I am having an exposure time problem with one of my colors. 647 Alexaluor
My other colors expose for less than 1 second or at the most 3 seconds.
the 647 can take 10+ to even 20 seconds.
what the heck is going on? My vials are within their expiration dates, So ir seems unlikely it it bad,
I want to test my marker in both colors at once as stand alone stains to see if it comes up quickly at say 488. I also want to try a different scope to see if its scope dependent.
thoughts? I do image the area last at 647, would imaging it first make a difference? seems like it wouldn't since my dapi/488 are less than 1 second and my 555 is 2-4 seconds.
At this point I don't know if its the marker, the color or the scope, or something else.
Hi Patrick,
Alexafluor647 maximum excitation is @ 647, and maximum emission is @ 660nm
Do you have the right filter combination ? For example excitation 630 BP, dichroic mirror 650, emission 655 LP. If you use a standard "rhodamine" filter set, it will be far from optimal with a too low excitation efficiency, and then a highly reduced emission. And as emission filter has a broader wavalength range, you will capture more unspecific signal (noise).
Another point is that many cameras have anti IR filter put in front of the CMOS captor. Do you have such filter ? If you do, you have to remove it, but it may be tricky.
Check the signal intensity with your eyes, but considering that for many people 660nm is near to the limit of color perception, it may be difficult for you to see it properly.
Hope these points help you.
Oliv
It should be the correct filter cube, but I will check that.
I am pretty sure it only fits one way.
So its unlikely that it is not in correctly. I will look at that today.
The type of lamp you are using for your excitation can make a big difference too. Mercury lamps have a much lower intensity in the near-infrared range compared to xenon lamps. (see: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/fluorescence/fluorosources/)
In my experience, the signal obtained from AF647 is much more visible using laser scanning confocal microscopy than regular fluorescence microscopy.