I actually visualize the attached bacteria to surfaces by Using EM.
And I would like to have the explanation of the green color I obtain instead the blue one. In the all the articles I have read, they obtain a blue color of bacterial cells which were stained using DAPI.
DAPI is a nuclear stain that could bind to the AT regions of DNA. Based on its excitation(358nm) and emission(461nm) spectra of DAPI it is not possible to obtain a green fluorescence. If the whole cell have a green fluorescence glow then my guess is that its be due to bacterial cell auto-fluorescence (1,2). May I know whether you permeablise the bacterial cell so that DAPI could interact with the bacterial DNA.
(1) Cytometry. 1997 Jun 15;30(3):151-6. Reducing cellular autofluorescence in flow cytometry: an in situ method. Mosiman VL1, Patterson BK, Canterero L, Goolsby CL.
(2) Anal. Chem. 84, 3, 1526-1532 Detection and Quantification of Bacterial Autofluorescence at the Single-Cell Level by a Laboratory-Built High-Sensitivity Flow Cytometer. Lingling Yang, Yingxing Zhou, Shaobin Zhu, Tianxun Huang, Lina Wu, and Xiaomei Yan.
if you are using a fluorescence microscope, yes it can be possible to get different colour due to the filter or the colour specified in the software. Check the filter and software settings, this might solve your problem.
Abhijeet Singh Khaddouj Amzil I guess, when we use blue filter to observe DAPI through Epifluorescence microscope we can green colored cells. However when we use special DAPI filter , then we get blue color.
THis is what I understood .Please state if i am wrong.
We have conventional fluorescent microscope and it has filters described on coilours like blue, green, uv, and bright field. How can we choose filter for dapi or lysotracker dye
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