My inclination is to use subresolution size beads 
(< 200 nm) of different colors. As Jim P. pointed 
out the z-res. is easily twice as bad as the 
theoretical values. Further, objectives that are 
nominally of the same high NA (1.4 and above) can 
yield quite different z-resolutions even factors 
of 2. In addition, the z-axis PSF can be very 
asymmetric, which becomes obvious when using 
sub-res. beads and may be less obvious using very 
large beads. Making sure you use the correct 
coverslip thickness and be aware that the z-res. 
is strongly dependent on the mediums RI. 
Depending on the amount of SA of you particular 
objective it will also get worse as you focus 
deeper into the sample, particularly as you get 
more than 10 um from the coverslip. Some 
deconvolution software can at least partially 
correct for the z-axis spread caused by SA if 
good calibration data is available. It just 
depends on how fussy you want to be.

Mario


>For specific "spacers" our 15 micron fluorescent 
>beads were designed for CLSM based Z (depth) 
>calibration.  We also have 6 micron ones if you 
>prefer. 
>
>These also show the effect of color and RI 
>mismatch on Z depth accuracy.  Simple protocol 
>included. 
>
>FocalCheck� microspheres, 15 µm, fluorescent 
>green ring stainŽdark red throughout
>Cat. No. F-7238
>Or
>FocalCheck� microspheres, 15 µm, fluorescent 
>dark-red ring stainŽgreen throughout
>Cat. No. F-7239
>
>Kind Regards,
>
>Mike Ignatius
>Molecular Probes/Life Technologies
>
>-----Original Message-----
>From: Confocal Microscopy List 
>[mailto:[log in to unmask]] On 
>Behalf Of Patrick Van Oostveldt
>Sent: Tuesday, May 11, 2010 10:24 AM
>To: [log in to unmask]
>Subject: Re: Optical Section Thickness-Leica SP5
>
>If you really want to determine the thickness of a section you should 
>take account of the fact that the refractive index of the medium can 
>change the estimated thickness. Cfr. Paper of Visser, Oud and 
>Brakenhoff in the early days of CSLM. Just google to "visser 
>refractive index" and you will find the pdf print.
>
>My experience is that fixation can also affect effective thickness of 
>the section due to shrinking or swelling.
>
>A test is possible by using a fluorescent dye mounted under a 
>coverglass with specific spacers and measure the number of steps 
>necessary to image the whole object.
>
>A simple test is the use of some fluorescentive index and axial 
>...Quoting "Linda Barthel" <[log in to unmask]>: textile fiber of PE 
>which is round and where you can directly check if XY diameter 
>corresponds to XZ distances.
>
>Bye
>
>Patrick Van Oostveldt
>
>
>
>
>>  Seems to be a simple enough question-but having trouble getting a simple
>>  answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.
>>  2.0.0 software.  How do we determine the optical sections thickness of our
>>  images?  This must be a simple oversight on our part.
>>  Thanks,
>>  Linda Barthel
>>  Gregg Sobocinski
>>  University of Michigan
>>  Dept of MCDB
>>  Ann Arbor
>>
>
>
>
>--
>Dep. Moleculaire Biotechnologie
>Coupure links 653
>B 9000 GENT
>
>tel 09 264 5969
>fax 09 264 6219


-- 
________________________________________________________________________________
Mario M. Moronne, Ph.D.
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