Paul and Martin,
    Thank you for your observations and comments. In answer to your
questions/remarks: We have checked for spectral bleedthrough and
find that under our experimental conditions that we do not observe
any. I also know that the mis-registration of which Paul speaks
could be a problem but using 210 nm diameter fluorescent beads
I have determined that is not a big problem (The mis-registration
we see is less than the resolution and smaller than the structures
we are observing).
   As I was listing these controls I realized that the control that
should solve are problem is one that is stained in the same manner
but has more fine structure to be able to determine the amount
of out-of-focus signal contribution to our signal.
 
   Jim