How can I simulate two fluorophores with a reticle? Fluorophores typically emit in an incoherent fashion.  I thought I could simulate two fluorophores (e.g. single molecule fluors) with a reticle.  My concept was to make two small holes in a chrome photomask very close together (e.g. 200 - 500 nm).  I was going to use this to characterize an optical system that would eventually be used for measuring single molecule emission.
However, if I back illuminate the reticle with an LED, I'm concern that the two holes in the mask will act coherently; they will be spatially coherent with a constant phase offset.  This will produce an interference pattern between the two holes (i.e. spatial coherence).  This doesn't happen when two fluorophores emit.


*

*Am I right to be concerned?

*Is there a way to make them incoherent?

 A: 1) I think you are right to be worried. You would have to illuminate your two apertures with a light source with a spatial coherence smaller than 200 - 500 nm. It is I am afraid impossible to get a spatial coherence below the diffraction limit. Simply imagine you would image one single molecule with a very high NA objective on one of the holes, it would be impossible to illuminate just one hole.
2) I can think of a - not so practical - way to get around it. The ingredients you need are A) illumination below the diffraction limit B) the two sources must be incoherent. You could make two metal coated waveguides (like the ones used in aperture NSOM measurements) that end at the two holes. You could for instance make a long waveguide on top of a glass plate and illuminate it from two sides. In the middle of the waveguide you could make a deep but narrow trench that you fill with a metal with a very low penetration length. In that way you have two waveguides that end at the same place but are isolated. You will probably have quite low throughput and quite some work to do to get it working....
Maybe it is easier to just take two single molecules?
