[OpenSPIM] Fluorescent solution for light sheet alignment

[David Potter]

Emmanuel,

I use both a fluorophore in the bath and beads in agarose. I find the
approximate focal point and in plane tilt of the beam/sheet with the bath.
For sheet tilt out of the detection focal plane, and setting cylindrical
lens rotation angle, I use free floating beads in the bath or beads held in
an agarose plug. Beads can be quite a problem to get out of the bath,
though this may not impact some imaging sessions, so I use an agarose plug.
I accept that lensing occurs when shooting through the agarose cylinder so
I shoot through the centre as best I can.

I tend to use Alexa dyes in the bath as I think the dyes are less likely to
get taken up into cells. TetraSpeck beads are great for fine aligning
second or third lasers to the alignment laser.

Cheers,

- David

David Potter
Lattice Light Sheet Microscopy Manager
Monash Micro Imaging - Advanced Optical Microscopy
Monash University Clayton, VIC 3800, Australia



On 23 August 2017 at 03:00, <openspim-request at openspim.org> wrote:

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>    1. Fluorescent solution for light sheet alignment (Emmanuel Reynaud)
>    2. Re: Fluorescent solution for light sheet alignment
>       (Nikita Vladimirov)
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> Message: 1
> Date: Tue, 22 Aug 2017 11:28:53 +0100
> From: Emmanuel Reynaud <emmanuel.reynaud at ucd.ie>
> To: openspim at openspim.org
> Subject: [OpenSPIM] Fluorescent solution for light sheet alignment
> Message-ID:
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>
> Hi,
>
> Did anybody ever used a chamber filled with fluorescent solution (e.g.
> fluorescein) to align the light sheet? Got a request from another group who
> planned to use such method to align detection vs illumination objectives?
>
> Any ideas welcome,
>
> Emmanuel
>
> *Dr Emmanuel G. Reynaud*
> Principal Investigator, Integrative Biology
> Conway Institute of Biomolecular and Biomedical Science
> UCD Centre for Biomedical Engineering (
> http://www.ucd.ie/biomedicalengineering/)
>
> e-mail: emmanuel.reynaud at ucd.ie <matthias.wilm at ucd.ie>
> Website:http://cellbiology.ucd.ie/
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> Date: Tue, 22 Aug 2017 14:23:29 +0200
> From: Nikita Vladimirov <nvladimus at gmail.com>
> To: Emmanuel Reynaud <emmanuel.reynaud at ucd.ie>
> Cc: openspim at openspim.org
> Subject: Re: [OpenSPIM] Fluorescent solution for light sheet alignment
> Message-ID:
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> QZOsw at mail.gmail.com>
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>
> Yes, I did, it worked nicely with fluorescein, for finding the beam waist
> in the field of view, and checking the XY tilt.
>
> Nikita Vladimirov
>
> On Tue, Aug 22, 2017 at 12:28 PM, Emmanuel Reynaud <
> emmanuel.reynaud at ucd.ie>
> wrote:
>
> > Hi,
> >
> > Did anybody ever used a chamber filled with fluorescent solution (e.g.
> > fluorescein) to align the light sheet? Got a request from another group
> who
> > planned to use such method to align detection vs illumination objectives?
> >
> > Any ideas welcome,
> >
> > Emmanuel
> >
> > *Dr Emmanuel G. Reynaud*
> > Principal Investigator, Integrative Biology
> > Conway Institute of Biomolecular and Biomedical Science
> > UCD Centre for Biomedical Engineering (http://www.ucd.ie/
> > biomedicalengineering/)
> >
> > e-mail: emmanuel.reynaud at ucd.ie <matthias.wilm at ucd.ie>
> > Website:http://cellbiology.ucd.ie/
> >
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