Killing cells in a zebra fish

[Jeremy Adler-4]

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We have a user who wants to kill specific GFP labelled single cells in a zebra fish and follow the developmental consequences.
Multiphoton or confocal used to zap a selected area was the suggestion but

Is bleaching the GFP sufficient to damage the cell.
How can we determine if the cell is dead/damaged - trypan blue in fish.
The extent to which surrounding cells are also damaged.
Are there any photoactivatable toxins ?

Ideas, references, suggestions would be greatly appreciated.


Jeremy Adler
===============================================
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                   Light & EM microscopy / FlowCytometry & Cell Sorting / Image Analysis ===============================================
Jeremy Adler   PhD - Senior research engineer Light, Confocal Microscopy, Image Analysis
E-mail: [hidden email]
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[Rowlands, Christopher J]

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Photodynamic therapy does exactly this - I've used verteporfin in the past (http://dx.doi.org/10.1088/1612-2011/11/11/115605), but there's plenty of alternative PDT agents. Regarding the assessment of the cell, sacrifice the fish and LIVE/DEAD stain? Or if you need it still alive, bleach the GFP and see whether the GFP intensity recovers over time (suggesting that protein synthesis is still occurring).

Chris

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jeremy Adler
Sent: 15 August 2019 12:41
To: [hidden email]
Subject: Killing cells in a zebra fish

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We have a user who wants to kill specific GFP labelled single cells in a zebra fish and follow the developmental consequences.
Multiphoton or confocal used to zap a selected area was the suggestion but

Is bleaching the GFP sufficient to damage the cell.
How can we determine if the cell is dead/damaged - trypan blue in fish.
The extent to which surrounding cells are also damaged.
Are there any photoactivatable toxins ?

Ideas, references, suggestions would be greatly appreciated.


Jeremy Adler
===============================================
                    B i o V i s   P l a t f o r m of  Uppsala University
                   Light & EM microscopy / FlowCytometry & Cell Sorting / Image Analysis ===============================================
Jeremy Adler   PhD - Senior research engineer Light, Confocal Microscopy, Image Analysis
E-mail: [hidden email]
070-1679349

Dag Hammarskjölds v 20
751 85 UPPSALA, SWEDEN
http://biovis.uu.se/
===============================================
















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Dear Jeremy,
you might consider using "Killer-GFPs" like Killer-Red or Supernova. They
are used for CALI, chromophore assisted laser inactivation. If the
cytoplasm is full of it, then the iiradiated cell should die. You might be
able to use really low laser powers. I don´t know about fish, but in
culture cells Calcein is a good life/dead stain.
Best
Elisa

On Thu, Aug 15, 2019 at 1:42 PM Jeremy Adler <[hidden email]> wrote:

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>
> We have a user who wants to kill specific GFP labelled single cells in a
> zebra fish and follow the developmental consequences.
> Multiphoton or confocal used to zap a selected area was the suggestion but
>
> Is bleaching the GFP sufficient to damage the cell.
> How can we determine if the cell is dead/damaged - trypan blue in fish.
> The extent to which surrounding cells are also damaged.
> Are there any photoactivatable toxins ?
>
> Ideas, references, suggestions would be greatly appreciated.
>
>
> Jeremy Adler
> ===============================================
>                     B i o V i s   P l a t f o r m of  Uppsala University
>                    Light & EM microscopy / FlowCytometry & Cell Sorting /
> Image Analysis ===============================================
> Jeremy Adler   PhD - Senior research engineer Light, Confocal Microscopy,
> Image Analysis
> E-mail: [hidden email]
> 070-1679349
>
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> 751 85 UPPSALA, SWEDEN
> http://biovis.uu.se/
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[Tim Feinstein]

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Hi Jeremy,

I'd recommend using a mitochondrial or nuclear-targeted KillerRed for that purpose.  You'd need a pretty modest 555nm light flux to zap targeted cells into apoptosis.  There are probably newer and better genetically targeted photosensitizing agents, but that's the one that I've used.  

Best,


T

Timothy Feinstein, Ph.D.
Research Scientist
Department of Developmental Biology
University of Pittsburgh

 



On 8/15/19, 7:42 AM, "Confocal Microscopy List on behalf of Jeremy Adler" <[hidden email] on behalf of [hidden email]> wrote:

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    We have a user who wants to kill specific GFP labelled single cells in a zebra fish and follow the developmental consequences.
    Multiphoton or confocal used to zap a selected area was the suggestion but
   
    Is bleaching the GFP sufficient to damage the cell.
    How can we determine if the cell is dead/damaged - trypan blue in fish.
    The extent to which surrounding cells are also damaged.
    Are there any photoactivatable toxins ?
   
    Ideas, references, suggestions would be greatly appreciated.
   
   
    Jeremy Adler
    ===============================================
                        B i o V i s   P l a t f o r m of  Uppsala University
                       Light & EM microscopy / FlowCytometry & Cell Sorting / Image Analysis ===============================================
    Jeremy Adler   PhD - Senior research engineer Light, Confocal Microscopy, Image Analysis
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[Drummond, Iain A.]

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Yes we have photosensitized cells with GFP. We used a 405 laser line to ablate pronephric kidney tubules. You can use propidium iodide to track the cell death. Here's one of our papers on this:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092191/


--
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Associate Professor, Nephrology Division
Massachusetts General Hospital
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On 8/15/19, 7:42 AM, "Confocal Microscopy List on behalf of Jeremy Adler" <[hidden email] on behalf of [hidden email]> wrote:

            External Email - Use Caution        
   
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    *****
   
   
    We have a user who wants to kill specific GFP labelled single cells in a zebra fish and follow the developmental consequences.
    Multiphoton or confocal used to zap a selected area was the suggestion but
   
    Is bleaching the GFP sufficient to damage the cell.
    How can we determine if the cell is dead/damaged - trypan blue in fish.
    The extent to which surrounding cells are also damaged.
    Are there any photoactivatable toxins ?
   
    Ideas, references, suggestions would be greatly appreciated.
   
   
    Jeremy Adler
    ===============================================
                        B i o V i s   P l a t f o r m of  Uppsala University
                       Light & EM microscopy / FlowCytometry & Cell Sorting / Image Analysis ===============================================
    Jeremy Adler   PhD - Senior research engineer Light, Confocal Microscopy, Image Analysis
    E-mail: [hidden email]
    070-1679349
   
    Dag Hammarskjölds v 20
    751 85 UPPSALA, SWEDEN
    http://biovis.uu.se/
    ===============================================
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
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[Henrik Persson]

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Hi Jeremy,

I have not done this but in this paper I read a while back (Liu et al.,
Immunity 2016
<https://www.sciencedirect.com/science/article/pii/S1074761316300978>),
they used 2-photon microscopy (800 nm) to kill single endothelial cells in
zebra fish brain to study blood vessel repair using intravital microscopy.

From their methods:
*"Laser Microsurgical Cell Ablation, *
Photoconversion, In Vivo Time- Lapse, and FRET Imaging For laser
microsurgical cell ablation, the target endothelial cell or macrophage was
focused in a single confocal plane and irradiated for 3 s by a multi-photon
laser at 800 nm (Chameleon Vision2 Laser System, Coherent)."

Hope this helps

Best wishes
/Henrik

______________________
Henrik Persson, Ph.D.
Translational Biology & Engineering Program
Mechanical and Industrial Engineering
University of Toronto, Canada
LinkedIn <http://www.linkedin.com/in/henrikcpersson>




On Thu, 15 Aug 2019 at 11:07, Feinstein, Timothy N <[hidden email]> wrote:

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> *****
>
> Hi Jeremy,
>
> I'd recommend using a mitochondrial or nuclear-targeted KillerRed for that
> purpose.  You'd need a pretty modest 555nm light flux to zap targeted cells
> into apoptosis.  There are probably newer and better genetically targeted
> photosensitizing agents, but that's the one that I've used.
>
> Best,
>
>
> T
>
> Timothy Feinstein, Ph.D.
> Research Scientist
> Department of Developmental Biology
> University of Pittsburgh
>
>
>
>
>
> On 8/15/19, 7:42 AM, "Confocal Microscopy List on behalf of Jeremy Adler"
> <[hidden email] on behalf of [hidden email]>
> wrote:
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> and include the link in your posting.
>     *****
>
>
>     We have a user who wants to kill specific GFP labelled single cells in
> a zebra fish and follow the developmental consequences.
>     Multiphoton or confocal used to zap a selected area was the suggestion
> but
>
>     Is bleaching the GFP sufficient to damage the cell.
>     How can we determine if the cell is dead/damaged - trypan blue in fish.
>     The extent to which surrounding cells are also damaged.
>     Are there any photoactivatable toxins ?
>
>     Ideas, references, suggestions would be greatly appreciated.
>
>
>     Jeremy Adler
>     ===============================================
>                         B i o V i s   P l a t f o r m of  Uppsala
> University
>                        Light & EM microscopy / FlowCytometry & Cell
> Sorting / Image Analysis ===============================================
>     Jeremy Adler   PhD - Senior research engineer Light, Confocal
> Microscopy, Image Analysis
>     E-mail: [hidden email]
>     070-1679349
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>     751 85 UPPSALA, SWEDEN
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[Advanced Imaging Lab]

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 Dear Jeremy,

You can try using a laser-capture microdissection microscope.  We have used
it to cut a zebrafish
axon and the fish survived the procedure.

Best regards,
Esther

*Dr. Esther G.L. Koh* :: Head, Advanced Imaging Laboratory :: Life Sciences
Institute Immunology Programme :: National University of Singapore :: Centre
for Life Sciences, 28 Medical Drive #03-06E, Singapore 117456

On Thu, 15 Aug 2019 at 19:42, Jeremy Adler <[hidden email]> wrote:

> *****
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> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>
> We have a user who wants to kill specific GFP labelled single cells in a
> zebra fish and follow the developmental consequences.
> Multiphoton or confocal used to zap a selected area was the suggestion but
>
> Is bleaching the GFP sufficient to damage the cell.
> How can we determine if the cell is dead/damaged - trypan blue in fish.
> The extent to which surrounding cells are also damaged.
> Are there any photoactivatable toxins ?
>
> Ideas, references, suggestions would be greatly appreciated.
>
>
> Jeremy Adler
> ===============================================
>                     B i o V i s   P l a t f o r m of  Uppsala University
>                    Light & EM microscopy / FlowCytometry & Cell Sorting /
> Image Analysis ===============================================
> Jeremy Adler   PhD - Senior research engineer Light, Confocal Microscopy,
> Image Analysis
> E-mail: [hidden email]
> 070-1679349
>
> Dag Hammarskjölds v 20
> 751 85 UPPSALA, SWEDEN
> http://biovis.uu.se/
> ===============================================
>
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[pkshah]

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Hi Jeremy,

It depends a great deal on how deep the GFP+ cells are into the animal and
whether the goal is to kill individual or all GFP+ cells.

We had great success using blue light output from a dye laser (Andor
Micropoint) in killing hair cells in 2-3 day old larvae:
https://www.cell.com/developmental-cell/fulltext/S1534-5807(17)30864-X
But these are cells are quite superficial.

For broader killing, the ROS generating fluorescent proteins others have
mentioned should work well as long as the light exposure required doesn't
also cause off-target toxicity.

For killing individual cells deep inside of tissue, your only real bet is a
femtosecond laser. For a recent implementation of this, see this paper from
the Ahrens and Keller labs:
https://www.nature.com/articles/s41592-018-0221-x

Our experience both with femtosecond and nanosecond lasers for killing
single cells has been to use damage accumulation- a long train of low
energy pulses enough to bleach a visible spot in the nucleus, but not so
much that the entire nucleus or cell bleaches. In our hands this has been
quite important for limiting off-target damage. It's important for there to
be residual signal so one can actually assess cell death- typically one
would expect to see the nucleus either condense or fragment anywhere from 5
minutes to 45 minutes after ablation. Hair cells seem to be cleared within
minutes after death so, depending on your timelapse imaging rates, the
corpse can simply disappear between timepoints. Acute insults like
laser-induced cell killing can also cause significant off-target damage
on-axis with the laser, so it's also important to watch cells above and
below the target for signs of death or damage as this can potentially
confound your experiment.

Please feel free to reach out if I can provide any more detailed advice!

Best,
Pavak


On Thu, Aug 15, 2019 at 4:42 AM Jeremy Adler <[hidden email]> wrote:

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> We have a user who wants to kill specific GFP labelled single cells in a
> zebra fish and follow the developmental consequences.
> Multiphoton or confocal used to zap a selected area was the suggestion but
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> Is bleaching the GFP sufficient to damage the cell.
> How can we determine if the cell is dead/damaged - trypan blue in fish.
> The extent to which surrounding cells are also damaged.
> Are there any photoactivatable toxins ?
>
> Ideas, references, suggestions would be greatly appreciated.
>
>
> Jeremy Adler
> ===============================================
>                     B i o V i s   P l a t f o r m of  Uppsala University
>                    Light & EM microscopy / FlowCytometry & Cell Sorting /
> Image Analysis ===============================================
> Jeremy Adler   PhD - Senior research engineer Light, Confocal Microscopy,
> Image Analysis
> E-mail: [hidden email]
> 070-1679349
>
> Dag Hammarskjölds v 20
> 751 85 UPPSALA, SWEDEN
> http://biovis.uu.se/
> ===============================================
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