[OpenSPIM] About the calibration of the lightsheet

[Jan Huisken]

Hi Edgar

> -----Weitergeleitet von Edgar Escobar Nieto/Fraunhofer IPT am 05.08.2013 11:54 -----
> An: openspim at openspim.org
> Von: Edgar Escobar Nieto/Fraunhofer IPT
> Datum: 31.07.2013 15:02
> Betreff: (Unbenannt)
> 
> Hi dear all,
> 
> I reached to the point where I need to calibrate the light-sheet. And I was wondering
> what is the optical density of the ND filter recommended to perform the calibration.

Since you are reflecting the laser right onto the camera you need to be sure that you can turn down the laser intensity far enough that you do not damage your camera. Therefore the ND depends on the lowest power you can set your laser to and the sensitivity of your camera. The ND filter can be introduced in the illumination or detection path. Some fluorescence emission filter may also do the job.

> 
> I would like to know also what is the Frequency (lp/mm) recommended for the Opal Glass Ronchi Ruling Slides.
> I think that high a frequency would be better, but there must be a limit.

The grid is used to (1) have some features on which you can focus and (2) to have some reflective parts the give you a profile of the beam. The distance does not matter very much. It is usually good if you have ca. 10 reflective lines across the entire FOV, which depends on your magnification, camera, etc. 
In addition it is nice if you also have an adjacent region on this piece of glass that is fully reflective where you can inspect the full beam profile (after focusing on it with the grid part). Here you can align for sheet thickness and uniformity.

> 
> Only to be sure, what I understood is that these Ruling Slides have already a reflective surface, so there is no
> need to glue a mirror to the ruling slides.

No. The whole piece needs to be fairly thin to fit between the lenses. So you would have to cut out a small piece anyway. Cutting is not so easy but the cut does not need to be precise.  Just smash it into pieces and pick the best piece ;-)

> 
> Thank you in advance for your comments.

Sure.

Best
Jan

Dr. Jan Huisken
MPI of Molecular Cell Biology and Genetics
Pfotenhauerstr. 108, 01307 Dresden, Germany

> 
> Kind regards,
> Edgar Escobar Nieto
> _________________________________________________________________________
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> 
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