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That's odd.  Doesn't Zeiss have a blue correction optic in the microscope to
keep things chromatically aligned?

Craig


On Mon, Jan 10, 2011 at 3:32 PM, Rosemary White <[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
> *****
>
> Hi Lloyd,
>
> I've found that the red and blue emission aren't quite lined up vertically,
> at least with our "blue" 63x objective, so have to cut the top one or two
> slices off a blue vertical series (depending on depth of slice), and the
> bottom slices off a red series and reassemble to get the emissions aligned
> -
> i.e. from the same depth in the tissue.  The objectives do vary a bit,
> perhaps ours isn't as well corrected as some.
>
> cheers,
> Rosemary
>
>
> On 11/01/11 9:16 AM, "Lloyd Donaldson" <[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
> > *****
> >
> > Hi Rosemary
> >
> > We don't really work with live tissue but we do have a Leica 63x water
> lens on
> > our old TCS NT. I never really found it particularly useful. I haven't
> tried a
> > direct comparison on our new SP5 II but I would expect the glycerol lens
> to be
> > much better unless you have to be in water.
> > If your blue 63x is the same as ours I have imaged blue green and red
> both
> > sequentially and simultaneously without any obvious problem. We image
> lignin
> > at blue and or green emission and combine with alexa568 or alexa647 for
> immuno
> > work. There is sometimes some quenching going on so we separate the
> lignin and
> > alexa as far as we can. We have to mount in oil for this work which is
> why I
> > am using the blue lens.
> >
> > Lloyd
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[log in to unmask]]
> On
> > Behalf Of Rosemary White
> > Sent: Tuesday, 11 January 2011 10:48 a.m.
> > To: [log in to unmask]
> > Subject: Re: Glycerol Objectives - experience with
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear Lloyd,
> >
> > Does the 63x glycerol objective provide substantial improvements over the
> > 63x water (with correction collar) for live tissues?  We have one of the
> > "blue" Leica 63x objectives, corrected for the blue-UV end of the
> spectrum
> > (though only have 405 laser), which is great except when you want to
> image
> > both red and blue emission....
> >
> > thanks,
> > Rosemary White
> >
> >
> > Dr Rosemary White
> > CSIRO Plant Industry
> > GPO Box 1600
> > Canberra, ACT 2601
> > Australia
> >
> > T 61 2 6246 5475
> > F 61 2 6246 5334
> > E [log in to unmask]
> >
> >
> >
> > On 11/01/11 6:21 AM, "Lloyd Donaldson" <
> [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
> >> *****
> >>
> >> Jeremy
> >>
> >> We have a Leica 63x glycerol objective with adjusting ring which gives
> very
> >> noticeable improvement over 63x oil which we also have. We also have a
> 20x
> >> multi immersion objective that can use glycerol. We have both oil and
> >> glycerol
> >> objectives, although we prefer glycerol as a mounting medium it is
> >> incompatible with some of our samples because of induced swelling so we
> have
> >> to mount in oil for those samples. We also have the oil lens because it
> is UV
> >> compatible - we have a 355 nm laser.
> >>
> >>
> >> Dr Lloyd Donaldson
> >>
> >> Senior Scientist, Project Leader - Microscopy/Wood Identification
> >> Scion - Next Generation Biomaterials
> >> Private Bag 3020, Rotorua
> >> New Zealand 3010
> >>
> >> Ph: 64 7 343 5581
> >>
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On
> >> Behalf Of Jeremy Adler
> >> Sent: Monday, 10 January 2011 9:39 p.m.
> >> To: [log in to unmask]
> >> Subject: Glycerol Objectives - experience with
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> We are considering buying a new confocal for examining fixed and
> >> potentially thick biological specimens.
> >> Given the need to have to an RI match between the specimen, mounting
> >> medium and immersion medium, I want to ask about the pros and cons of
> >> glycerol objectives.
> >>
> >> Oil objectives appear to have no obvious advantages, the higher RI
> >> will only apply if specimens are mounted in a medium with a RI close
> >> to that of oil - while most media seem to have lower RIs. Why would
> >> anyone choose an oil objective  and which mounting media work for
> >> thick specimens ?
> >>
> >> A possible disadvantage of glycerol objectives is that glycerol is
> >> hygroscopic which could change its RI. Are there any RI equivalent
> >> immersion media ?.
> >>
> >> So the pros and cons of oil or glycerol objectives.
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >> Jeremy Adler
> >> Genetics & Pathology
> >> Rudbeckslaboratoriet
> >> Daghammersköljdsväg 20
> >> 751 85 Uppsala
> >> Sweden
> >>
> >> 0046 (0)18 471 4607
> >>
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