Wide field system for optogenetics

[Alessandro Esposito]

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Dear all,
     we are preparing the lab to adventure in the new field of
optogenetics and I have to consider which microscope to purchase. Let's
not consider budget limits for a moment and focus on wide field
microscopes as I have just updated one of our confocals for
optogenetics.

Does anybody have experience with wide field microscopes designed for
these types of applications? Although not strictly necessary, I am
considering patterned illumination, for instance with a DMD. I will
certainly equip the system with one or two EM-CCD or sCMOS for fast
imaging.

Being overwhelmed with other engineering projects, I would prefer to
purchase a fully commercial solution in this case, with a user-friendly
software, but I would not mind to have some flexibility in customization.

I am aware that a perfect system does not exist, but I wished to ask
the invaluable opinion of the community.

Kind regards,

Alessandro

[Kristen O'Connell]

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If by "wide-field" you mean a through-the-lens approach rather than a fiber
optic, I use a 488 LED from Thor Labs on an Olympus BX41 with a 40x water
immersion lens for this purpose. The LED is driven by the LEDD1B cube
interfaced via BNC to an analog IN on a Digidata 1440 and controlled by
pClamp.

We use it to control ChR2 in brain slices with simultaneous patch clamp. It
works well for my purposes. Because it's controlled by an analog channel,
Clampex can modulate both amplitude and frequency very quickly, with the
flexibility to design complex experimental protocols. I don't use this
system for imaging, but I see no reason why you couldn't (although
fiber-delivered light would be required if you wanted to stimulate away
from the area you want to image).

Spatial control is obtained by moving the lens (this is a fixed-stage slice
patch rig). It's not perfect, but it works. Control experiments using
synaptic blockers and light-stimulation of adjacent but non-synaptically
connected regions indicate that we have pretty good isolation (i.e., no
IPSCs when the light is on).

A coherent blue light source would probably be better, but this was (very)
inexpensive, easy to set up, and simple to interface with the rest of the
rig and ThorLabs was very helpful in finding a cost-effective solution that
actually works.

*absolutely no commercial relationship to Thorlabs, I'm just a happy
customer*




On Tue, Apr 9, 2013 at 5:34 AM, Alessandro Esposito <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>      we are preparing the lab to adventure in the new field of
> optogenetics and I have to consider which microscope to purchase. Let's
> not consider budget limits for a moment and focus on wide field
> microscopes as I have just updated one of our confocals for
> optogenetics.
>
> Does anybody have experience with wide field microscopes designed for
> these types of applications? Although not strictly necessary, I am
> considering patterned illumination, for instance with a DMD. I will
> certainly equip the system with one or two EM-CCD or sCMOS for fast
> imaging.
>
> Being overwhelmed with other engineering projects, I would prefer to
> purchase a fully commercial solution in this case, with a user-friendly
> software, but I would not mind to have some flexibility in customization.
>
> I am aware that a perfect system does not exist, but I wished to ask
> the invaluable opinion of the community.
>
> Kind regards,
>
> Alessandro
>

--
Kristen M.S. O'Connell, Ph.D.
Assistant Professor
University of Tennessee Health Science Center
Department of Physiology
Memphis, TN 38163
Ph: 901-448-2648

[Alessandro Esposito]

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Dear Kristen,
     thank you very much for your reply. I notice I should have better specified
my question as optogenetics start to mean different things to different people. I
am planning to use light-inducible enzymes using LOV domain or PIF6/PhyB
domains in cells. The work will be carried out in cell culture. I have already a two
photon system that will do the job, but I need also a wide field microscope )non
scanning) for faster and more gentle imaging.  

Cheers,

Alessandro


On Tue, 9 Apr 2013 11:11:53 -0500, Kristen O'Connell
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>If by "wide-field" you mean a through-the-lens approach rather than a fiber
>optic, I use a 488 LED from Thor Labs on an Olympus BX41 with a 40x water
>immersion lens for this purpose. The LED is driven by the LEDD1B cube
>interfaced via BNC to an analog IN on a Digidata 1440 and controlled by
>pClamp.
>
>We use it to control ChR2 in brain slices with simultaneous patch clamp. It
>works well for my purposes. Because it's controlled by an analog channel,
>Clampex can modulate both amplitude and frequency very quickly, with the
>flexibility to design complex experimental protocols. I don't use this
>system for imaging, but I see no reason why you couldn't (although
>fiber-delivered light would be required if you wanted to stimulate away
>from the area you want to image).
>
>Spatial control is obtained by moving the lens (this is a fixed-stage slice
>patch rig). It's not perfect, but it works. Control experiments using
>synaptic blockers and light-stimulation of adjacent but non-synaptically
>connected regions indicate that we have pretty good isolation (i.e., no
>IPSCs when the light is on).
>
>A coherent blue light source would probably be better, but this was (very)
>inexpensive, easy to set up, and simple to interface with the rest of the
>rig and ThorLabs was very helpful in finding a cost-effective solution that
>actually works.
>
>*absolutely no commercial relationship to Thorlabs, I'm just a happy
>customer*
>
>
>
>
>On Tue, Apr 9, 2013 at 5:34 AM, Alessandro Esposito <
>[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>>      we are preparing the lab to adventure in the new field of
>> optogenetics and I have to consider which microscope to purchase. Let's
>> not consider budget limits for a moment and focus on wide field
>> microscopes as I have just updated one of our confocals for
>> optogenetics.
>>
>> Does anybody have experience with wide field microscopes designed for
>> these types of applications? Although not strictly necessary, I am
>> considering patterned illumination, for instance with a DMD. I will
>> certainly equip the system with one or two EM-CCD or sCMOS for fast
>> imaging.
>>
>> Being overwhelmed with other engineering projects, I would prefer to
>> purchase a fully commercial solution in this case, with a user-friendly
>> software, but I would not mind to have some flexibility in customization.
>>
>> I am aware that a perfect system does not exist, but I wished to ask
>> the invaluable opinion of the community.
>>
>> Kind regards,
>>
>> Alessandro
>>
>
>
>
>--
>Kristen M.S. O'Connell, Ph.D.
>Assistant Professor
>University of Tennessee Health Science Center
>Department of Physiology
>Memphis, TN 38163
>Ph: 901-448-2648

[samuel connell]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Requested Commercial Response:

Good Evening Alessandro,

Intelligent Imaging Innovations (3i) has invested a large amount of R&D and
engineering resources to create flexible turnkey optogenetics systems. I
would be happy to provide some references off list for scientists who are
using our tools for their optogenetics research. At the heart of most of
these systems is Phasor, 3i's 3D digital holography system. I'll provide
some thoughts on the technology below, but feel free to connect with me off
list and we can talk shop on a configuration that would work for your
biology.

Phasor is the world's first commercial system for optogenetic photo-
stimulation using digital holography. It offers the ability to deliver
significant power to a user-defined pattern (or multiple patterns) at once,
and can actually illuminate at different 3D positions simultaneously.

Because Phasor uses diffraction to shape the waveform of light entering the
specimen, it is far more efficient than systems that use a digital
micromirror device (DMD), which can only block undesired light rather than
redirect it where it is needed. Further, those systems are restricted to
operating in a single plane of focus.

In addition to optogenetic photostimulation, Phasor has many other uses in
photomanipulation:

• fluorescence recovery after photobleaching (FRAP)
• photoconversion (e.g., Kaede)
• photoactivation (e.g., paGFP)
• uncaging (e.g., MNI-glutamate)

Phasor can be used with widefield, spinning disk confocal, and multi-photon
systems, and can also be combined with point-scanning
photostimulation such as 3i's Vector. It is available in visible light and
multiphoton versions, and both versions can be used together in the same
system. Coupled with 3i's LaserStack, more than six different wavelengths
can be selected and even alternated in rapid succession, with different
patterns generated for each wavelength.

Best Regards,
--
Sam

Samuel A. Connell

Senior Applications Scientist

Intelligent Imaging Innovations, Inc

Office: [phone] x6926

Cell:    [phone]

[hidden email]




On Tue, Apr 9, 2013 at 3:34 AM, Alessandro Esposito <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>      we are preparing the lab to adventure in the new field of
> optogenetics and I have to consider which microscope to purchase. Let's
> not consider budget limits for a moment and focus on wide field
> microscopes as I have just updated one of our confocals for
> optogenetics.
>
> Does anybody have experience with wide field microscopes designed for
> these types of applications? Although not strictly necessary, I am
> considering patterned illumination, for instance with a DMD. I will
> certainly equip the system with one or two EM-CCD or sCMOS for fast
> imaging.
>
> Being overwhelmed with other engineering projects, I would prefer to
> purchase a fully commercial solution in this case, with a user-friendly
> software, but I would not mind to have some flexibility in customization.
>
> I am aware that a perfect system does not exist, but I wished to ask
> the invaluable opinion of the community.
>
> Kind regards,
>
> Alessandro
>

[Julio Vazquez]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Alessandro,

If you are looking for a widefield system capable of photoactivation, in addition to 3i, there are turnkey systems from (in no special order):

PerkinElmer (spinning disk)
Prairie Technologies (swept field)
Applied Precision (deconvolution)
Andor (spinning disk, maybe other...)
and probably others (Zeiss, Leica, etc...)

Perhaps you can look up their web sites to see what they have to offer. Their products will vary in price, technology offered (spinnig disk, etc...), and software. Once you have narrowed down your needs, you may want a demo... My guess is that these diferent systems will probably all do the job, so it may come down to price, which type of system is best for your needs (spining disk, etc, ), which system has the best tailored capabilities/functionality for you,  and which software you prefer.

We have systems from several of these vendors... you may email me for specifics if you like.

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

http://www.fhcrc.org




On Apr 9, 2013, at 9:35 AM, Alessandro Esposito wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Kristen,
>     thank you very much for your reply. I notice I should have better specified
> my question as optogenetics start to mean different things to different people. I
> am planning to use light-inducible enzymes using LOV domain or PIF6/PhyB
> domains in cells. The work will be carried out in cell culture. I have already a two
> photon system that will do the job, but I need also a wide field microscope )non
> scanning) for faster and more gentle imaging.  
>
> Cheers,
>
> Alessandro
>
>
> On Tue, 9 Apr 2013 11:11:53 -0500, Kristen O'Connell
> <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> If by "wide-field" you mean a through-the-lens approach rather than a fiber
>> optic, I use a 488 LED from Thor Labs on an Olympus BX41 with a 40x water
>> immersion lens for this purpose. The LED is driven by the LEDD1B cube
>> interfaced via BNC to an analog IN on a Digidata 1440 and controlled by
>> pClamp.
>>
>> We use it to control ChR2 in brain slices with simultaneous patch clamp. It
>> works well for my purposes. Because it's controlled by an analog channel,
>> Clampex can modulate both amplitude and frequency very quickly, with the
>> flexibility to design complex experimental protocols. I don't use this
>> system for imaging, but I see no reason why you couldn't (although
>> fiber-delivered light would be required if you wanted to stimulate away
>> from the area you want to image).
>>
>> Spatial control is obtained by moving the lens (this is a fixed-stage slice
>> patch rig). It's not perfect, but it works. Control experiments using
>> synaptic blockers and light-stimulation of adjacent but non-synaptically
>> connected regions indicate that we have pretty good isolation (i.e., no
>> IPSCs when the light is on).
>>
>> A coherent blue light source would probably be better, but this was (very)
>> inexpensive, easy to set up, and simple to interface with the rest of the
>> rig and ThorLabs was very helpful in finding a cost-effective solution that
>> actually works.
>>
>> *absolutely no commercial relationship to Thorlabs, I'm just a happy
>> customer*
>>
>>
>>
>>
>> On Tue, Apr 9, 2013 at 5:34 AM, Alessandro Esposito <
>> [hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear all,
>>>     we are preparing the lab to adventure in the new field of
>>> optogenetics and I have to consider which microscope to purchase. Let's
>>> not consider budget limits for a moment and focus on wide field
>>> microscopes as I have just updated one of our confocals for
>>> optogenetics.
>>>
>>> Does anybody have experience with wide field microscopes designed for
>>> these types of applications? Although not strictly necessary, I am
>>> considering patterned illumination, for instance with a DMD. I will
>>> certainly equip the system with one or two EM-CCD or sCMOS for fast
>>> imaging.
>>>
>>> Being overwhelmed with other engineering projects, I would prefer to
>>> purchase a fully commercial solution in this case, with a user-friendly
>>> software, but I would not mind to have some flexibility in customization.
>>>
>>> I am aware that a perfect system does not exist, but I wished to ask
>>> the invaluable opinion of the community.
>>>
>>> Kind regards,
>>>
>>> Alessandro
>>>
>>
>>
>>
>> --
>> Kristen M.S. O'Connell, Ph.D.
>> Assistant Professor
>> University of Tennessee Health Science Center
>> Department of Physiology
>> Memphis, TN 38163
>> Ph: 901-448-2648

[Michael Mohammadi]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

[Commercial Response]

Dear Alessandro,

Welcome to the field of optogenetics!

I have some experience (grad school work) with building a DMD based system for uncaging/optogenetics.  You can find a description of the process we employed at the following link:
http://www.jove.com/video/2003/patterned-photostimulation-with-digital-micromirror-devices-to

There are advantages and disadvantages to a home-built system that I would be happy to discuss outside of the mailing list.

I am now working with Andor Technology Plc where we have that a DMD based approach is ideal for optogenetics.  Our DMD-based system, the Andor Mosaic 3, meets both the high speed requirements of the probes/readout and is highly flexible to a variety of light sources and microscopes.

Very key unique features of our DMD-based approach (Mosaic 3) for optogenetics:
        -4 GB of RAM on the Mosaic head to store patterns and trigger through TTL.  Optogenetics requires speed and this approach allows for very fast activation, sequencing and pattern changing of single or multiple wavelengths
        -Gray scale capability (that is, you can change pixel by pixel intensities of light so multiple cells in a field of view can get the same or very different intensities of light)
        -Full integration with any light source of your choosing from LED to Lasers.  We have many systems that utilize commercial LED systems that are generally more than sufficient (e.g. we achieve light intensity of >30 mW / mm^2 with Mosaic 3 + 460nm commercial LED) for optogenetics.  (Mattis et al. 2012 gives a great overview of the power density needed for common probes and the majority are highly sensitive at <10 mW / mm^2) especially in cultured cells/thin tissue preparations removing the need for high powered lasers).  For those applications needing more power (FRAP, etc) we can provide high power lasers and dozens of references where the DMD approach works well.
        -Very precise flat field of illumination optics which can be an issue in other designs (be sure to measure this!)
        -Mosaic Duet system available for simultaneous two color illumination
        -Highly flexible solution with integration on all current generation microscopes as well with a variety of software platforms, as well as SDK available for integration in MATLAB, Python, etc.
        -Key to your request is that we can fully integrate our own cameras (EMCCD and sCMOS) to deliver a full optogenetics solution from the light source/delivery through to the photodetector!

Please feel free to reach out to me directly with any questions you might have about pursuing your own home-based system, our Mosaic 3, or even other application questions regarding your experiments.

Aside: I recently published a brief overview on the (very) basics of optogenetics on my (non-commercial) blog which might give those new to the field a very brief historical perspective which includes some key references as well:
http://sciencedrivenlife.wordpress.com/2013/03/25/optogenetics-history-research-clinical/

I look forward to hearing from you!

Cheers,

Michael

Andor Technology
Michael Mohammadi, PhD
Business Development Manager, AI/DSD, Americas
[hidden email]
Cell: 860-335-2808


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alessandro Esposito
Sent: Tuesday, April 09, 2013 6:35 AM
To: [hidden email]
Subject: Wide field system for optogenetics

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To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear all,
     we are preparing the lab to adventure in the new field of
optogenetics and I have to consider which microscope to purchase. Let's
not consider budget limits for a moment and focus on wide field
microscopes as I have just updated one of our confocals for
optogenetics.

Does anybody have experience with wide field microscopes designed for
these types of applications? Although not strictly necessary, I am
considering patterned illumination, for instance with a DMD. I will
certainly equip the system with one or two EM-CCD or sCMOS for fast
imaging.

Being overwhelmed with other engineering projects, I would prefer to
purchase a fully commercial solution in this case, with a user-friendly
software, but I would not mind to have some flexibility in customization.

I am aware that a perfect system does not exist, but I wished to ask
the invaluable opinion of the community.

Kind regards,

Alessandro


[http://www.andor.com/newsletter/footer/sig1.jpg]<http://www.andor.com/microscopy-systems/revolution/wd-system>