Amount of shear needed for DIC imaging (and provided by Zeiss objectives)

Posted by Shalin Mehta on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Amount-of-shear-needed-for-DIC-imaging-and-provided-by-Zeiss-objectives-tp590130.html

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

What does the shear required by DIC depend on? Magnification or NA? To me it appears that only magnification should matter as DIC measures the phase gradient in direction of shear between two points in space. Any good reference that quantitatively relates magnification/NA to the shear required?

I wish to do time-lapse of live cells in DIC. I have a 40x 0.75 NA and 40x 1.3 NA oil objectives from Zeiss. For the 0.75 NA objective we do not have DIC prism but for 1.3NA we have (as per Zeiss nomencleture DIC III). During time-lapse I see lot of focal drift with oil objective but with air objective focus is relatively stable. Is it alright to use 1.3 NA objective's prism with 0.75 NA objective?

I have taken some images with such configuration and I think the match is not perfect - because when changing the bias retardation by moving prism I do not see transition from positive gradient to zero contrast to negative gradient.

Thanks for all inputs

Best regards
Shalin

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My co-ordinates:
Shalin Mehta, Graduate student
Graduate Programme in Bioengineering, NUS, Singapore
Email: shalin {dot} mehta {at} gmail {dot} com
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