Thanks for your response Kevin.

How does one deal with the imaginary term for the case of an oil
immersion lens with an aqueous specimen, i.e. when NA > n2, can the
sqrt(-1) just be thrown out?  The numbers seem to work out that way.

Esteban


2008/2/15 Kevin Braeckmans <[log in to unmask]>:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Esteban,
>
> A simple ray-tracing diagram in combination with Snell's law will readily
> give you the following formula:
>
> Dz = Dz_motor * sqrt((n2^2 - NA^2)/(n1^2 - NA^2))
>
> Where Dz is the actual axial displacement in the sample with refractive
> index n2, Dz_motor is the axial displacement as indicated by the microscope
> focus motor, n1 is the refractive index of the immersion medium and NA is
> the numerical aperture of the objective lens.
>
> Note that, as expected, for a perfectly matched system (n1=n2): Dz =
> Dz_motor. Also, for small NA (NA<<n1 & n2) this leads to the much used
> approximate correction n2/n1 as you have mentioned in your message. It is
> not valid for large NA though.
>
> I have used this formula with good results in my own studies. I seem to
> remember there has been a publication during the 90's which compares this
> formula with a more rigorous treatment starting from wave optics. I cannot
> seem to find the reference, though. Maybe someone else on this list can ...
>
> Hope this helps.
>
> Kevin
>
>
>
> Kevin Braeckmans, Ph.D.
> Lab. General Biochemistry and Physical Pharmacy
> Ghent University
> Harelbekestraat 72
> 9000 Ghent
> Belgium
> Tel: +32 (0)9 264.80.78
> Fax: +32 (0)9 264.81.89
>
> > -----Oorspronkelijk bericht-----
> > Van: Confocal Microscopy List [mailto:[log in to unmask]]
> > Namens G. Esteban Fernandez
> > Verzonden: vrijdag 15 februari 2008 0:11
> > Aan: [log in to unmask]
> > Onderwerp: How to correct z distance in RI mismatch?
>
> >
> > Hi everyone,
> >
> > A user needs to measure the volume of fluid inclusions (mostly water)
> > embedded in a clear mineral of RI ~2.4.  We did this by collecting
> > z-stacks of reflected light on our LSM 510 confocal and rendering a 3D
> > model, which turned out pretty reasonable.  We used an oil-immersion
> > 63x Plan-Apo and oil on either side of the coverslip.
> >
> > I know that the z distances are not accurate because of the grossly
> > different RIs but I'm not sure how to correct them.  Can I just
> > multiply the z step size by an n2/n1 factor to get the real Δz (with
> > different factors for oil/mineral and mineral/water)?  In the
> > literature I've seen less simplistic correction models that seem more
> > accurate but no practical ways to implement them that I could decipher
> > were presented.  Any help would be much appreciated.
> >
> > Thanks,
> > Esteban
> >
> > --
> > G. Esteban Fernandez, Ph.D.
> > Associate Director
> > Molecular Cytology Research Core Facility
> > University of Missouri
> > 1201 E. Rollins St.
> > Columbia, MO 65211
> >
> > 573-882-4895
> > 573-884-9676 fax
> >
> > http://www.biotech.missouri.edu/mcc/
>



-- 
G. Esteban Fernandez, Ph.D.
Associate Director
Molecular Cytology Research Core Facility
University of Missouri
1201 E. Rollins St.
Columbia, MO 65211

573-882-4895
573-884-9676 fax

http://www.biotech.missouri.edu/mcc/