Nanoscopy of bacterial cells immobilized by holographic optical tweezers.

Robin Diekmann,Thomas Huser,Deanna L Wolfson,Christoph Spahn,Mark Schüttpelz,Mike Heilemann

doi:10.1038/ncomms13711

Abstract

Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension. Holographic optical tweezers (HOTs) enable the ability to freely control the number and position of optical traps, thus facilitating the unrestricted manipulation of cells in a volume around the focal plane. Here we show that immobilizing non-adherent cells by optical tweezers is sufficient to achieve optical resolution well below the diffraction limit using localization microscopy. Individual cells can be oriented arbitrarily but preferably either horizontally or vertically relative to the microscope's image plane, enabling access to sample sections that are impossible to achieve with conventional sample preparation and immobilization. This opens up new opportunities to super-resolve the nanoscale organization of chromosomal DNA in individual bacterial cells.

Highlights

Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension
The same applies to labelled structures that are visualized with subdiffraction resolution by single emitter localizations. This corresponds to the direct stochastic optical reconstruction microscopy (dSTORM) image of the structure, as it might be determined for an immobilized sample, convolved with the position distribution experienced within the optical trap
In case of the microsphere bead edges, this fact is illustrated by their average full width at half maximum (FWHM) becoming broader in the dSTORM images as the trap power decreases and roughly follpowffiffiiffiffinffiffigffiffiffi the theoretically expected dependence of FWHM / 1= PTrap (Fig. 1a and Supplementary Note 1)

Summary

Introduction

Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension. Localization microscopy techniques, such as direct stochastic optical reconstruction microscopy (dSTORM) and photoactivated localization microscopy (PALM), typically require several thousands of image frames for the reconstruction of a single super-resolved image, leading to very long acquisition times on the scale of seconds to minutes[2,3] This entails a high demand on the spatial position stability of the sample, which is typically achieved by chemical fixation and attachment of biological samples such as cells to glass coverslips. Beam shaping by a spatial light modulator (SLM) enables the generation and dynamic control of multiple, independent optical traps[8] We combined this system with dSTORM to facilitate the immobilization of the sample at specific, pre-defined positions and orientations in suspension during the nanoscopic imaging process

Methods

Results

Conclusion