A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores

R M P Breedijk,S Brul,N O E Vischer,T Bernas,E M M Manders,V Krishnaswami,P Setlow,J Wen

doi:10.1038/s41598-020-62377-1

Abstract

Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited. Current super-resolution techniques require either the use of special exogenous probes, high illumination doses or multiple image acquisitions with post-processing or combinations of the aforementioned. Here, we describe a new approach by combining annular illumination with rescan confocal microscopy. This optics-only technique generates images in a single scan, thereby avoiding any potential risks of reconstruction related artifacts. The lateral resolution is comparable to that of linear structured illumination microscopy and the axial resolution is similar to that of a standard confocal microscope. As a case study, we present super-resolution time-lapse imaging of wild-type Bacillus subtilis spores, which contain low numbers of germination receptor proteins in a focus (a germinosome) surrounded by an autofluorescent coat layer. Here, we give the first evidence for the existence of germinosomes in wild-type spores, show their spatio-temporal dynamics upon germinant addition and visualize spores coming to life.

Highlights

Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited
The optimum corresponds to M = 1 + (Wem/Wex)[2], where ‘Wem’ is the full width at half maximum (FWHM) of the emission point spread function (PSF) and ‘Wex’ is the FWHM of the excitation PSF
While previous studies reported a correlation between the amounts of germinant receptor (GR) and the time to germination at a population level, we found no clear correlation at a single spore level[44]

Summary

Introduction

Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited. We describe a new approach by combining annular illumination with rescan confocal microscopy This optics-only technique generates images in a single scan, thereby avoiding any potential risks of reconstruction related artifacts. An artifact-free super-resolution image can be generated in a single scan, by using an annular aperture in the excitation path of a rescan confocal microscope This technique scores high on its ease of use and can be applied to any common confocal imaging scenarios with standard probes and sample preparation routines. No mathematical post-processing is required and our technique is immune to any image reconstruction artifacts By addressing these challenges, we perform time-lapse, super-resolution imaging of wild-type bacterial spore germination using our new imaging approach

Methods

Results

Conclusion