Hi researchers,

I am constructing a Prism TIRF, and trying to visulize signals from Cy3 or Cy5 labeled DNA immobilixed on PEG/PEG biotin surface with low camera exposure time. However, the signal-to-noise ratio seem low and sometimes it is hard to detect signals.

The samples are new and channe quartz slides are made fresh, too. I am thinking the reason could be due to inappropriate filters preventing the excitation light from coming to camera. We are using the dichroic mirror ZT561rdc and T660lpxr from the filter sets 49909 and 49006 Chroma for Cy3 and Cy5, respectively. Because they are available in my lab. Do they think it could be a problem? Should we change to a different type of blocking filters as Notch?

Besides, we are using CMOS Prime BSI camera, would it also cause high background because this camera does not have gain control as EMCCD, the type many researchers use in TIRF microscope.

Please give us some advice. Thank you