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Iain,

I guess the occurrence of FRET concerns me to some extent because there might be a (wavelength-dependent) lateral magnification difference of the bead images onto each camera. The camera lenses I use have an adjustable focal length and it is possible to achieve pixel-to-pixel registration by making appropriate adjustments of these lenses, but I want to be sure that the magnification difference I'm seeing is for a certain excitation/emission combination.

A similar argument for wanting to remove the FRET effect might be made when trying to match the axial focus of both cameras.

But maybe my concern is trivial? Perhaps the magnification difference between 488 nm excitation / 700 nm emission compared to 640 nm excitation / 700 emission is too small to detect (ie: sub-pixel difference). I'm not sure, but I seem to recall one instance where I thought I could see this difference. 

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



On 2014-09-24, at 10:20 PM, Iain Johnson wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
> 
> John:
> 
> I'm sure the TetraSpeck beads exhibit FRET.  Even the single-color
> Molecular Probes beads (FluoSpheres) exhibit FRET (homo-FRET in this
> case), deduced from the fact that they have very low fluorescence
> polarization, contrary to the predictions of hydrodynamics.  Whether the
> red signal you are seeing with 488 nm is actually FRET or just direct
> excitation of the red and far red dyes at 488 nm is another question.  The
> latter effect is inevitably present to some extent, as the dyes have a
> non-zero absorption cross-sections at all wavelengths below the 0-0
> transition (as does every other fluorescent dye ever invented).   The
> spectra of the dyes used to make the tetraspeck beads are available in the
> Molecular Probes Spectraviewer utility.
> 
> I guess I'm wondering why you need to use two different lasers.  Is there a
> significant difference in the illumination distribution delivered by the
> two lasers that invalidates aligning the cameras using green and red
> signals derived from 488 nm excitation only?
> 
> Iain
> 
> On Tue, Sep 23, 2014 at 12:19 PM, John Oreopoulos <
> [log in to unmask]> wrote:
> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>> 
>> 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