I observe dramatic differences in E. coli GFP-tagged protein localization when I analyze non-stained cells and cells stained with membrane dye FM 4-64
GFP-tagged proteins show no specific localization in non-stained cells. When stained with fm 4-64, however, these same GFP-tagged proteins show clear membrane localization. When I excite GFP-tagged proteins (at 475 nm) I believe I may slightly excite fm 4-64 (its excitation peak is at 515 nm), but I observe GFP emission at 523 nm and FM 4-64 emits at 640 nm . Therefore I see no reason why, although it could result slightly excited, I observe FM 4-64 derived signal at the GFP channel. It is not a culture related problem neither, as I do this comparison with samples taken from one single culture.
I attach a PP slide where two images showing GFP-tagged protein localization in FM 4-64-stained and non-stained cells are compared.
Has any of you ever faced similar problems? I would appreciate some hints regarding this phenomenon. Thanks!
Hi, I think your protein in non stain cells is still membrane localized, from your image, I suppose you put the focus on membrane rather than going further,i.e., middle. I think if you let focus go further, you will see membrane localized and similar to your second image.
good luck
@ Feng Lin: Thanks for your reply. I do actually acquire slides along the z axis in order to study the localization of my proteins in the three dimensional space. However, at no single focus, starting from "top" to "bottom" of the cell, do I observe membrane localization for my GFP-tagged proteins in the non-stained cells. On the contrary, when stained the GFP signal truly resembles the one emitted by the FM4-64 dye, clearly mimicking membrane localization at several focus points.
Maybe you are right and this is the actual localization of my proteins, but I must clarify why this is happening and why I cannot observe such clear localization in non-stained cells. I dig and dig in the net but I found no reasonable explanation yet.
I just found a reference (https://pages.wustl.edu/levin/book-chapter-fluorescence-microscopy-approaches-bacteria-2002, pages 18 and 20) stating that, although its emission peak is at red colored wavelengths, FM4-64 is actually faintly visible with most GFP filter sets. So I guess what I observe at my stained cells is just this weak green fluorescence emited by FM4-64 rather than actual fluorescence emitted by my GFP-tagged proteins.
FM 4-64 has a very broad excitation spectrum (as you can see in the attached link), making it hard to use in multi-dye fluorescence . While its excitation peak is actually around 508 nm where it has an extinction of 46000 1/M/cm, at 515 and 475 nm it has an extinction of 45500 and 38000 1/M/cm respectively. So you don't just slightly excite FM 4-64 at 475 nm, but you excite it almost as efficient as you do at 515 nm.
I assume you or the microscope changes the emission filter when switching from FM 4-64 to GFP. So if you do use FM 4-64 together with GFP you can only hope that the GFP emission filter can block all of the FM 4-64's emission, which has a peak around 750 nm, but also emits some photons around 640 and below, down to your excitation wavelength.
To efficiently filter out most of FM 4-64's emission in the GFP channel, the GFP emission filter should filter out all photons above around 550 nm. If this is not the case, then you will see FM 4-64 in the GFP channel. As this cuts of a piece of the GFP emission this might not be the case very often.
https://www.lifetechnologies.com/nl/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html?ICID=svtool&UID=3166ch2
@ Pomp:
Thanks for your clear explanation. It is so often used in combination with GFP, I did not expect FM4-64 emission could possibly be observed at such wavelengths. I am still surprised of how I can observe that much FM4-64 emission with my GFP observation filter set, considering it is optimized for emission observation at 523 nm.
Anyway, your explanation and additional information I have found highlight this is an issue to consider when working on GFP in combination with FM4-64, especially in protein localization experiments like mine where rather low expression of the protein of interest is often desired. In my case, I will just use a different membrane dye.
Which concentration of FM4-64 and which solvent have you used? FM4-64 as lipohilic electrochemically active molecule can work as player itself.
The concentration must be very low (1 µM is maximal).
My best wishes!
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