High-Resolution Confocal Imaging of Pathogen-Infected Drosophila Hemocytes

Samenvatting

Source: Hiroyasu, A., et al. Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis. J. Vis. Exp. (2018)

This video demonstrates high-resolution confocal imaging of Drosophila melanogaster hemocytes infected with Coxiella burnetii to visualize pathogen localization in 3D, enabling detailed analysis of intracellular infection dynamics and host-pathogen interactions.

Protocol

1. Confocal Imaging
   1. Configure the confocal microscope for three-color imaging of DAPI (4',6-Diamidino-2-phenylindole), EGFP (enhanced green fluorescent protein), and mCherry. Use the following setting: DAPI excitation (ex) 405 nm, emission (em) 415-480 nm; EGFP ex 488 nm, em 493-564 nm; mCherry ex 587 nm, em 597-700 nm.
   2. Place the sample on the microscope and focus on the sample using a 63X/1.4 numerical aperture (NA) objective. Locate desired hemocytes in the field of view for imaging.
   3. Adjust laser power and detector gains to achieve appropriate exposure of the sample. Check multiple z-planes to ensure the exposure level is appropriate for the entire sample thickness.
   4. Find the top and bottom positions on the z-axis of a whole hemocyte. Set these positions as the start and end positions for z-sectioning.
   5. Only use scanning zoom to image the area containing the hemocyte. Zoom factors of 3X are often used.
   6. Collect the image series at appropriate resolution such as 1024 x 1024 pixels in the x-y plane, and 0.3 µm spacing in the z dimension.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Coxiella burnetii - mCherry	Dr. Heinzen, R.
Microscope cover glass	Fisher Scientific	12-545-80
4',6-diamidino-2-phenylindole	Thermo Fisher Scientific	62247
Confocal microsope	Leica	TCS SP8-X White Light Confocal Laser Scanning Microscope
Microscope slides	Fisher Scientific	12-552-3