[OpenSPIM] Alignment / Calibration Slides

[Krieger, Jan]

Dear Neil!

We use a slightly simpler alignment scheme: I designed a mount for a simple 5x5x1mm³ surface mirror that fits in our SPIM (see Fig. 6.12d on p. 94 in http://www.jkrieger.de/dissertation_final.pdf). If you hvae just a little bit of dirt on the mirror (e.g. by touching it and then cleaning it with a fine cloth, you can easily focus such a bit of dirt and then know where the sharp line on your mirror is. If you're interested, I can send you the design for the mirror mount, which is pretty simple to make for any mechanics workshop.

Also we have a simple plugin for a beadscan evaluation (PSF determination from z-stack) and to characterize a lightsheet in our SPIM-FCS/FCCS software:
http://www.dkfz.de/Macromol/quickfit/
or as a Matlab script:
https://github.com/jkriege2/B040_BeadScanEvaluation

Detailed instructions for this eval scheme will appear in a few weeks (I hope) in Nature Protocols in a (just-accepted) paper on imaging FCS/FCCS on a SPIM or TIRF. If you're interested, I can send you a preprint-version privately.

Best,
JAN




Dr. Jan W. Krieger
German Cancer Research Center (dkfz)
Department B040 - Biophysics of Macromolecules (Prof. J. Langowski)
Im Neuenheimer Feld 580
69120 Heidelberg

fon: +49 / 6221 / 42-3395
fax: +49 / 6221 / 42-3391
e-mail: j.krieger at dkfz.de
www: http://www.dkfz.de/Macromol/

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Von: openspim-bounces at openspim.org [openspim-bounces at openspim.org] im Auftrag von Michael Weber [weber at mpi-cbg.de]
Gesendet: Freitag, 2. Oktober 2015 16:22
An: openspim at openspim.org
Betreff: Re: [OpenSPIM] Alignment / Calibration Slides

Dear Neil,

hope you enjoyed reading the paper :). We use a home-made mirror/grid for aligning our setups. The "mirror/grid" part is cut from a stage micrometer:

Edmund Optics "Multi-Grid Standard Stage Micrometer"
http://www.edmundoptics.com/test-targets/image-analysis-test-targets/multi-grid-standard-stage-micrometer/2700/

Cutting the glass is tricky - maybe you have access to a workshop. You want to have both mirror and grid on top of each other on the same piece of glass, such that you just have to move the sample up and down to switch between them. One end of this piece of glass is then mounted in the tip of a 1 ml Braun Omnifix-F syringe using glue. That's the second difficult step. Ideally, the glass surface ends up straight in the rotation axis of your setup. Again, if you have access to a workshop, they might be able to help you with that. The syringe is then mounted in the sample holder. Make sure that the syringe has the right length for positioning the glass in the focal plane. One can also think of a machined or 3D-printed adapter (or entire holder) for the piece of glass.

Another tool that is really useful for alignment are fluorescent beads / microspheres in 1.5% agarose. This is especially good for the fine tuning of the light sheet position, PSF measurements and so on. Mirror/grid are more suitable for a coarse alignment.

Best,
Michael


On Oct 1, 2015, at 6:35 PM, "Anthony, Neil" <nantho2 at EMORY.EDU<mailto:nantho2 at EMORY.EDU>> wrote:

Hi all,

I was just reading the paper suggested by Jan in Aug (Weber, Michael, Michaela Mickoleit, and Jan Huisken. 2014. “Light Sheet Microscopy.” Methods in Cell Biology 123: 193–215. doi:10.1016/B978-0-12-420138-5.00011-2.), thinking ahead to the calibration/alignment part of the setup.

Does anybody have part numbers and/or websites for the required pieces?  Also, I’d be interested in how you’ve positioned and held them at the objective focus, and ensured their correct orientation.

Thanks
Neil

Neil Anthony, PhD  | Research Imaging Specialist
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Michael Weber
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Max Planck Institute of Molecular Cell Biology and Genetics
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