Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Super-resolution structured illumination microscopy, 3D-SIM, is an innovative technique for the visualization of fluorescent reporter proteins of germination receptor clusters in spore membranes. Using these procedures, unsurpassed resolution of germinosome localization and spore inner membrane lipid domains can be achieved. The technique will be demonstrated by PhD student Juan Wen and Master of Science student Raymond Pasman of our laboratory.

Before beginning the procedure, clean high-precision coverslips with one-molar hydrochloric acid for 30 minutes in a gently shaking water bath, followed by two, five-minute washes in ultrapure Type 1 water. After the second wash, place the coverslips in 100%ethanol for about three minutes before drying the coverslips and verifying their clarity. Then, clean glass microscope slides with 70%ethanol for one minute before allowing the slides to dry and verifying their clarity.

For fluorescent microsphere and spore imaging, first pre-warm two slides on a 70-degree Celsius heating block for a few seconds before adding a 70-degree Celsius, 65-microliter droplet of sterilized 2%agarose onto one of the slides. Place the other slide on top to spread the agarose between the slides. After five minutes, remove one of the slides and cut the dried agarose into a one-by-one-centimeter section.

Add one times 10 to the eighth fluorescent microspheres or spores in 0.4 microliters of sterile, ultrapure Type 1 water to the agarose, and place a high-precision coverslip onto the patch using the coverslip to slide the patch off of the slide onto the coverslip surface. Then, fix a 65-microliter, 1.5-by-1.6-centimeter Gene Frame onto a dried slide, and place the coverslip onto the frame, closing all of the corners of the frame. For imaging of the samples, place the slide onto a structured illumination microscope equipped with a 100X oil objective, and focus on the 100-nanometer fluorescent microspheres.

Adjust the correction ring on the 100x objective until a symmetric point spread function is obtained to minimize blurring of the images, and select a field of view with approximately 10 round fluorescent microspheres. Apply a grating focus adjustment for the designated excitation wavelengths, in this case, 561 and 488 nanometers, as a guide for the image analysis software before focusing on the spores with the transmission light. Capture a transmission light image in the 16x average mode with 20-millisecond exposures for each image.

Then, capture 3D-structured illumination microscope raw fluorescent images of the spores with the 3D-SIM illumination mode. For slice reconstruction, click Param on the structured illumination pad tab sheet to open the N-SIM Slice Reconstruction window. Follow the suggested instructions, and click on the appropriate controls to set the illumination modulation contrast to auto, the high-resolution noise suppression to one, and the out of the focus blur suppression to 0.05 as starting points.

Then, click Reconstruct Slice to reconstruct the image and evaluate the quality of the reconstructed images by the fast Fourier-transformed images and reconstruction score that are displayed after the reconstruction. Adjust the high-resolution noise from 0.1 to five and the out of the focus blur suppression from 0.01 to 0.5 until the best parameter settings are obtained. Then, click Apply to apply the changes.

Click Close to close the window. Open an FM4-64 stained PS4150 spores raw image. Then, click Reconstruct Slice to execute the slice reconstruction, and save the reconstructed image.

To convert the 3D-SIM raw images of the KGB80 geminosome into pseudo-widefield images, left-click on the ImageJ SIMcheck plugin and select Raw Data SI and Pseudo Widefield. Randomly select about 25 spores in each inverted transmission image for a later germinosome analysis in the fluorescent pseudo-widefield images until approximately 350 spores have been selected. Then, use ImageJ to assess the maximum intensity for each fluorescent signal expressed by each group of spores and the integrated intensity of each 3D spore image.

To analyze the pseudo-widefield images of the KGB80 germinosome, use the mean integrated intensity value of seven stacks as the integrated signal intensity of the KGB80 spore. Then, determine the background intensities by imaging the PS4150 background strain using identical settings, and regard the fluorescent spots in individual KGB80 spores as germinosome foci when they are clearly distinguishable from the background. In this representative experiment, two foci of the GerD-GFP fluorescing spores appeared in different stacks with three total GerD-GFP foci observed in the compositive Z3 stack.

Here, a spore with only one GerD-GFP focal point in the spore was observed. In total, around 40 to 50%of the spores had two or one GerD-GFP and GerKB-mCherry cluster, respectively. While the integrated intensity of the GerD-GFP scaffold protein was different between different populations, the integrated intensity of GerKB-mCherry was about the same in different populations.

When the spore had multiple foci, the maximum fluorescence intensity of GerD-GFP and GerKB-mCherry foci tended to decrease, and the maximum fluorescence of all of the bright spots, regarded as the germinosome foci, was higher than the maximum auto-fluorescence of the PS4150 spores. Notably, brighter FM4-64 spots similar to germinosome foci appear in both intact and decoated PS4150 spores, suggesting that these brighter FM4-64 spots might be involved in the clustering of germinosome proteins in the inner membrane. Adding spores to the agarose and placing agarose into the frame requires a continuously and careful fluid movement for these minis materials.

The conversion of 3D-SIM raw images of the KGB80 germinosome into pseudo-widefield images and their interpretation requires expert knowledge on spore biology. Our structured illumination microscopy procedure can be applied to the imaging of low abundant proteins or dim fluorescence reporters in different bacterias or other band materials. The organization of the germinosome can now be studied using double labeling procedures to assess co-localization of Bacillus germination proteins and specific membrane domains.