[OpenSPIM] Follow-up: [Open-SPIM] Is anyone using digital scanning to reduce banding?
Frédéric BRAU
brau at ipmc.cnrs.fr
Tue Jul 5 16:49:30 CDT 2016
Dear Jan and Tim,
Sorry to come into your discussions but I noticed your comment Jan
concerning this point : 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.
In our lab we built a home-made ultramicroscope (and reproduce an
open-spim :
http://unice.fr/plateformes/mica/ressources/developpements-1/developpements)
and I was wondering about the best method to fuse images taken from the
left+those taken from the right from our ultramicroscope (without any
rotation). At the moment we only do averaging which maybe not the best
solution, I suppose. Do you know a paper mentionning the different ways
to do image fusion, or any advice about that (I also knew about a method
consisting to cut the image in 3 part, but I don't really remember all
the steps)?
Thanks in advance,
Best regards,
Frédéric
Le 05/07/2016 à 20:35, Jan Huisken a écrit :
> 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
>> <mailto: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
>>
>> 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]
>> *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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--
Frédéric Brau <mailto:%20brau at ipmc.cnrs.fr>
Institut de Pharmacologie Moléculaire et Cellulaire - UMR7275 CNRS/UNS
<http://www.ipmc.cnrs.fr>
660, Route des Lucioles
06560 VALBONNE
Tel: 33 (0)4 93 95 77 83 Bureau / 77 87 / 77 88 / 77 89 Confocaux
Fax: 33 (0)4 93 95 77 08
Plateforme MICA: Microscopie Imagerie Côte d'Azur <http://mica.unice.fr>
Réseau Technologique de Microscopie de Fluorescence Multidimensionnelle
<http://rtmfm.cnrs.fr>
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