I'm implementing RPA assay using fluorescence microscope.
Protocol of its assay mentions that "The red fluorescence of RPA is excited at λ exc.=543 nm and collected through a long-pass filter near λ exc.=601 nm.".
When I choose wavelength in microscopy, I don't know what I choose either 543 nm or 601 nm.
Which fluorescence wavelength should I choose?
Regards,
Daiha Shin
Hi Daiha,
My recommendation:
Read up on fluorescence physics! You'll see very quickly that both wavelengths (or spectra, really) are relevant here. The smaller peak wavelength (543nm) denotes the maximal excitation point for this fluorophore (although again, there's a whole spectrum and this is just the peak). The larger one is the maximal emission (same caveats regarding spectrum here).
In practice, you want to illuminate your fluorophore with light around 543nm (green/yellow ish), and look for your fluorescent signal around 601nm (red/far-red).
Fluorescence microscopes usually have filter sets designed for certain excitation/emission wavelength pairs. Check your microscope for a filter set with something close to 543/601 nm. It doesn't have to be exactly the same. If there is nothing suitable, you may have to borrow or purchase a suitable filter set. If you use incorrect filters, you may not be able to see the fluorescence very well, or at all.
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