This article describes a method for imaging bacterial spores using structured illumination microscopy (SIM). The technique allows for the precise mapping of germination proteins within the spores.
Take a slide containing dormant bacterial spores and fluorescent microspheres as a reference.
The spores carry red and green fluorescent reporters for germination proteins, with the inner membrane stained using a lipophilic dye.
Mount the slide on a structured illumination microscope or SIM equipped with an oil immersion objective.
Locate the fluorescent microspheres and adjust the focus to minimize image blurring.
Next, use transmitted light to locate the bacterial spores.
Switch to SIM mode and illuminate the spores to excite the fluorophores.
Acquire high-resolution fluorescence images across the spore depth.
On the lipophilic dye-stained inner membrane, the germination proteins appear as distinct red and green foci. This enables precise mapping of the location of the germination proteins within bacterial spores.
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.
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