Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT

Florian Schueder,Hiroshi M Sasaki,Maximilian T Strauss,Juanita Lara-Gutiérrez,Heinrich Grabmayr,Peng Yin,Ralf Jungmann,Sinem K Saka,Brian J Beliveau,Johannes B Woehrstein

doi:10.1038/s41467-017-02028-8

Abstract

Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.

Highlights

While the stochastic switching in stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) is enabled by modulating the emission of fixed, target-bound organic fluorophores or fluorescent proteins, a different approach called points accumulation for imaging in nanoscale topography (PAINT)[10] rely on freely diffusing dyes or dye-labeled entities that either bind statically to target molecules or transiently interact with them
We have demonstrated that sub-20-nm lateral and sub-80-nm axial resolution using DNA-PAINT in combination with a minimally modified commercial spinning disk confocal (SDC) microscope is possible, resulting in roughly a 10-fold improvement over the diffraction limit in both the lateral and axial direction
We show that super-resolution with DNA-PAINT can be achieved in whole cells away from the coverslip for a broad range of biological targets while maintaining a good signal-to-noise ratio (SNR) ratio

Summary

Introduction

In STORM, this impacts the ability to switch dyes into the dark state (as this is a photoinduced effect), limiting the applicability to areas with low labeling densities, as most fluorescent molecules need to reside in the dark state in any given imaging frame to allow for efficient single-molecule detection at such targets. While this is less of a concern for PALM microscopy, bleaching of fluorescent proteins outside of the detection volume prior to detection in the confocal volume

Methods

Results

Conclusion