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Scan acceptor required

Fred fluorescence resonance energy transfer offer can be performed in several different ways I'm going to run methods of Fred to fix a detection which is called acceptor photo bleaching this is my negative control actual this is free wife B and freeze and see if be expressed in the leaves of nicotine up into mana and I would like to confirm that there is no threat between the CFP and wife in this case first of all if I claim that I'm detecting Fred in the nuclei I should be able to see the whole nucleus I can use tools in graphics to measure what is the size of the structure I'm looking at I select line to draw a line to have the longest axis of the nucleus and then I can select their measurement checkbox here and it tells me that this structure is approximately nine point zero two micrometers and I can make sure that this is a reproducible size okay so the size of the nucleus is approximately from some 6 to 10 micrometers no I don't need that anymore in the channels I can see what is the thickness of my optical section and currently is thirteen point six micrometers this thickness will be set by the size of the pinhole if I decrease the pinnacle size the thickness of optical section also decreases I will keep it fully open to make sure that you will be able to see new clay which are even bigger than this size acceptor photobleaching methods relies on differences in the intensities of donor which in this case is safety an acceptor which in this case is wife B depending on whether our acceptor is present or not that is that the emission light of CFP is used as excitation light of wife B and if this happens I cannot detect very strong emission of CV because it's immediately absorbed by the wife B protein if I photobleach the wife P protein it will not be able to absorb a mission of the sieve P and in this case I will be able to detect higher intensity of CF p so now I'm going to detect both GFP and wife B channels and then I will photobleach wife B and see whether the signal in the cfp channel becomes stronger since I'm working with a quantitative intensities I need to make sure that I don't have any oversaturated pixels in either channels I will go to live dimensions and range indicator and obviously I have some in the wife B Channel and I need to get rid of them by for example pulling down the laser intensity or gain or offsets I'm also detecting chlorophyll just for the fun of it and in the CFP Channel I have very few diversity rated pixels that's okay and I'm also detecting a transmitted light channeled using this data please note that you would like to detect donor and acceptor simultaneously...