Dear Colleagues.
I am trying to do fluorescence imaging of DAPI/Phalloidin double-stained cells.
However, DAPI/Phalloidin Images look almost identical.
Channel for DAPI :495 Dic +448/20 BandPass(430~460, blue)
Channel for Phalloidin: after 495 Dic, 564 Dic +525/50 Bandpass(500~550, green)
System: Custom-built Two-photon microscope, excited at 740nm (140fs)
I want to check the two-photon emission spectrum overlapping, But I cannot find any emission spectrum reference of dyes.
If you have experience with Phalloidin/DAPI double imaging with TPM, could you give me a clue to solve these issues?
Dear Yong,
while excitation spectra are different for single photon and two photon excitation, emission spectra are the same. This means, you can just use any only spectra viewer app to check the emission spectrum overlapping, for example here: https://www.fpbase.org/spectra/.
I never worked with simultaneous Dapi/Phalloidin staining, but while it seems your blue filter should only see Dapi, you have quite a lot of spectrum overlapping in your green filter band. This means that, since you have quite a lot of Dapi signal in your sample, it seems to overwhelm the signal from Phalloidin.
Maybe try exciting with a longer wavelength, and see if the signal in the blue channel decreases, in this case any remaining signal in the green channel would be generated by Phalloidin, hopefully.
Paolo Pozzi
Dear Dr. Paolo Pozzi
Thank you very much for your comments.
I did not know that the emission spectra of DAPI/Phalloidin fluorophores overlap so much. (Fig 1)
It's a shame I didn't know this until now. Haha..
As advised, I tried to emit in the 750 nm and 800 nm bands, respectively. As expected, the relative intensity of the DAPI channel decreased more than that of the Phalloidin channel. but still not well differentiated. (Fig 2)
Unfortunately I don't have narrower bandpass filter for green wavelength for now. I will test after it arrived.
In my opinion, when imaging DAPI/Phalloidin co-staining in a single photon (confocal), lasers of different wavelengths (eg, 355 nm and 488 nm) are used, so each fluorescence is selectively excited, so there is no mixing between channels.
However, when excited with a wavelength of 740~800 nm for two-photon imaging, DAPI and Phalloidin are excited at the same time, which seems to be a problem.
I wonder if my thinking is correct.
Regards
Hello Yong,
yes, your thinking is indeed correct. The fact that in two photon excitation you can excite most fluorophores efficiently simultaneously is indeed a "feature" of two photon microscopy, which is often very convenient, but in cases like yours can generate problems.
Anyway, since DAPI seems to emit the vast majority of green light in your sample, maybe try to have a look at it with a confocal or simple epifluorescence microscope with 488 excitation, and verify that the Phalloidin staining worked correctly. Maybe your sample for some reason only has DAPI, and that's why you still don't see much difference with longer excitation wavelengths.
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