Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Ian,

I've been using pol-l-lysine with water like medium with some success.

Just do the following:

1- Spread a bit o pol-l-lysine in you coverslip and let it dry
2- Add your beads and let them dry (I use PS-Speck from M. Probes..this
is probably familiar to you!)
3- Add some water and seal it

If you look at the ones near the coverslip, you will have several ones
fixed. Nevertheless if you don't have an objective with correction
collar, be sure that your sample is as close as it can be from the
coverslip, but I usually use a glycerol 1.3 with correction for
spherical aberration...it makes a difference!

Notwithstanding, if some one has that transparent gel similar to 1.3 RI,
it would be welcome.

All the best,
______________________________
Nuno Moreno
Phone: +351 214464606
Fax  : +351 214407970
www.igc.gulbenkian.pt
Cell Imaging Unit
Instituto Gulbenkian de Ciência
Portugal
_______________________________
# No anti-virus - Using Linux #


Ian Clements wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello All
> I've been toying with these issues a bit of late.
>
> Embedding the beads in optical cements that can have an RIs near 1.51
> will certainly help correct spherical problems for oil immersion lenses
> but it will make matters a lot worse for water immersion lenses.
>
> You can get optical cements that have RIs ~1.59 similar to the material
> of the beads.  However this results in axial distortion due to the
> surrounding higher RI even with oil lenses.  It makes imaging really
> difficult with water lenses when you embed the beads in something like
> this.
>
> Ideally you'd probably want to put the beads in something with an RI
> similar to the RI of your standard sample (whatever that is) and image
> with the appropriate immersion lens.  The only problem is that lower RI
> (water-like) immersion media aren't particularly viscous so the beads
> move around.
>
> I'd be interested in hearing from anyone with suggestions on a low RI
> gel which stays optically clear but is firm enough to stop small beads
> moving.  I've tried various PVA mixes but they tend to shrink or cure to
> an RI about 1.4-1.45 range.  Matrigel gets awfully scattering when it
> sets up.
>
> I haven't tried QDots but in theory this would seem to be an ideal
> application but knowing whether you have clusters or individual dots
> would be a challenge.
>
> Ian Clements
> Imaging Technology
> Molecular Probes Inc.
>
>
>
> Lutz Schaefer wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi Peter,
>>
>> >
>> >       Question: is it possible (and has it been tried) to calculate the
>> > PSF from a "supra-resolution" target?  If you know the precise
>> dimension
>> > of the "ideal sphere", then dividing the Fourier transform of the
>> acquired
>> > image by the transform of the "ideal sphere" (i.e. deconvolving) should
>> > yield the transform of the PSF.  Then the inverse will be the PSF image
>> > itself, which can then be used for deconvolution using 3rd party
>> software
>> > packages.
>> >
>>
>> Yes, it had been done. The (relatively old) Zeiss KS400 software uses
>> relatively large beads (~1 micron or larger depending on objective
>> lens) to
>> calculate a measured PSF. An iterative maximum likelihood algorithm is
>> used
>> for that. The only disadvantage of the method is that larger beads may
>> not
>> contain enough high frequencies to be restored, leaving you with a
>> slightly
>> too large restored PSF.
>>
>> Cheers
>> Lutz
>>
>> ____________________________________
>> Lutz Schaefer
>> Advanced Imaging Methodology Consultation
>> 16-715 Doon Village Rd.
>> Kitchener, Ontario, N2P 2A2
>> Website: http://home.golden.net/~lschafer/
>> ____________________________________
>>
>>
>