[OpenSPIM] Follow-up: [Open-SPIM] Is anyone using digital scanning to reduce banding?

Tue Jul 5 13:35:19 CDT 2016

Hi Tim,

great! I like your setup.

One remark to the last figure caption in your document: You need to adjust the amplitude to get the maximum efficiency of the mSPIM. You start with a small amplitude and increase it until the light sheet intensity (and fluorescence) drops. Now you have found the amplitude where you pivot the sheet as much as the NA of your illumination lens allows and you are getting the best performance. Going any further, the beam will be cut off and you loose laser power.

Best
Jan

> On Jul 5, 2016, at 3:18 PM, Feinstein, Timothy N <tnf8 at PITT.EDU> wrote:
> 
> Hi folks, 
> 
> Many thanks for your help.  After much testing I have concluded that anti-striping along the lines of Huisken & Stainier (2007) is extremely important for imaging large structures like a zebrafish embryo.  This adds significant complexity and cost (~$2,000) to the basic openSPIM design but in my opinion it makes the instrument dramatically more useful for segmentation and quantiative imaging.  Importantly the fact that I could make it suggests that it is within the engineering abilities of most people who can build an openSPIM.  We have not started using it yet as our lasers are also out for an upgrade; I will update the list when I have had a chance to test it.  
> 
> I had to fill in some details from the 2007 paper on my own, so it is likely that more elegant/less kludgy designs exist.  Still, this guide I wrote for our group may be useful for those thinking of adding anti-striping in the future.  
> 
> https://www.dropbox.com/s/6r1z13pggat7v76/Anti-striping%20mirror.docx?dl=0 <https://www.dropbox.com/s/6r1z13pggat7v76/Anti-striping%20mirror.docx?dl=0>
> 
> All the best, 
> 
> 
> Tim
> 
> Timothy Feinstein, Ph.D. 
> Research Scientist
> University of Pittsburgh Department of Developmental Biology
> 
> 
> From: <openspim-bounces at openspim.org <mailto:openspim-bounces at openspim.org>> on behalf of Timothy Feinstein <tnf8 at pitt.edu <mailto:tnf8 at pitt.edu>>
> Date: Tuesday, May 3, 2016 at 9:27 AM
> To: Jan Huisken <huisken at mpi-cbg.de <mailto:huisken at mpi-cbg.de>>
> Cc: "openspim at openspim.org <mailto:openspim at openspim.org>" <openspim at openspim.org <mailto:openspim at openspim.org>>
> Subject: Re: [OpenSPIM] Is anyone using digital scanning to reduce banding?
> 
> Correction, I meant 0.05% tricaine. 
>  
> Best,
> 
> 
> T
>  
> From: Jan Huisken [mailto:huisken at mpi-cbg.de <mailto:huisken at mpi-cbg.de>] 
> Sent: Friday, April 29, 2016 3:01 AM
> To: Feinstein, Timothy N
> Cc: openspim at openspim.org <mailto:openspim at openspim.org>
> Subject: Re: [OpenSPIM] Is anyone using digital scanning to reduce banding?
>  
> Dear Tim,
>  
> I think you are mixing up two issue.
>  
> 1. Getting rid of stripes.
>  
> Yes, you can do a little bit in post-processing but the stripes can be quite complex and are not necessarily straight. Oftentimes you see a lot of lensing and I do not think you can easily remove it with image processing.
>  
> a) Double-sided illumination helps. Depending on the sample you may want to do simultaneous or sequential double-sided illumination. There are various ways of merging the two images.
> b) A resonant mSPIM mirror is cheap and easy to integrate. You will get rid of most stripes, but again this depends on the sample. The details for a) and b) are in our Opt. Lett. paper (Huisken & Stainier, 2007). Basically, you are pivoting the light sheet around the center of your field of view, e.g. with 1kHz. The stripes are “washed out” during the exposure time of your camera. We use this on most of our systems and it helps a lot to reduce stripes.
> c) Multi-view fusion can also help to some extent.
>  
> 2. DSLM vs. SPIM
>  
> Yes, DSLM should also help reducing stripes but I have no experience with that. Basically, your light sheet is not coherent anymore and the sheet does not “interfere with itself”. However, the setup is more expensive and requires you to power and control another element: the galvo scanner to sweep the beam up and down. You need a proper scan mirror and not just the cheap mSPIM mirror. The additional benefit is that the resulting light sheet is more uniform than the cropped Gaussian light sheet. However, you need to illuminate each line with higher intensity which may result in saturation and worse dynamic range. Obviously you need to wait for the scan and need to synchronize your readout. Depending whether you have a global shutter or rolling shutter camera you need to take some precaution.
>  
> Best
> Jan
>  
>  
>  
> —
> Jan Huisken
> Head of Max Planck Research Group
>  
> Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)
>  
> On Apr 27, 2016, at 4:13 PM, Feinstein, Timothy N <tnf8 at PITT.EDU <mailto:tnf8 at PITT.EDU>> wrote:
>  
> Hello all, 
> 
> I found the Selzer group's solution (Keller et al. 2013) quite interesting
> and would love to know if anyone had a positive experience doing that.
> The banding from my basic openSPIM is definitely a problem for
> segmentation and analysis.
> 
> As far as I can tell fixing the banding will mostly involve buying a scan
> mirror.  I don't have a quote yet for the Cambridge VM500 scanning mirror
> that the Selzer group used but its ThorLabs equivalent (#GVS101?) costs
> about $1k.  That seems reasonable and not that hard for regular folks to
> implement.  
> 
> In related news I just noticed that the TeraStitcher plugin for Vaa3D now
> has an option to fight banding in light sheet data.  I have not tested it
> yet but potentially that feature could be useful.  I will just post my
> usual warning for folks trying TeraStitcher for the first time: it
> stitches really well but it could be a hair more user-friendly.
> 
> Best, 
> 
> 
> Tim
> 
> Timothy Feinstein, Ph.D.
> Research Scientist
> University of Pittsburgh Department of Developmental Biology
> 
> 
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