Dear all,
For those interested, a rigorous treatment
of how to correct for z-measurements in case of a mismatch in refractive
indices can be found in the Handbook by Pawley, 3rd edition, chapter 20 by Egner
and Hell. (I believe this chapter is also in the 2nd edition of the Handbook).
With regard to Esteban’s situation (see
thread ‘How to correct z distance in RI mismatch’), yesterday I
gave the suggestion to use the geometrical formula for an effective NA of
approx. 1 (i.e. half the illumination light cone). Comparing this with the
values reported in table 2 of the chapter by Egner and Esteban, a reasonable
correspondence is found for a focus depth of 15 to 20 micron (less than a
micron difference). After that the geometrical formula (with adjusted effective
NA) does not give an adequate prediction anymore. In any case, in this chapter
you can find everything needed for accurate calculations.
Best regards,
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
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