High-Resolution Confocal Imaging of Pathogen-Infected Drosophila Hemocytes

Place a slide containing fixed hemocytes onto the confocal microscope stage.

These hemocytes are obtained from Drosophila melanogaster larvae and have been infected with a bacterial pathogen.

The hemocytes are labeled with green fluorescent protein, their nuclei are stained with a blue dye, and the bacteria express red fluorescent protein for visualization.

Configure the microscope to detect all three fluorophores to track cellular compartments and pathogens simultaneously.

Using a high-resolution objective, focus on the target hemocyte to capture subcellular details.

Adjust the laser power and detector gains to achieve appropriate sample exposure and optimize signal clarity.

Scan multiple Z-planes to ensure the exposure level is appropriate.

Set the top and bottom Z positions to image the entire hemocyte.

Use scanning zoom to isolate the hemocyte of interest and acquire a high-resolution 3D image.

This method enables visualization of pathogen localization within the hemocyte.

Configure the confocal microscope for three-color imaging of DAPI, EGFP, and mCherry.

Place the sample on the microscope and focus on the sample using a 63X/1.4 numerical aperture objective. Locate desired hemocytes in the field of view for imaging. Then, adjust the laser power and detector gains to achieve the appropriate exposure of the sample.

Check multiple Z planes to ensure the exposure level is appropriate for the entire sample thickness. Next, find the top and bottom position on the Z axis of a whole hemocyte. Set these positions as the start and end positions for Z sectioning.

Use scanning zoom to image the area containing the hemocyte. Collect the image series at the appropriate resolution, such as 1024 by 1024 pixels in the X-Y plane, and 0.3 micrometer spacing in the Z dimension.