Occurrence of FRET in Invitrogen TetraSpeck beads?

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Occurrence-of-FRET-in-Invitrogen-TetraSpeck-beads-tp7582666.html

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I have a question for the listserver pertaining to the application of Invitrogen TetraSpeck fluorescent beads (no commercial interest):

http://www.lifetechnologies.com/order/catalog/product/T7282

I often use fluorescent beads like these for aligning two cameras (ie: simultaneous detection) on a widefield microscope or a spinning disk confocal microscope system. The spectral channels are usually green (488 nm excitation, 525/50 nm emission filter) and red (561 nm excitation, 593/40 nm emission filter) or far red (642 nm excitation, 700/75 nm emission filter). A high-quality 565 nm cut-on wavelength or dichroic mirror or a 620 nm dichroic mirror splits the multi-color emission between the two cameras.

My issue is that as of late, I've noticed that with just 488 nm excitation alone, I can get quite a bit of fluorescence in my red or far red channel. It's possible that this signal is due to cross-talk or bleedthrough (I don't know the exact spectra of the beads), but I'm also wondering if there is a FRET effect going on here since the beads contain multiple dyes that are likely in close proximity within the bead. I find that I have to bias (increase) the EM gain on the green channel camera to get the green fluorescence signal comparable/balanced to the much brighter red or far-red fluorescence signal (which requires no EM gain and very little 561 nm or 642 nm excitation power). I can usually see from the image histogram that a good portion of the signal in the red or far red channel is due to this undesired signal induced by 488 nm excitation (which is undesirable for two camera alignment procedures).

Does anyone out there have a similar experience with these beads and can someone suggest some ways avoid this undesired signal (that don't involve changing the filters). What I really want is multi-color beads that are spectrally distinct in these channels and only respond to the corresponding laser lines.

Much thanks,



John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca