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Photostimulation: Andor MicroPoint or Mosaic

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Christian Kukat

Photostimulation: Andor MicroPoint or Mosaic

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Dear confocal list community,

We have an Andor Dragonfly spinning disk confocal, but no FRAP scanner, yet.

We are interested in performing experiments to recruit fuorescently-tagged proteins to DNA damage sites using laser microirradiation and live-cell imaging. For this, we plan to microirradiate points but also line patterns, as described for example in the publications mentioned below (below signature). We would prefer 405 nm for the wavelenght to induce DNA damage.

Andor offered to us now 2 different tools for photostimulation:
- MicroPoint https://andor.oxinst.com/products/photostimulation/micropoint <https://andor.oxinst.com/products/photostimulation/micropoint>
- Mosaic https://andor.oxinst.com/products/photostimulation/mosaic <https://andor.oxinst.com/products/photostimulation/mosaic>

We have now several questions and hope that someone of you can help us:
- Does anybody have experiences with one or the other tool regarding the recruitment of proteins to DNA damage sites?
- How fast is the MicroPoint system for the creation of line patterns?
- As far as we know, none of them is integrated in the Andor Fusion software. So how well works this using the iQ software or swapping between Fusion and iQ?
- What are the pros and cons of them?

We appreciate any input from the community!

Best wishes and stay safe!

Christian

_______________________________________________________________________

Christian Kukat, PhD
Head of FACS & Imaging Core Facility
Max Planck Institute for Biology of Ageing
- ISAC SRL Emerging Leader 2016-2020 -
Joseph-Stelzmann-Str. 9b, D-50931 Köln / Cologne, Germany

E-mail: [hidden email]
www.age.mpg.de
_______________________________________________________________________

Examples in publications:

Fig. 1 in Kim J.J., Kumbhar R., Gong F., Miller K.M. (2019) In Time and Space: Laser Microirradiation and the DNA Damage Response. In: Balakrishnan L., Stewart J. (eds) DNA Repair. Methods in Molecular Biology, vol 1999. Humana, New York, NY
https://link.springer.com/protocol/10.1007/978-1-4939-9500-4_3#Sec14 <https://link.springer.com/protocol/10.1007/978-1-4939-9500-4_3#Sec14>

Fig. 1 in Tampere, M. and Mortusewicz, O. (2016). DNA Damage Induction by Laser Microirradiation. Bio-protocol 6(23): e2039. DOI: 10.21769/BioProtoc.2039 <https://doi.org/10.21769/BioProtoc.2039>.
https://bio-protocol.org/e2039 <https://bio-protocol.org/e2039>

Fig. 2 in Haince JF, McDonald D, Rodrigue A, et al. PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites. J Biol Chem. 2008;283(2):1197-1208. doi:10.1074/jbc.M706734200
https://www.jbc.org/content/283/2/1197.full <https://www.jbc.org/content/283/2/1197.full>

PAVAK SHAH

Re: Photostimulation: Andor MicroPoint or Mosaic

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Hi Christian,

The Mosaic is really the system intended for patterns, although I don't
have much experience with it. It's a projector, so you may trade off a
little bit of sharpness in your pattern and some limitation in peak
intensity for truly simultaneous illumination.

The micropoint is a dye laser that maxes out at a 15 Hz repetition rate,
not really practical for quickly painting shapes.

A few vendors sell 355 nm pulse lasers with kHz rep rates that can paint
shapes well, but I don't know how software integration works with a
Dragonfly. Gataca's iLas and Rapp's Firefly systems come to mind. I moved
from a micropoint to a Rapp system and have been extremely impressed with
its capabilities. 355 nm illumination will also produce DNA damage more
readily than 405 nm. We mostly do point ablations, but it also handles
painting very well.

Best,
Pavak

On Wed, Jun 17, 2020, 6:09 AM Christian Kukat <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear confocal list community,
>
> We have an Andor Dragonfly spinning disk confocal, but no FRAP scanner,
> yet.
>
> We are interested in performing experiments to recruit fuorescently-tagged
> proteins to DNA damage sites using laser microirradiation and live-cell
> imaging. For this, we plan to microirradiate points but also line patterns,
> as described for example in the publications mentioned below (below
> signature). We would prefer 405 nm for the wavelenght to induce DNA damage.
>
> Andor offered to us now 2 different tools for photostimulation:
> - MicroPoint
> https://andor.oxinst.com/products/photostimulation/micropoint <
> https://andor.oxinst.com/products/photostimulation/micropoint>
> - Mosaic
> https://andor.oxinst.com/products/photostimulation/mosaic <
> https://andor.oxinst.com/products/photostimulation/mosaic>
>
>
> We have now several questions and hope that someone of you can help us:
> - Does anybody have experiences with one or the other tool regarding the
> recruitment of proteins to DNA damage sites?
> - How fast is the MicroPoint system for the creation of line patterns?
> - As far as we know, none of them is integrated in the Andor Fusion
> software. So how well works this using the iQ software or swapping between
> Fusion and iQ?
> - What are the pros and cons of them?
>
> We appreciate any input from the community!
>
> Best wishes and stay safe!
>
> Christian
>
> _______________________________________________________________________
>
> Christian Kukat, PhD
> Head of FACS & Imaging Core Facility
> Max Planck Institute for Biology of Ageing
> - ISAC SRL Emerging Leader 2016-2020 -
> Joseph-Stelzmann-Str. 9b, D-50931 Köln / Cologne, Germany
>
> E-mail: [hidden email]
> www.age.mpg.de
> _______________________________________________________________________
>
>
> Examples in publications:
>
> Fig. 1 in Kim J.J., Kumbhar R., Gong F., Miller K.M. (2019) In Time and
> Space: Laser Microirradiation and the DNA Damage Response. In: Balakrishnan
> L., Stewart J. (eds) DNA Repair. Methods in Molecular Biology, vol 1999.
> Humana, New York, NY
> https://link.springer.com/protocol/10.1007/978-1-4939-9500-4_3#Sec14 <
> https://link.springer.com/protocol/10.1007/978-1-4939-9500-4_3#Sec14>
>
> Fig. 1 in Tampere, M. and Mortusewicz, O. (2016). DNA Damage Induction by
> Laser Microirradiation. Bio-protocol 6(23): e2039. DOI:
> 10.21769/BioProtoc.2039 <https://doi.org/10.21769/BioProtoc.2039>.
> https://bio-protocol.org/e2039 <https://bio-protocol.org/e2039>
>
> Fig. 2 in Haince JF, McDonald D, Rodrigue A, et al. PARP1-dependent
> kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage
> sites. J Biol Chem. 2008;283(2):1197-1208. doi:10.1074/jbc.M706734200
> https://www.jbc.org/content/283/2/1197.full <
> https://www.jbc.org/content/283/2/1197.full>
>

Zdenek Svindrych-2

Re: Photostimulation: Andor MicroPoint or Mosaic

In reply to this post by Christian Kukat

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Christian,

we have both on the same spinning disk (not a Dragonfly, though), but I
haven't used them for DNA damage.

The micropoint (basically a dye laser pumped by a nitrogen pulsed laser)
produces quite strong (50-100 uJ) nanosecond pulses that will ablate
anything. But in our configuration it can only do single shot ablation, the
laser is focused to a small spot on the sample, no galvo. The repetition
rate of the laser itself is limited to 15 Hz, so it may not be suitable for
your application.

Mosaic is a 100-200 mW 405 nm laser with a DMD projection, so arbitrary
patterns are possible, but if you want to irradiate a small point or thin
line, the power is of course much lower.

Mosaic can be controlled from MetaMorph (I haven't tried) or IQ. IQ should
be able to control Dragonfly quite well, I don't remember if there's any
critical functionality missing (maybe the ASI stage? Perfect Focus?). I use
the Fusion software almost exclusively. But IQ is also capable of grabbing
the screen output of another program (instead of getting data directly from
the camera), so it should be possible to run both IQ and Fusion
simultaneously, control the Mosaic through IQ and everything else through
Fusion...

Andor also offers (or used to offert) the FRAPPA - a galvo-based unit for
illuminating arbitrary shapes, this can deliver much stronger illumination
into small regions (point, line), but you need second (single mode) fiber
output from your laser module, and it's again controlled through IQ. I used
it with the good old Yokogawa X1, and since the FRAPPA sits in between the
spinning disk unit and the microscope stand, it's quite possible that it
limits your field of view...

Best, zdenek

On Wed, Jun 17, 2020 at 9:08 AM Christian Kukat <[hidden email]>
wrote:

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

BROWNE Mark

Re: -|EXT|- Re: Photostimulation: Andor MicroPoint or Mosaic

In reply to this post by Christian Kukat

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

COMMERCIAL RESPONSE

Hello all,

Zdenek's remarks are mostly spot on, but we wanted to correct a few minor points for accuracy.

The Micropoint has two versions - one with galvos and one provides fixed beam, which Zdenek references. It has been widely used for DNA damage studies mostly at 365 nm but covers about 20 wavelengths (select suitable dye cells) reaching out to 650 nm. Longer wavelengths make it suitable for cutting deeper into tissue. You can cut points and lines with the galvo version very effectively. I will not delve deepr in this post, but would be happy to discuss further via direct email if you wish.

Mosaic is a DMD based device available in two configurations. One with a large field of illumination (17 mm diagonal) mainly for optogenetics and photoswitching and the other with a smaller field of 8 mm diagonal which is used mainly for bleaching and higher power density needs. Either can be supplied with 1100 mW 405 laser and/or 445 1300 mW laser. As Zdenek says, arbitrary regions can be illuminated simultaneously and with the high power lasers, features as small as 1 or 2 microns can be effectively bleached or irradiated for user defined periods, inducing damage.

I hope this proves useful and appreciate your interest.

Mark

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Zdenek Svindrych
Sent: Wednesday, June 17, 2020 10:00 AM
To: [hidden email]
Subject: -|EXT|- Re: Photostimulation: Andor MicroPoint or Mosaic

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Christian,

we have both on the same spinning disk (not a Dragonfly, though), but I haven't used them for DNA damage.

The micropoint (basically a dye laser pumped by a nitrogen pulsed laser) produces quite strong (50-100 uJ) nanosecond pulses that will ablate anything. But in our configuration it can only do single shot ablation, the laser is focused to a small spot on the sample, no galvo. The repetition rate of the laser itself is limited to 15 Hz, so it may not be suitable for your application.

Mosaic is a 100-200 mW 405 nm laser with a DMD projection, so arbitrary patterns are possible, but if you want to irradiate a small point or thin line, the power is of course much lower.

Mosaic can be controlled from MetaMorph (I haven't tried) or IQ. IQ should be able to control Dragonfly quite well, I don't remember if there's any critical functionality missing (maybe the ASI stage? Perfect Focus?). I use the Fusion software almost exclusively. But IQ is also capable of grabbing the screen output of another program (instead of getting data directly from the camera), so it should be possible to run both IQ and Fusion simultaneously, control the Mosaic through IQ and everything else through Fusion...

Andor also offers (or used to offert) the FRAPPA - a galvo-based unit for illuminating arbitrary shapes, this can deliver much stronger illumination into small regions (point, line), but you need second (single mode) fiber output from your laser module, and it's again controlled through IQ. I used it with the good old Yokogawa X1, and since the FRAPPA sits in between the spinning disk unit and the microscope stand, it's quite possible that it limits your field of view...

Best, zdenek

On Wed, Jun 17, 2020 at 9:08 AM Christian Kukat <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear confocal list community,
>
> We have an Andor Dragonfly spinning disk confocal, but no FRAP
> scanner, yet.
>
> We are interested in performing experiments to recruit
> fuorescently-tagged proteins to DNA damage sites using laser
> microirradiation and live-cell imaging. For this, we plan to
> microirradiate points but also line patterns, as described for example
> in the publications mentioned below (below signature). We would prefer 405 nm for the wavelenght to induce DNA damage.
>
> Andor offered to us now 2 different tools for photostimulation:
> - MicroPoint
> https://andor.oxinst.com/products/photostimulation/micropoint <
> https://andor.oxinst.com/products/photostimulation/micropoint>
> - Mosaic
> https://andor.oxinst.com/products/photostimulation/mosaic <
> https://andor.oxinst.com/products/photostimulation/mosaic>
>
>
> We have now several questions and hope that someone of you can help us:
> - Does anybody have experiences with one or the other tool regarding
> the recruitment of proteins to DNA damage sites?
> - How fast is the MicroPoint system for the creation of line patterns?
> - As far as we know, none of them is integrated in the Andor Fusion
> software. So how well works this using the iQ software or swapping
> between Fusion and iQ?
> - What are the pros and cons of them?
>
> We appreciate any input from the community!
>
> Best wishes and stay safe!
>
> Christian
>
> ______________________________________________________________________
> _
>
> Christian Kukat, PhD
> Head of FACS & Imaging Core Facility
> Max Planck Institute for Biology of Ageing
> - ISAC SRL Emerging Leader 2016-2020 - Joseph-Stelzmann-Str. 9b,
> D-50931 Köln / Cologne, Germany
>
> E-mail: [hidden email]
> www.age.mpg.de
> ______________________________________________________________________
> _
>
>
> Examples in publications:
>
> Fig. 1 in Kim J.J., Kumbhar R., Gong F., Miller K.M. (2019) In Time
> and
> Space: Laser Microirradiation and the DNA Damage Response. In:
> Balakrishnan L., Stewart J. (eds) DNA Repair. Methods in Molecular Biology, vol 1999.
> Humana, New York, NY
> https://link.springer.com/protocol/10.1007/978-1-4939-9500-4_3#Sec14 <
> https://link.springer.com/protocol/10.1007/978-1-4939-9500-4_3#Sec14>
>
> Fig. 1 in Tampere, M. and Mortusewicz, O. (2016). DNA Damage Induction
> by Laser Microirradiation. Bio-protocol 6(23): e2039. DOI:
> 10.21769/BioProtoc.2039 <https://doi.org/10.21769/BioProtoc.2039>.
> https://bio-protocol.org/e2039 <https://bio-protocol.org/e2039>
>
> Fig. 2 in Haince JF, McDonald D, Rodrigue A, et al. PARP1-dependent
> kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA
> damage sites. J Biol Chem. 2008;283(2):1197-1208.
> doi:10.1074/jbc.M706734200 https://www.jbc.org/content/283/2/1197.full
> < https://www.jbc.org/content/283/2/1197.full>
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth
___________________________________________________________________________
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