How to define the presence/absence of an object in immunofluorescence

Posted by Ke Peng on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-photon-microscope-questions-tp7583010p7583025.html

Dear lister:
I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
I'm looking forward and I'll be very grateful to your opinions.
Best wishes,
Aro