I have tried the Alexa 405 with very little success. I tried to detect it with both standard epifluorescence and confocal microscopy and couldn't find a reliable signal even though the 488, 568, 594, 633 secondaries all worked in the particular staining I was doing. I was told that the blue dyes are very problematic and finicky but if anyone has a protocol that works for these dyes I would also like to know about it.

If you are looking to use something out of the "normal" (green and red) fluorescence range I have had a lot of success with Alexa633 (just make sure that if you have a red dye it is 568 not 594; the 594 bleeds through to the 633 channel a bit so if your red signal is really strong you will get crosstalk).

I have used Cascade Blue, AMCA and Alexa 405 with success. They are all uncomfortable (to me) on the eyes and often are associated with autofluorescent background in tissues, because of the use of a mercury burner for excitation. With a confocal microscope that has a 405 diode laser; there is less of a problem with visualization. The dyes are very bright with that laser because of the greater absorption at 405 and lower autofluorescence of cells excited at 405. I would not use to label something of low expression.

Thanks a lot for quick replies! May I ask a bit more from both of you:

- Audrey, can you see Alexa633 by eye (in standard epifluorescence) or is it getting too far towards IR? I have tried Alexa647 and can't see a thing. And do you know if the color is different enough from Alexa546 to be distinguished? I'm trying to do triple labelling with Alexa488, Alexa546 and something for the third a and I unfortunately don't have a BW camera either, so I'm stuck to the visible range.

- Brian, I have noticed the blue background too, was hoping that the signal might be strong enough to be visible anyhow (and maybe Alexa/DyLIght 405 is also violet enough to be a slightly different color?). But may I ask what exactly do you mean by uncomfortable, do you mean it's difficult to see it or that it's in some other way irritating or unpleasant?

With best regards, Mikael

Must you do your examinations visualy? I would think a high qualty camera would be very good at picking it out. CCD is much more sensitive than human vision. Assuming that background is not too much of an issue.

Oh, I just read you don't have a camera. Sorry about that. You should get a camera if you can. Digital image processing makes life so much better, for so many reasons.

the 405 dyes are not very efficient and the cameras as well as the human eye could be better there... if we use 3 combinations, we rather go for 660 instead of 405, since there are quite strong ones around (CF660R, Atto665, Alexa 660), all working with the Cy5 filter and can be excited by 633 laser for confocal

ou, sorry, i just read that you have no monochromatic camera... i would strongly suggest that you buy one, even an old iCCD would do. 660 of course the human eye cannot see much there but since most of the tissues and material is low in background there, this would be the purest solution.

I find looking at the blue emission through the eyepieces uncomfortable. I don't care for DAPI either. It's just me. I would not use the Alexa 546, not enough separation from the 488. I would use Alexa 405, Alexa 488 and Alexa 594.

at least some of problems that are discussed here are not with dyes alone but with poor match between dyes and filter sets. Filter sets are often unlabeled and neglected; and what exactly do they pass and not pass remains a mystery, leading to wrong conclusion about dyes "bad" properties. A filter with exactly matching good quality optics can give you a "single molecule" detection regardless of the wavelength.

Yeah, we have a very good RGB camera, but I realized too late that color cameras filter off UV and IR signals. Well, I guess I'll give the 405 a try, and if it doesn't I'll indeed have to get a new camera. It's funny actually, someone bought a filter cube for Alexa647 and we spent quite some time wondering why on Earth can't we see anything at all. Until we obtained a Licor Odyssey infrared instrument for western blots, and I realized what was "wrong"....

I often compare the optical path with an electric circuit; really there is no mystery to what happens to light at the end point, it is a result of everything that is placed on its way...

We have used 405 dyes. Not very visible to the eye, but digital cameras can "see the fluorescence". What works best s to amplify the 405 fluorescence by using the biotiyl-tyramine-streptavidine: 405 labeled method.

Blue Red and Green Dyes are all visible to the human eye and the spectra are sufficiently separated to uniquely identify objects stained with each. If you can not see the signal from Alexa 405 or DyeLight 405 dye you probably do not have an appropriate filterset and/or light source for it. The peak absorption for 405 dye is very narrow compared to DAPI or Hoechst so you need to be sure your light source is compatible with it.

At 420 nm one may "see" the light.

In fluorescence microscopy of the kind your experiments seem to be you may use more sensitive detectors, camera included. You have to check with the microscope producer and identify ways to complete your equipment.