axial resolution of surface features (was Re: reflection mode in material science)

[Stanislav Vitha]

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

Dear conofcalists,
how would I estimate or calculate what is the smallest z-difference of the
sample surface that can be visualized and/or accurately measured?
The sample would either have uniform reflectivity, or be coated with a
very thin fluorescent layer, or be made of clear resin containing a
fluorescent dye. These samples are resin replicas of human teeth from
archaelogical excavations. The researcher is interested in diet-related
microwear 3D pattern on the surface of these teeth.

It is my understanding that for topographical projection we need to find
the position of the signal peak intensity along Z (for each XY pixel
position), which we can do with much better accuracy than is the FWHM of
the peak. - so height steps on the surface that are smaller then the axial
resolution of the objective should be visible and measurable.

But how small is too small?
Is there a simple way to calculate or estimate this?

I would be using a 40x/0.6 long working distance dry objective, and 488 or
543 nm laser. The z-motor on our Olympus scope has minimum step of 10 nm
(but does not have an encoder) and the pinhole would be closed to about
0.6 Airy.

Thanks in advance!

Stan Vitha



Dr. Stanislav Vitha
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257

tel: [phone] (main desk)
tel: [phone] (direct link)
fax: [phone]


On Tue, 6 Nov 2007 15:41:51 -0500, MODEL, MICHAEL <[hidden email]> wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>It's typically done using interferometry. Reflection mode in confocal can
be tricky because the signal depends on the tilt of the surface (see
P.C.Cheng's articles in Confocal Handbook)
>
>Mike
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Juan Luis Ribas

>Sent: Tuesday, November 06, 2007 1:19 PM
>To: [hidden email]
>Subject: reflection mode in material science
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear all,
>Several groups in our University are moving now to the confocal to
>perform z profiles of materials. I would like to get more information
>about the way of doing a correct adquisition in reflection mode to
>ensure a realistic depth map. I've found that a z profile in the same
>region with different air objectives give very different measurements,
>so I'm not sure which is the real value. And is very difficult to obtain
>some useful information about this technique.
>I'll be very grateful if someone could recomend me  some link, book or
>paper about reflection mode and z profile caracterization in materials.
>All the best
>
>Juan Luis
>
>--
>Juan Luis Ribas, PhD
>Servicio de Microscopía
>Centro de Investigación, Tecnología e Innovación
>Universidad de Sevilla
>Av. Reina Mercedes 4b
>41012 Sevilla
>
>Tfno: 954559983
>========================================================================