Technical Reference #3066

3066.

3D reconstruction of granulomas from transmitted light images implemented for long-time microscope applications Amélie Delaby; Leon Espinosa; Catherine Lépolard; Christian Capo; Jean-Louis Mège, Universite de la Mediterranee, Journal of Immunological Methods, 360(3066), (2010)
Link To Paper

Abstract:
Image analysis tools are essential to describe and quantify dynamic biological phenomena such as early stages of granuloma formation. Granulomas are constituted of a collection of immune cells that contain pathogens leading to their elimination. We presented here a new method to obtain granuloma 3D reconstruction from transmitted light images. Granulomas were generated by incubating peripheral blood mononuclear cells with beads coated with sonicated Coxiella burnetii a bacterial pathogen. Biological samples were observed under a confocal microscope and recorded during several hours providing a large set of data of several gigabytes. Our image processing called Focus Detection Plugin (FDP) allowed to extract relevant images from large datasets and to perform a deblurring of image stacks. This FDP method that was implemented as an ImageJ plugin did not require powerful computer resources and was simple to use. To validate our FDP method we compared our results with 3D reconstruction of fluorescent images. Both methods yielded comparable results. We concluded that our FDP method was able to generate processed images yielding robust 3D reconstruction of whole cell bodies and presented major advantages for long-time recordings since no cell labeling was needed. This method was convenient to study the early stages of granuloma formation and may be applied to other complex biological systems.

Keywords:
Granulomas Transmitted light Live imaging 3D reconstruction

Materials & Methods:
PBMCs from healthy donors were isolated from leukopacks (Etablissement Français du Sang Marseille France) by Ficoll gradient centrifugation as previously described (Honstettre et al. 2003). PBMCs were suspended in RPMI 1640 containing 20mM HEPES 10% fetal calf serum (FCS) 2mM L-glutamine 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen Cergy Pointoise France). Monocytes and T lymphocytes were isolated by positive selection onMACS CD14 and CD3 microbeads respectively (Miltenyi Paris France). For fluorescence experiments 106 CD14+monocyteswere incubated with 25 μg ofFM4–64 (Invitrogen) a lipophilic tracer known to insert into the outer leaflet of the surface membrane for 5 min and 106 T lymphocytes were incubated with 5 μmol carboxyfluorescein diacetate succinimidyl ester (CFSE Invitrogen)which passively diffuses into cells for 10 min. PBMCs (2×106 per assay) or fluorescent monocytes and T lymphocytes (ratio of 1/4) were incubated with 800 beads coated with C. burnetii extracts in RPMI 1640 containing 10% FCS 2mM L-glutamine and antibiotics in 35mm glass bottom culture Petri dish coated with poly-D-lysine (MatTek Corp. Ashland MA USA). Granuloma formation was observed under an inverted microscope.

Microscopic Technique
Inverted microscope

Cell Type(s)
Monocytes and T lymphocytes