Adding images for illustration, including far-red channel images once unmanipulated and once after heavy ImageJ processing, and unmanipulated green channel image (laminin).  

One thing to consider here is that many cameras used for microscopy are not very sensitive in the far red range. Your eye isn't either, which is why you can't see the Alexa647 image in the eyepiece very well unless you are labeling a really abundant target in that channel.

   Here are a couple things I would recommend trying:

1) Get into the camera settings (on the camera or in the software, depending on the system you are using) and see what the camera gain is set to. The sensitivity of cameras varies. We use a QiCAM camera (which is not an extended range camera with extra sensitivity in the deep red) and we can usually get decent images in the AlexaFluor647 with a gain of 3-5. As you increase the gain, you may also increase the background grey level. You'll need to balance these two things to ensure a decent image.

2) Swap your labels around. If you have an antigen that is very abundant and bright in the green or far red channels, try labeling that one in the Alexa647 channel, and moving your other target that is currently in the Alexa647 channel into the green or the near red.

3) If you don't need DAPI for nuclear staining in the blue channel, you might try tagging your target with ALexa350 instead of Alexa647. Our experience with Alexa350 is that it can work, but we often experience higher nomn-specific autofluorescence in that channel than in others.

4) You might try this as a last resort and ONLY with approval from whoever is in charge of your microscope. Some cameras have an infrared filter placed in front of the camera chip, which will cut some of your far red signal. Some people open the camera and pop that filter out (it looks like a clear plastic block glued over the chip. If you feel the need to monkey with that, talk to whoever is in charge of the system BEFORE doing anything. (In our case, we've left that filter in place and can still get images in this channel most of the time if we arrange our fleuorescent labeling scheme carefully. I really don't like the idea of opening up the camera and prying bits out of it).

Thank you very much for the suggestions, David. I had a hunch it might be the camera. Unfortunately the setup is owned by someone else so there is a limit to how much I can fiddle around with it. Swapping around the labels and increasing the gain is probably the best course of action for then! 

The human eye is not very good at perceiving infra red light, so in the 647 channel you have to rely pretty much on the camera.

As David suggests, swap the fluorescent tags if possible.

Good luck!

It should (has to) be somehow possible to get access to a proper confocal microscope at your institution (that would also allow you to reduce background, to z-scans, and so on) if your investigations rely on fluorescence microscopy. This is especially true when your current microscope has a (weird) issue with the detection of AF647. That should not at all be a problem for a standard microscope, and since you are pretty sure that it is not the AB, the microscope is to blame. If you are not even allowed to look for the error in depth, you simply should not use that instrument. Swapping the labels may improve your signal for one epitope, but you just pay for it with a decreased signal for another epitope, which is far from optimal. Maybe talk to your PI about that sometime;)

I stumbled on this thread while trying to troubleshoot my own problem with Alexa Fluor 647.

You case is camera. Check out the spec sheet:

https://www.lumenera.com/media/wysiwyg/resources/documents/datasheets/microscopy/infinity-2-1R-datasheet.pdf

According to this, 2-1RC refers to a tri-color version. Your camera is not meant for far-red.

So this is quite late, but you are all correct in that the problem was the camera simply not being set up to detect far red wavelengths. I have switched to a proper microscope (not confocal, but it can detect more than three colours) and not had a problem since. Thanks for all your replies!