http://confocal-microscopy-list.275.s1.nabble.com/Occurrence-of-FRET-in-Invitrogen-TetraSpeck-beads-tp7582666p7582681.html
Wavelength dependent magnification differences would be seen as blurring toward the edge of the field in transmitted white light images for a monochrome camera and colour fringing in a colour camera.
> *****
> 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.
> *****
>
> 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:
>
>> *****
>> 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.
>> *****
>>
>> 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 <
>>
[hidden email]> 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
Mark B. Cannell Ph.D. FRSNZ