Posted by
Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/7th-Course-on-Optical-Microscopy-Imaging-for-Biosciences-20-24-April-2015-IBMC-Porto-tp7583912p7583944.html
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Ah, you are using far red dyes with a confocal. That's a relatively new
thing. In this case the NIR corrected lenses normally used for 2-photon are
actually useful in a confocal situation since you are working with very red
light both for excitation and emission. If you also want to image GFP then
your system has to be very broadly corrected.
Also, hopefully someone can chime in here, how sensitive are the Perkin
Elmer APDs to 800 to 900nm light? Many detectors are very insensitive this
far down. You typically want to use a multi-alkali PMT for those sorts of
ranges. I find the Hamamatsu 10721-20 works well out to about 850-900nm yet
is still sensitive 'enough' in the blue and green as well (I use it for GFP
or DAPI and very red things). Here's the product sheet:
http://www.hamamatsu.com/eu/en/product/alpha/P/3003/3044/H10721-20/index.htmlGetting back to your objective lens, in this case you need something that
is very broadly chromatically corrected. You will want to consult your
vendor as to which lenses they offer that meet this need. You will also
want to avoid working on the edge of your field of view, and might have to
crop down a bit as chromatic aberration gets worse near the edge of your
full field.
Best of luck!
Craig Brideau
On Mon, Jun 29, 2015 at 4:27 PM, Vitaly Boyko <
[hidden email]>
wrote:
> Hi Craig,
>
> thank you for your reply. How would one image Alexa 790/Cy7 together with
> let say CeFP/dapi, GFP through the one photon confocal system (with Perkin
> Elmer APDs as detectors). Are there many IR APOCHROMAT lens that are
> corrected between 400 nm (tough, I guess, at least 500 nm) and 900nm at 40x
> NA 1.0-1.1 or 60x NA 1.2?Multi-photon collect scattered (non-linear,
> non-chroma corrected) light via NDDs...
>
> Vitaly
>
>
>
>
> On Monday, June 29, 2015 6:16 PM, Craig Brideau <
[hidden email]>
> wrote:
>
>
> Your question is fairly vague, but here are some general answers: With 2
> photon the important thing is how well the laser is focused. This means
> your objective has to be well corrected for the NIR beam from the laser.
> The resulting visible fluorescence is directed towards wide-aperture PMTs,
> which don't care about aberration very much.
> For single-photon confocal, you have to get your fluorescence through the
> confocal pinhole, which will be *very* picky about aberration. Since you
> are trying to get the light through a very small aperture any aberration
> will cause signal loss.
>
> Craig
>
> On Mon, Jun 29, 2015 at 4:04 PM, Vitaly Boyko <
[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.
> *****
>
> Dear List,
> I would greatly appreciate if someone could shed light on the extent of
> lateral and axial chromatic aberrations correction, and its limitations in
> the VIS portion of spectrum when image has been projected into the
> non-descanned detectors (one and two photon imaging)?
> Many thanks in advance.
> Vitaly
>
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