I have a protein (conjugated with FITC fluorophore) that may adsorb and cover the bacterial cell surface or be taken up into the cytoplasm.
Using confocal (fluorescence) microscopy, how can you tell the difference with any confidence? (LSM700)
The cells are rather small at 200 x 1200 nm.
A high-resolution image should be able to distinguish between labeling of just the surface or accumulation inside the cytoplasm. Surface labeling will appear as a bright ring at the edge of the cells. If the fluorophore is accumulated in the cytoplasm, the entire cell should be stained essentially evenly. If it is a Gram-negative bacterium, it may be difficult or impossible to distinguish between surface labeling and periplasmic labeling.
Dear Thomas Førland Oftedal ,
I would recoment to try to quenche the signal. with TrypanBlue (TB) By theory the FITC labeld protein should not be quenched by the TB when it is within a compartment (bacterial cell) where the TB can not reach it.
Kind regards Soenke
Mr. Oftedal,
By mass spectrometry.
Please, consider detail in the following discussion and the corresponding reference sections, therein. It is shown quantification approach as well sielding to reliability at r = 1. In addition, the mass spectrometry also provides imaging tool.
Discussion:
https://www.researchgate.net/post/any_expert_in_endotoxin_quantification_by_fluorescent_assay
I agree with Sönke Weinert . Probably the easiest is trypan blue. This is a common technique often employed with phagocytosis assays, and a technique we use with our Vybrant Phagocytosis assay kit (link below). Since the FITC is on the surface, the trypan blue works through absorbance quenching to quench the FITC signal. Since trypan blue is not cell permeant and not taken up via phagocytosis, the phagocytoses bacteria then becomes unquenched and fluoresces green upon entering the cells.
So, all you'd need to do is put a subset of the labeled bacteria into a solution of trypan blue, compared to a control with no trypan blue, and see if the signal is quenched. If it isn't quenched, then the dye is internalized in the bacteria. If it is quenched, then the dye is on the outside of the bacteria surface.
Be aware, though, that if the bacterial cell wall and/or plasma membrane are compromised, the trypan blue will also enter the cell and quench internal signal.
Trying to determine the localization of signal via high-resolution imaging is reliant upon having a very high-performance microscope or super-resolution system. Most standard widefield or even confocal microscopes are not reliably capable of determining it with enough precision:
Vybrant kit link:
https://www.thermofisher.com/order/catalog/product/V6694#/V6694
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