hardware systems are often costly and scarce, and the acquisition time for each volume still remains constrained by the Nyquist criterion [1,2,3]

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