Imaging Bacillus subtilis germination proteins

Mariliis Tark-Dame, PhD. COO at Confocal.nl

Imaging highly dynamic proteins in cells is challenging, and it is even more challenging if these proteins are present in low quantities, form below resolution clusters and surrounded by autofluorescent structures.   

In order to image fast protein movement in a cell without motion blur, imaging speed in the range of 20ms is required.

Common approaches for overcoming the limitations are:

• Increasing the expression level of the fluorophore-fusion 
• Increasing the exposure time
• Switching to super resolution imaging techniques that rely on image reconstruction

All are prone to generating artifacts, such as non-native protein localization and motion-blur.

Here, we have imaged Bacillus subtilis sporulation and germination proteins SpoVAEa fused to GFP during germination using 2 imaging modalities: 

1. Wide-field microscopy 
2. Re-scan confocal microscopy (RCM)

A. Wide-field	B. RCM

In wide-field, a decent image was acquired in a snapshot with exposure time of 2 seconds (Figure 1, A). This image revealed three distinct protein clusters. With RCM (Figure 1, B), that has improved sensitivity and resolution, the same sample can be imaged with higher temporal resolution: 45 frames per second, which translates to 1.35 msec exposure time at the molecular resolution – the time every single sub diffraction level molecule is excited during scanning. Due to fast scanning the motion blur is minimized. When following the germinosome, highly dynamic protein structures moving continuously through the spore were identified (Figure 2, A). The average intensity projection based on these 1000 images shows preferential positioning of the protein during this image acquisition time (Figure 2, B).

A. Montage of every 1/40 single frames from 20 second imaging	B. Average intensity projection of 1000 frames

Why RCM allows seeing such fast-moving structures?

1. High sensitivity 
   1. Camera based detection allows for detecting every photon 
   2. Camera detector has very high signal to noise ratio even in dim samples
   3. Due to signal to noise, high contrast images can be recorded
2. Improved resolution
   1. RCM allows for factor 1.4 improved resolution. By combining RCM with deconvolution post-processing another factor of 1.4 can be won, reaching 120nm resolution. This extra resolution is crucial for imaging small organisms or organelles.

Imaging system and conditions:

Confocal.nl RCM equipped with Nikon TiE, Toptica CLE and Hamamatsu Flash4 V3 camera.
Imaging: Erik Manders, Confocal.nl
Image analysis by Desiree Salas, Confocal.nl
Sample courtesy of J. Wen, S. Brul, University of Amsterdam

References:

doi: 10.1038/s41598-020-62377-1
A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores.

R. M. P.   Breedijk, J. Wen, V. Krishnaswami, T. Bernas, E. M. M. Manders, P. Setlow, N. O. E. Vischer & S. Brul; March 24 2020, Scientific Reports.