Capturing Images by Confocal Microscopy
I repeated the transfection of HL-60 cells last week and captured images on the confocal microscope today. The brightfield images are on the left. Fluorescent images are on the right. The images on top are HL-60 cells one week after transfection with a red fluorescent protein tag. Normal untransfected HL-60 cells are on the bottom.
All cells were fixed in 2% paraformaldehyde and mounted with an antifade media. A lot of cellular degradation has occurred in the transfected cells, but you can see the lone intact cell expressing the red fluorescent protein tag in the center of the frame. Untransfected HL-60 cells do not have any red fluorescent protein, but if you look closely you can see a tiny bit of autofluorescence. All images were captured on the same settings. All images are 45X and the fluorescent images were obtained using a 552nm laser.
I need to repeat the experiment again with new cells from ATCC. I have been doing all of the preliminary studies on HL-60 cells that were frozen in Liquid Nitrogen (LN2) 20 years ago. It will be a few weeks before my new shipment of plasmid for the transfection arrives as well.
So, until next time my fine fellow geek friends! Eureka!
The Leica TCS SP8 Confocal Microscope
I used the Leica TCS SP8 Confocal Microscope to obtain these images. The SP8 is an inverted laser powered single-point True Confocal Scanning system (TCS) allowing the user to produce publication quality high resolution images of fluorescently labeled histologically fixed tissues and cells, as well as cultured live cells.