Visualization of Germination Proteins in the Inner Membrane of Bacterial Spores

Abstract

Source: Wen, J., Pasman, et. al., Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores. J. Vis. Exp. (2019)

This video demonstrates the use of structured illumination microscopy to acquire three-dimensional fluorescence images that reveal the organization of germination proteins within the bacterial spore.

Protocol

1. Germination Protein Imaging (Timing: 1 H)
   1. Capture the transmission and fluorescence images of spores as well as a mixture of red and yellow-green carboxylate-modified fluorescent microspheres on a Structured Illumination Microscope (see Table of materials) equipped with a 100x oil objective (Numerical Aperture = 1.49), a CCD (Charge-Coupled Device) camera, and image analysis software (see Table of materials). Generate all images at room temperature without the disturbance of ambient light. Make sure to always clean the 100x objective and the slide with 75% ethanol before imaging.
   2. Focus on 100 nm (diameter) fluorescent microspheres and optimize the point spread function (psf) by adjusting the correction ring on the 100x objective until a symmetric psf is obtained, thus minimizing blurring of the image. The psf is the impulse response or the response of an imaging system to a point source or point object.
   3. Select a field of view with approximately 10 round fluorescent microspheres. Apply a grating focus adjustment for both 561 nm and 488 nm excitation wavelengths as the guide for the image analysis software.
   4. Focus the spores with the transmission light and capture a transmission light image in the 16× average mode with 200 ms exposures for each image.
   5. Capture 3D-SIM raw fluorescent images of the spores with the illumination mode “3D-SIM”, the camera settings to readout mode Electron Multiplying (EM) Gain 10MHz at 14-bit, and EM gain at 175. Excite the FM4-64 probe in PS4150 spores with 561 nm laser light at 20% laser power, and an illumination time of 400 ms.
   6. Excite the GerKB-mCherry and GerD-GFP in KGB80 spores with, respectively, 561 nm laser light at 30% laser power for 1 s, and 488 nm laser light at 60% laser power for 3 s. Z-Stack settings are in the top-to-bottom mode, 0.2 µm/ step.
      NOTE: These laser parameters were applied to assure a maximum brightness value of around 4,000 for the histogram window.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Air dried glass slides	Menzel Gläser	630-2870
APO TIRF N20R8 100× oil objective (NA=1.49)
Bacillus subtilis KGB80 (PS4150 gerKA gerKC gerKB-mCherry cat, gerD-gfp kan)
Bacillus subtilis PS4150 (PS832 ΔgerE::spc, ΔcotE::tet)
FluoSpheres carboxylate-modified microspheres	Invitrogen, 0.1 μm	F8803
FM4-64	Thermo Fisher Scientific	F34653
Histodenz nonionic density gradient medium	Sigma-Aldrich	D2158
Image J
iXON3 DU-897 X-6515 CCD camera	Andor Technology		https://imagej.net/Welcome
Microscope imaging software	Nikon, Japan	NIS-Element AR 4.51.01
MilliQ Ultrapure Deminerilzed Water	Millipore	Milli-Q IQ 7003
Nikon Eclipse Ti microscope
Precision Coverslips	Paul Marienfeld	117650
Screw cap tubes 50 mL	Thermo Fisher Scientific		Nunc TM