Confocal movies of BBB co-cultures
Over the last couple of weeks, I’ve been doing confocal fluorescence microscopy over at the Med Ctr. I was hoping this tool would clean up some of the background fluorescence in my immunofluorescent images. More importantly, the confocal can take ‘slices’ of images in the z-plane and stack them on top of each other to make a 3D cross-section of your sample. I thought this would illustrate the thickness advantages of pnc-Si over PET for co-cultures. Slices can be as little as 200nm thick. I have tons of data to analyze from my time over there but here are a couple of highlights.
This movie shows a stack of 400nm z-slices which has been animated to show the cross section. Here, the glial cells were stained red and the bEnd3 cells were stained blue (nucleus). These cells were cultured on pnc-Si and the 2 fluorescent signals were essentially on top of each other.
60x zoom 2.2 1(2)-3DAnimation-avi movie
Here, the endothelial cells were stained green with calcein AM (since the nuclei didn’t show up well in the first movie). The z-slice thickness is the same as the first movie but these cells are on PET membranes. You can clearly see a huge gap between the fluorescent signals – this is the membrane thickness.