[OpenSPIM] changing "objectives"

[Feinstein, Timothy N]

Thank you Fredrik and Peter!  Right now I am looking at either an Olympus U-AEC 1x/2x magnification changer or else the DE20BXC 2x magnifying camera adapter from SPOT Imaging.

I checked the Nyquist sampling frequency for deconvolution at www.svi.nl/NyquistCalculator, and it looks like magnification is a good idea.  The optimal sampling frequency for widefield with a 0.5 NA water lens is 0.26 um per px.  I am imaging at 0.33 um/px with a 1x camera adapter.  Therefore I have a little resolution to gain, though a 2x adapter at 0.16 um/px will be grossly oversampling.  It might be a good idea to use a 1.5x adapter instead, as 0.22 um/px is pretty close to the theoretical limit.  As I understand it SPIM improves the axial resolution of images but does not change lateral resolution relative to widefield.  Is this correct?

All the best,


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology


From: Fredrik Ek <fredrik.ek at med.lu.se<mailto:fredrik.ek at med.lu.se>>
Date: Wednesday, September 23, 2015 at 4:34 AM
To: Timothy Feinstein <tnf8 at pitt.edu<mailto:tnf8 at pitt.edu>>, openspim <openspim at openspim.org<mailto:openspim at openspim.org>>
Subject: SV: changing "objectives"

Hi,

We are using a Olympus magnifier 1x,2x and 4x with c-mount together with a Flash4.0 v2 and it works very well with the SPIM setup (20x 0.5 objective).

Best,

Fredrik

Fredrik Ek, PhD,
Researcher, Chemical Biology & Therapeutics
Department of Experimental Medical Science
Lund University, BMC D10
S-221 84 Lund, SWEDEN
------------------------
Fredrik.Ek at med.lu.se<mailto:Fredrik.Ek at med.lu.se>
Phone: +46-727 17 22 05



Från: openspim-bounces at openspim.org<mailto:openspim-bounces at openspim.org> [mailto:openspim-bounces at openspim.org] För Feinstein, Timothy N
Skickat: den 22 september 2015 17:35
Till: openspim
Ämne: [OpenSPIM] changing "objectives"

Hi folks,

Thanks to the indispensable help of many users here, our SPIM is up and working.  You are all the best.

Since a SPIM makes it quite hard to change objectives, I bought 1x and a 0.5x C-mount camera adapters to get the equivalent of a 20x and a 10x objective.  It seems to work quite well.  Now I wonder whether anyone has tried a magnifying tube lens like the Olympus U-CA or U-ECA to get a higher power image.  It all depends on whether a 'vanilla' openSPIM design is already imaging near the Nyquist limit.  We get 0.33 um/px using the Olympus 20x/0.5 and a Hamamatsu Flash4.0 v2, and even with deconvolution it may be pointless to magnify the image any further.  Any thoughts appreciated.

Alternatively, has anyone tried keeping a second sample chamber with a higher power objective to swap in as needed?  It seems like a pain but less cumbersome than building a second instrument.

Thanks,


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology


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