I was wondering if anyone of you has looked into the background generated by autofluorescence of standard (TIRF) objectives.
I have set up a TIRF microscope for single molecule imaging with laser lines 488, 561 and 638nm. While the imaging of single emitters excited with the 561 laser (e.g. Cy3B, ATTO565) and 638nm (ATTO655) is easily possible with good signal-to-background, I have troubles getting similar quality signals from ATTO488 or Alexa488, even though the dye should perform even better on paper.
It also appears that the signal level of the 488 dye is not the problem, but the increased background is. It even spills over onto the 650-750nm emission channel.
Filters, Dichros and Immersion oil could already be ruled out, so I looked into autofluorescence of the objective and found a short paragraph in Brunstein et al:
*Furthermore, upon 488-nm excitation with low μW power and EMCCD detection, all three tested objectives displayed measurable yellow-green autofluorescence (Fig. S2). This instrument fluorescence passes undetected as a background offset when imaging the sample-plane. BFP imaging with a Bertrand lens allowed us to identify the origin of this fluorescence inside the objective.*
Besides choosing narrower emission filter bandpasses or using different modes of illumination, how are people tackling this? Please also let me know if you have no idea to solve it, but experienced the same issues