Building a STORM/FPALM

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David Burk David Burk
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Building a STORM/FPALM

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Since the topic of home built super-resolution systems has been brought up I was wondering if anyone had a very simple "Super Res for Dummies" type manual for building such a system.  While I feel quite capable of following directions and assembling components together I have a certain degree of trepidation when it comes to figuring out how to drive all the components software-wise.  I will admit I lack programming experience and limit myself to making ImageJ macros (poorly) or Cell Profiler pipelines - not writing code in Matlab to open shutters and such.  I am aware that many articles describe their setups and give overview diagrams but, from a true schematic standpoint, they lack sufficient detail for someone as rigidly OCD as myself.  Perhaps there is a class or course offered somewhere that covers this "roll your own" approach and I have yet to convince Google to divulge that information.  

Along the same lines, I am very interested in trying to construct an optical projection tomography system for our facility and, again, while I know many labs have built their own systems and published details of them, some critical details elude me.  Have any listers built their own OPT rig and, if so, could you provide a detailed component list as well as assembly instructions?  Personally, I would be more than willing to come visit a lab that has implemented their own solution to the FPALM/STORM and/or OPT method if they wouldn't mind spending a little time answering what they most likely would think are silly questions (and would allow me to take pictures of their system).

David

David H. Burk, PhD
Cell Biology and Bioimaging Core
Pennington Biomedical Research Center
Baton Rouge, LA 70808


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alberto Diaspro
Sent: Saturday, January 29, 2011 10:47 AM
To: [hidden email]
Subject: Re: Who has purchased a fluorescence nanoscope?

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

At the Italian Institute of Technology, we got a Leica STED-CW and home built an FPALM on an inverted Nikon thanks to software sharing from Sam Hess Lab, University of Maine. We home built a WLL and a CW STED controlled by MPI Nanobiophotonics software. We are currently interested n the Nikon N-STORM.
All the best
Alby
Il giorno 29/gen/2011, alle ore 17.19, Christophe Leterrier ha scritto:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The Bordeaux Imaging Center in Bordeaux, France has a STED and a
> TIRF/PALM http://www.bic.u-bordeaux2.fr/
>
> PALM/STORM setups are becoming more common (I'm aware of 2 setups
> already running in Marseille), because (at least this is what the
> people who did it told me) it is quite easy to add to an existing TIRF
> setup (provided you find software for the detection and localisation
> of individual fluorophores, but there is now  an available ImageJ plugin for that).
>
>
> --
> Christophe Leterrier
> Postdoc
> INSERM UMR641 // Ionic channels Lab
> IFR Jean Roche, Mediterranée University Marseille, France
> [hidden email]
>
>
>
>
> On Sat, Jan 29, 2011 at 16:52, G. Esteban Fernandez <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> UCLA has a STED
>>
>> http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791
>> <http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791>
>> http://www.cnsi.ucla.edu/staticpages/core-facilities#alms
>>
>>
>> <http://www.cnsi.ucla.edu/staticpages/core-facilities#alms>
>>
>> On Sat, Jan 29, 2011 at 7:32 AM, George McNamara
>> <[hidden email]>wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear Confocal listserv,
>>>
>>> Who has purchased a fluorescence nanoscope? What can you tell me
>>> about
>> your
>>> experiences - either here or privately?
>>>
>>> I see 11 OMX labs listed at http://www.api.com/omx-labs.asp (may be
>>> out
>> of
>>> date or some customers may be shy).
>>>
>>> I am aware of STED systems in the USA at Yale Univ, NIH and I was
>>> told
>> San
>>> Diego (have not found where), and possibly UC Denver. Paul French
>> apparently
>>> did his own upgrade of a Leica SP2 (
>>> http://www.focusonmicroscopy.org/2008/PDF/070_Auksorius.pdf and a
>>> Leica Scientific Forum video ... see also related work at
>>> ftp://ftp.espci.fr/incoming/Gilles/INSERM-STED-x6.pdf ).
>>>
>>> I am also aware of one 4pi microscope in the USA.
>>>
>>> So, who has N-SIM, N-STORM, SR-SIM, PALm, SR-200, and what can you
>>> tell
>> me
>>> about your experiences with your (purchased) nanoscope(s)?
>>>
>>> Sincerely,
>>>
>>> George
>>> p.s. Please no need to clutter up the listserv with other people's
>>> nanoscope references - I know how to use PubMed. For that matter,
>>> I've replicated the results of the following two papers on my confocal's:
>>>
>>> Subdiffraction fluorescence imaging of biomolecular structure and
>>> distributions with quantum dots. </pubmed/20600360>
>>>
>>> Heidbreder M, Endesfelder U, van de Linde S, Hennig S, Widera D,
>>> Kaltschmidt B, Kaltschmidt C, Heilemann M.
>>>
>>> Biochim Biophys Acta. 2010 Oct;1803(10):1224-9. Epub 2010 Jun
>>> 23.PMID: 20600360
>>>
>>>
>>> Quantum dot triexciton imaging with three-dimensional subdiffraction
>>> resolution. </pubmed/19453186>
>>>
>>> Hennig S, van de Linde S, Heilemann M, Sauer M.
>>>
>>> Nano Lett. 2009 Jun;9(6):2466-70.PMID: 19453186
>>>
>>> I have used <1 Airy unit pinhole to improve triexciton resolution
>> further.
>>> I have not tried doing 3D deconvolution on the data.
>>>
>>>
>>> --
>>>
>>>
>>> George McNamara, PhD
>>> Analytical Imaging Core Facility
>>> University of Miami
>>>
>>



ISTITUTO ITALIANO
DI TECNOLOGIA

Prof. Alberto Diaspro
Scientific Head
Nanophysics
Via Morego, 30 16163 Genova
Tel: +39-010.71.781.503
Fax +39-010-72.03.21
Mobile +39-3666719968
www.iit.it
[hidden email]
John Oreopoulos John Oreopoulos
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Re: Building a STORM/FPALM

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

David, when it comes to the software side of things for STORM/PALM, you might consider Henriques Ricardo's QuickPALM ImageJ/MicroManager plugin:

Henriques, R., et al., Quickpalm: 3d real-time photoactivation nanoscopy image processing in ImageJ. Nat Meth, 2010. 7(5): p. 339-340.

Henriques has also put together a few youtube videos guiding a user step-by-step on how to put it all together (hardware and software):

http://www.youtube.com/user/paxcal

Cheers!

John Oreopoulos
Research Assistant
Spectral Applied Research
9078 Leslie Street, Unit 11
Richmond Hill
Ontario, Canada
www.spectral.ca


On 2011-01-31, at 10:05 AM, David Burk wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Since the topic of home built super-resolution systems has been brought up I was wondering if anyone had a very simple "Super Res for Dummies" type manual for building such a system.  While I feel quite capable of following directions and assembling components together I have a certain degree of trepidation when it comes to figuring out how to drive all the components software-wise.  I will admit I lack programming experience and limit myself to making ImageJ macros (poorly) or Cell Profiler pipelines - not writing code in Matlab to open shutters and such.  I am aware that many articles describe their setups and give overview diagrams but, from a true schematic standpoint, they lack sufficient detail for someone as rigidly OCD as myself.  Perhaps there is a class or course offered somewhere that covers this "roll your own" approach and I have yet to convince Google to divulge that information.  
>
> Along the same lines, I am very interested in trying to construct an optical projection tomography system for our facility and, again, while I know many labs have built their own systems and published details of them, some critical details elude me.  Have any listers built their own OPT rig and, if so, could you provide a detailed component list as well as assembly instructions?  Personally, I would be more than willing to come visit a lab that has implemented their own solution to the FPALM/STORM and/or OPT method if they wouldn't mind spending a little time answering what they most likely would think are silly questions (and would allow me to take pictures of their system).
>
> David
>
> David H. Burk, PhD
> Cell Biology and Bioimaging Core
> Pennington Biomedical Research Center
> Baton Rouge, LA 70808
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alberto Diaspro
> Sent: Saturday, January 29, 2011 10:47 AM
> To: [hidden email]
> Subject: Re: Who has purchased a fluorescence nanoscope?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> At the Italian Institute of Technology, we got a Leica STED-CW and home built an FPALM on an inverted Nikon thanks to software sharing from Sam Hess Lab, University of Maine. We home built a WLL and a CW STED controlled by MPI Nanobiophotonics software. We are currently interested n the Nikon N-STORM.
> All the best
> Alby
> Il giorno 29/gen/2011, alle ore 17.19, Christophe Leterrier ha scritto:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> The Bordeaux Imaging Center in Bordeaux, France has a STED and a
>> TIRF/PALM http://www.bic.u-bordeaux2.fr/
>>
>> PALM/STORM setups are becoming more common (I'm aware of 2 setups
>> already running in Marseille), because (at least this is what the
>> people who did it told me) it is quite easy to add to an existing TIRF
>> setup (provided you find software for the detection and localisation
>> of individual fluorophores, but there is now  an available ImageJ plugin for that).
>>
>>
>> --
>> Christophe Leterrier
>> Postdoc
>> INSERM UMR641 // Ionic channels Lab
>> IFR Jean Roche, Mediterranée University Marseille, France
>> [hidden email]
>>
>>
>>
>>
>> On Sat, Jan 29, 2011 at 16:52, G. Esteban Fernandez <
>> [hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> UCLA has a STED
>>>
>>> http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791
>>> <http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791>
>>> http://www.cnsi.ucla.edu/staticpages/core-facilities#alms
>>>
>>>
>>> <http://www.cnsi.ucla.edu/staticpages/core-facilities#alms>
>>>
>>> On Sat, Jan 29, 2011 at 7:32 AM, George McNamara
>>> <[hidden email]>wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear Confocal listserv,
>>>>
>>>> Who has purchased a fluorescence nanoscope? What can you tell me
>>>> about
>>> your
>>>> experiences - either here or privately?
>>>>
>>>> I see 11 OMX labs listed at http://www.api.com/omx-labs.asp (may be
>>>> out
>>> of
>>>> date or some customers may be shy).
>>>>
>>>> I am aware of STED systems in the USA at Yale Univ, NIH and I was
>>>> told
>>> San
>>>> Diego (have not found where), and possibly UC Denver. Paul French
>>> apparently
>>>> did his own upgrade of a Leica SP2 (
>>>> http://www.focusonmicroscopy.org/2008/PDF/070_Auksorius.pdf and a
>>>> Leica Scientific Forum video ... see also related work at
>>>> ftp://ftp.espci.fr/incoming/Gilles/INSERM-STED-x6.pdf ).
>>>>
>>>> I am also aware of one 4pi microscope in the USA.
>>>>
>>>> So, who has N-SIM, N-STORM, SR-SIM, PALm, SR-200, and what can you
>>>> tell
>>> me
>>>> about your experiences with your (purchased) nanoscope(s)?
>>>>
>>>> Sincerely,
>>>>
>>>> George
>>>> p.s. Please no need to clutter up the listserv with other people's
>>>> nanoscope references - I know how to use PubMed. For that matter,
>>>> I've replicated the results of the following two papers on my confocal's:
>>>>
>>>> Subdiffraction fluorescence imaging of biomolecular structure and
>>>> distributions with quantum dots. </pubmed/20600360>
>>>>
>>>> Heidbreder M, Endesfelder U, van de Linde S, Hennig S, Widera D,
>>>> Kaltschmidt B, Kaltschmidt C, Heilemann M.
>>>>
>>>> Biochim Biophys Acta. 2010 Oct;1803(10):1224-9. Epub 2010 Jun
>>>> 23.PMID: 20600360
>>>>
>>>>
>>>> Quantum dot triexciton imaging with three-dimensional subdiffraction
>>>> resolution. </pubmed/19453186>
>>>>
>>>> Hennig S, van de Linde S, Heilemann M, Sauer M.
>>>>
>>>> Nano Lett. 2009 Jun;9(6):2466-70.PMID: 19453186
>>>>
>>>> I have used <1 Airy unit pinhole to improve triexciton resolution
>>> further.
>>>> I have not tried doing 3D deconvolution on the data.
>>>>
>>>>
>>>> --
>>>>
>>>>
>>>> George McNamara, PhD
>>>> Analytical Imaging Core Facility
>>>> University of Miami
>>>>
>>>
>
>
>
> ISTITUTO ITALIANO
> DI TECNOLOGIA
>
> Prof. Alberto Diaspro
> Scientific Head
> Nanophysics
> Via Morego, 30 16163 Genova
> Tel: +39-010.71.781.503
> Fax +39-010-72.03.21
> Mobile +39-3666719968
> www.iit.it
> [hidden email]
David Baddeley David Baddeley
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Re: Building a STORM/FPALM

In reply to this post by David Burk
I've built STORM/PALM system and can confirm that it's not t
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi David,

I've built STORM/PALM system and can confirm that it's not too difficult. That
said, every system will usually be quite different, depending on the specific
requirements and abilities making a 'one size fits all' instruction manual very
difficult to write. In general it'd be fair to say that some optics/laser design
& alignment experience is desirable when it comes to getting the hardware side
working, and it might be worth getting someone from the local physics department
to help you. Programming experience is also a plus, although something like
'QuickPALM' (as mentioned by John) might give you a leg up (you'd probably still
need to do a bit of tweaking to get everything working on your system). I'm
personally not a huge fan of ImageJ/umanager/ Java in general, so would probably
look for another solution, but this might be personal preference. We use custom,
in house, software which I think is a lot more mature, but which is currently
not particularly well documented.

It also pays to give some thought to what variants of PALM/STORM you want to
support as this has a huge influence on the setup. If your users are looking at
fixed specimens with antibody labelling, something like dSTORM is probably the
easiest to implement (single laser, no complicated switching patterns, can get
away without software control of shutters etc ...).

The two aspects of the hardware that you can't really get off the shelf
components for and are probably going to need to play with are:
-  the laser coupling (commercial TIRF couplers typically give too little power
over too large a field of view),
- the focus mechanism (most commercial z-drives show quite a lot of drift - so
you're going to either need some form of active stabilisation, or to redesign
the focus mechanism - we ended up using a PIFoc piezo focusser on a custom metal
bracket & doing away with the microscopes focus mechanism completely).

If you've got any specific questions, you're welcome to get in touch,
cheers,
David


----- Original Message ----
From: David Burk <[hidden email]>
To: [hidden email]
Sent: Tue, 1 February, 2011 4:05:53 AM
Subject: Building a STORM/FPALM

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

Since the topic of home built super-resolution systems has been brought up I was
wondering if anyone had a very simple "Super Res for Dummies" type manual for
building such a system.  While I feel quite capable of following directions and
assembling components together I have a certain degree of trepidation when it
comes to figuring out how to drive all the components software-wise.  I will
admit I lack programming experience and limit myself to making ImageJ macros
(poorly) or Cell Profiler pipelines - not writing code in Matlab to open
shutters and such.  I am aware that many articles describe their setups and give
overview diagrams but, from a true schematic standpoint, they lack sufficient
detail for someone as rigidly OCD as myself.  Perhaps there is a class or course
offered somewhere that covers this "roll your own" approach and I have yet to
convince Google to divulge that information.  


Along the same lines, I am very interested in trying to construct an optical
projection tomography system for our facility and, again, while I know many labs
have built their own systems and published details of them, some critical
details elude me.  Have any listers built their own OPT rig and, if so, could
you provide a detailed component list as well as assembly instructions?  
Personally, I would be more than willing to come visit a lab that has
implemented their own solution to the FPALM/STORM and/or OPT method if they
wouldn't mind spending a little time answering what they most likely would think
are silly questions (and would allow me to take pictures of their system).

David

David H. Burk, PhD
Cell Biology and Bioimaging Core
Pennington Biomedical Research Center
Baton Rouge, LA 70808


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Alberto Diaspro
Sent: Saturday, January 29, 2011 10:47 AM
To: [hidden email]
Subject: Re: Who has purchased a fluorescence nanoscope?

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

At the Italian Institute of Technology, we got a Leica STED-CW and home built an
FPALM on an inverted Nikon thanks to software sharing from Sam Hess Lab,
University of Maine. We home built a WLL and a CW STED controlled by MPI
Nanobiophotonics software. We are currently interested n the Nikon N-STORM.

All the best
Alby
Il giorno 29/gen/2011, alle ore 17.19, Christophe Leterrier ha scritto:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The Bordeaux Imaging Center in Bordeaux, France has a STED and a
> TIRF/PALM http://www.bic.u-bordeaux2.fr/
>
> PALM/STORM setups are becoming more common (I'm aware of 2 setups
> already running in Marseille), because (at least this is what the
> people who did it told me) it is quite easy to add to an existing TIRF
> setup (provided you find software for the detection and localisation
> of individual fluorophores, but there is now  an available ImageJ plugin for
>that).
>
>
> --
> Christophe Leterrier
> Postdoc
> INSERM UMR641 // Ionic channels Lab
> IFR Jean Roche, Mediterranée University Marseille, France
> [hidden email]
>
>
>
>
> On Sat, Jan 29, 2011 at 16:52, G. Esteban Fernandez <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> UCLA has a STED
>>
>> http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791
>> <http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791>
>> http://www.cnsi.ucla.edu/staticpages/core-facilities#alms
>>
>>
>> <http://www.cnsi.ucla.edu/staticpages/core-facilities#alms>
>>
>> On Sat, Jan 29, 2011 at 7:32 AM, George McNamara
>> <[hidden email]>wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear Confocal listserv,
>>>
>>> Who has purchased a fluorescence nanoscope? What can you tell me
>>> about
>> your
>>> experiences - either here or privately?
>>>
>>> I see 11 OMX labs listed at http://www.api.com/omx-labs.asp (may be
>>> out
>> of
>>> date or some customers may be shy).
>>>
>>> I am aware of STED systems in the USA at Yale Univ, NIH and I was
>>> told
>> San
>>> Diego (have not found where), and possibly UC Denver. Paul French
>> apparently
>>> did his own upgrade of a Leica SP2 (
>>> http://www.focusonmicroscopy.org/2008/PDF/070_Auksorius.pdf and a
>>> Leica Scientific Forum video ... see also related work at
>>> ftp://ftp.espci.fr/incoming/Gilles/INSERM-STED-x6.pdf ).
>>>
>>> I am also aware of one 4pi microscope in the USA.
>>>
>>> So, who has N-SIM, N-STORM, SR-SIM, PALm, SR-200, and what can you
>>> tell
>> me
>>> about your experiences with your (purchased) nanoscope(s)?
>>>
>>> Sincerely,
>>>
>>> George
>>> p.s. Please no need to clutter up the listserv with other people's
>>> nanoscope references - I know how to use PubMed. For that matter,
>>> I've replicated the results of the following two papers on my confocal's:
>>>
>>> Subdiffraction fluorescence imaging of biomolecular structure and
>>> distributions with quantum dots. </pubmed/20600360>
>>>
>>> Heidbreder M, Endesfelder U, van de Linde S, Hennig S, Widera D,
>>> Kaltschmidt B, Kaltschmidt C, Heilemann M.
>>>
>>> Biochim Biophys Acta. 2010 Oct;1803(10):1224-9. Epub 2010 Jun
>>> 23.PMID: 20600360
>>>
>>>
>>> Quantum dot triexciton imaging with three-dimensional subdiffraction
>>> resolution. </pubmed/19453186>
>>>
>>> Hennig S, van de Linde S, Heilemann M, Sauer M.
>>>
>>> Nano Lett. 2009 Jun;9(6):2466-70.PMID: 19453186
>>>
>>> I have used <1 Airy unit pinhole to improve triexciton resolution
>> further.
>>> I have not tried doing 3D deconvolution on the data.
>>>
>>>
>>> --
>>>
>>>
>>> George McNamara, PhD
>>> Analytical Imaging Core Facility
>>> University of Miami
>>>
>>



ISTITUTO ITALIANO
DI TECNOLOGIA

Prof. Alberto Diaspro
Scientific Head
Nanophysics
Via Morego, 30 16163 Genova
Tel: +39-010.71.781.503
Fax +39-010-72.03.21
Mobile +39-3666719968
www.iit.it
[hidden email]




Barbara Foster Barbara Foster
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Re: Building a STORM/FPALM

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

For those of you who might be interested in just
getting a quick overview of STORM/PALM as well as
several other superresolution techniques, I've
just posted the BioPhotonics article at our
website, www.MicroscopyEducation.com.  Just click
on the "Library" and look for the listing.

Best regards,
Barbara Foster, President and Sr. Consultant

Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: (972)924-5310  Skype: fostermme
W: www.MicroscopyEducation.com



At 05:59 PM 1/31/2011, you wrote:

>I've built STORM/PALM system and can confirm that it's not t
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi David, I've built STORM/PALM system and can
>confirm that it's not too difficult. That said,
>every system will usually be quite different,
>depending on the specific requirements and
>abilities making a 'one size fits all'
>instruction manual very difficult to write. In
>general it'd be fair to say that some
>optics/laser design & alignment experience is
>desirable when it comes to getting the hardware
>side working, and it might be worth getting
>someone from the local physics department to
>help you. Programming experience is also a plus,
>although something like 'QuickPALM' (as
>mentioned by John) might give you a leg up
>(you'd probably still need to do a bit of
>tweaking to get everything working on your
>system). I'm personally not a huge fan of
>ImageJ/umanager/ Java in general, so would
>probably look for another solution, but this
>might be personal preference. We use custom, in
>house, software which I think is a lot more
>mature, but which is currently not particularly
>well documented. It also pays to give some
>thought to what variants of PALM/STORM you want
>to support as this has a huge influence on the
>setup. If your users are looking at fixed
>specimens with antibody labelling, something
>like dSTORM is probably the easiest to implement
>(single laser, no complicated switching
>patterns, can get away without software control
>of shutters etc ...). The two aspects of the
>hardware that you can't really get off the shelf
>components for and are probably going to need to
>play with are: -  the laser coupling (commercial
>TIRF couplers typically give too little power
>over too large a field of view), - the focus
>mechanism (most commercial z-drives show quite a
>lot of drift - so you're going to either need
>some form of active stabilisation, or to
>redesign the focus mechanism - we ended up using
>a PIFoc piezo focusser on a custom metal bracket
>& doing away with the microscopes focus
>mechanism completely). If you've got any
>specific questions, you're welcome to get in
>touch, cheers, David ----- Original Message ----
>From: David Burk <[hidden email]> To:
>[hidden email] Sent: Tue, 1
>February, 2011 4:05:53 AM Subject: Building a
>STORM/FPALM ***** To join, leave or search the
>confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>***** Since the topic of home built
>super-resolution systems has been brought up I
>was wondering if anyone had a very simple "Super
>Res for Dummies" type manual for building such a
>system.  While I feel quite capable of following
>directions and assembling components together I
>have a certain degree of trepidation when it
>comes to figuring out how to drive all the
>components software-wise.  I will admit I lack
>programming experience and limit myself to
>making ImageJ macros (poorly) or Cell Profiler
>pipelines - not writing code in Matlab to open
>shutters and such.  I am aware that many
>articles describe their setups and give overview
>diagrams but, from a true schematic standpoint,
>they lack sufficient detail for someone as
>rigidly OCD as myself.  Perhaps there is a class
>or course offered somewhere that covers this
>"roll your own" approach and I have yet to
>convince Google to divulge that
>information.  Along the same lines, I am very
>interested in trying to construct an optical
>projection tomography system for our facility
>and, again, while I know many labs have built
>their own systems and published details of them,
>some critical details elude me.  Have any
>listers built their own OPT rig and, if so,
>could you provide a detailed component list as
>well as assembly instructions?  Personally, I
>would be more than willing to come visit a lab
>that has implemented their own solution to the
>FPALM/STORM and/or OPT method if they wouldn't
>mind spending a little time answering what they
>most likely would think are silly questions (and
>would allow me to take pictures of their
>system). David David H. Burk, PhD Cell Biology
>and Bioimaging Core Pennington Biomedical
>Research Center Baton Rouge, LA 70808
>-----Original Message----- From: Confocal
>Microscopy List
>[mailto:[hidden email]] On
>Behalf Of Alberto Diaspro Sent: Saturday,
>January 29, 2011 10:47 AM To:
>[hidden email] Subject: Re:
>Who has purchased a fluorescence nanoscope?
>***** To join, leave or search the confocal
>microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>***** At the Italian Institute of Technology, we
>got a Leica STED-CW and home built an FPALM on
>an inverted Nikon thanks to software sharing
>from Sam Hess Lab, University of Maine. We home
>built a WLL and a CW STED controlled by MPI
>Nanobiophotonics software. We are currently
>interested n the Nikon N-STORM. All the best
>Alby Il giorno 29/gen/2011, alle ore 17.19,
>Christophe Leterrier ha scritto: > ***** > To
>join, leave or search the confocal microscopy
>listserv, go to: >
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
> > ***** > > The Bordeaux Imaging Center in
>Bordeaux, France has a STED and a > TIRF/PALM
>http://www.bic.u-bordeaux2.fr/ > > PALM/STORM
>setups are becoming more common (I'm aware of 2
>setups > already running in Marseille), because
>(at least this is what the > people who did it
>told me) it is quite easy to add to an existing
>TIRF > setup (provided you find software for the
>detection and localisation > of individual
>fluorophores, but there is now  an available
>ImageJ plugin for >that). > > > -- > Christophe
>Leterrier > Postdoc > INSERM UMR641 // Ionic
>channels Lab > IFR Jean Roche, Mediterranée
>University Marseille, France >
>[hidden email] > > > > > On
>Sat, Jan 29, 2011 at 16:52, G. Esteban Fernandez
>< > [hidden email]> wrote: > >>
>***** >> To join, leave or search the confocal
>microscopy listserv, go to: >>
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
> >> ***** >> >> UCLA has a STED >> >>
>http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791 
> >>
><http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791>  
> >>
>http://www.cnsi.ucla.edu/staticpages/core-facilities#alms 
> >> >> >>
><http://www.cnsi.ucla.edu/staticpages/core-facilities#alms>  
> >> >> On Sat, Jan 29, 2011 at 7:32 AM, George
>McNamara >>
><[hidden email]>wrote: >> >>>
>***** >>> To join, leave or search the confocal
>microscopy listserv, go to: >>>
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
> >>> ***** >>> >>> Dear Confocal
>listserv, >>> >>> Who has purchased a
>fluorescence nanoscope? What can you tell me >>>
>about >> your >>> experiences - either here or
>privately? >>> >>> I see 11 OMX labs listed at
>http://www.api.com/omx-labs.asp (may be >>>
>out >> of >>> date or some customers may be
>shy). >>> >>> I am aware of STED systems in the
>USA at Yale Univ, NIH and I was >>> told >>
>San >>> Diego (have not found where), and
>possibly UC Denver. Paul French >>
>apparently >>> did his own upgrade of a Leica
>SP2 ( >>>
>http://www.focusonmicroscopy.org/2008/PDF/070_Auksorius.pdf 
>and a >>> Leica Scientific Forum video ... see
>also related work at >>>
>ftp://ftp.espci.fr/incoming/Gilles/INSERM-STED-x6.pdf
>). >>> >>> I am also aware of one 4pi microscope
>in the USA. >>> >>> So, who has N-SIM, N-STORM,
>SR-SIM, PALm, SR-200, and what can you >>>
>tell >> me >>> about your experiences with your
>(purchased) nanoscope(s)? >>> >>>
>Sincerely, >>> >>> George >>> p.s. Please no
>need to clutter up the listserv with other
>people's >>> nanoscope references - I know how
>to use PubMed. For that matter, >>> I've
>replicated the results of the following two
>papers on my confocal's: >>> >>> Subdiffraction
>fluorescence imaging of biomolecular structure
>and >>> distributions with quantum dots.
></pubmed/20600360> >>> >>> Heidbreder M,
>Endesfelder U, van de Linde S, Hennig S, Widera
>D, >>> Kaltschmidt B, Kaltschmidt C, Heilemann
>M. >>> >>> Biochim Biophys Acta. 2010
>Oct;1803(10):1224-9. Epub 2010 Jun >>> 23.PMID:
>20600360 >>> >>> >>> Quantum dot triexciton
>imaging with three-dimensional
>subdiffraction >>> resolution.
></pubmed/19453186> >>> >>> Hennig S, van de
>Linde S, Heilemann M, Sauer M. >>> >>> Nano
>Lett. 2009 Jun;9(6):2466-70.PMID:
>19453186 >>> >>> I have used <1 Airy unit
>pinhole to improve triexciton resolution >>
>further. >>> I have not tried doing 3D
>deconvolution on the data. >>> >>> >>>
>-- >>> >>> >>> George McNamara, PhD >>>
>Analytical Imaging Core Facility >>> University
>of Miami >>> >> ISTITUTO ITALIANO DI TECNOLOGIA
>Prof. Alberto Diaspro Scientific Head
>Nanophysics Via Morego, 30 16163 Genova Tel:
>+39-010.71.781.503 Fax +39-010-72.03.21 Mobile
>+39-3666719968 www.iit.it [hidden email]
Guy Cox-2 Guy Cox-2
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Re: Building a STORM/FPALM

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

Just to make it clear, his name is Ricardo Henriques.  He showed me an early version of his software and I was quite amazed by its speed.

 

                                                               Guy

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
      http://www.guycox.net
 

________________________________

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Tuesday, 1 February 2011 3:19 AM
To: [hidden email]
Subject: Re: Building a STORM/FPALM

 

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

David, when it comes to the software side of things for STORM/PALM, you might consider Henriques Ricardo's QuickPALM ImageJ/MicroManager plugin:

Henriques, R., et al., Quickpalm: 3d real-time photoactivation nanoscopy image processing in ImageJ. Nat Meth, 2010. 7(5): p. 339-340.

Henriques has also put together a few youtube videos guiding a user step-by-step on how to put it all together (hardware and software):

http://www.youtube.com/user/paxcal

Cheers!

John Oreopoulos
Research Assistant
Spectral Applied Research
9078 Leslie Street, Unit 11
Richmond Hill
Ontario, Canada
www.spectral.ca


On 2011-01-31, at 10:05 AM, David Burk wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Since the topic of home built super-resolution systems has been brought up I was wondering if anyone had a very simple "Super Res for Dummies" type manual for building such a system.  While I feel quite capable of following directions and assembling components together I have a certain degree of trepidation when it comes to figuring out how to drive all the components software-wise.  I will admit I lack programming experience and limit myself to making ImageJ macros (poorly) or Cell Profiler pipelines - not writing code in Matlab to open shutters and such.  I am aware that many articles describe their setups and give overview diagrams but, from a true schematic standpoint, they lack sufficient detail for someone as rigidly OCD as myself.  Perhaps there is a class or course offered somewhere that covers this "roll your own" approach and I have yet to convince Google to divulge that information.
>
> Along the same lines, I am very interested in trying to construct an optical projection tomography system for our facility and, again, while I know many labs have built their own systems and published details of them, some critical details elude me.  Have any listers built their own OPT rig and, if so, could you provide a detailed component list as well as assembly instructions?  Personally, I would be more than willing to come visit a lab that has implemented their own solution to the FPALM/STORM and/or OPT method if they wouldn't mind spending a little time answering what they most likely would think are silly questions (and would allow me to take pictures of their system).
>
> David
>
> David H. Burk, PhD
> Cell Biology and Bioimaging Core
> Pennington Biomedical Research Center
> Baton Rouge, LA 70808
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alberto Diaspro
> Sent: Saturday, January 29, 2011 10:47 AM
> To: [hidden email]
> Subject: Re: Who has purchased a fluorescence nanoscope?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> At the Italian Institute of Technology, we got a Leica STED-CW and home built an FPALM on an inverted Nikon thanks to software sharing from Sam Hess Lab, University of Maine. We home built a WLL and a CW STED controlled by MPI Nanobiophotonics software. We are currently interested n the Nikon N-STORM.
> All the best
> Alby
> Il giorno 29/gen/2011, alle ore 17.19, Christophe Leterrier ha scritto:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> The Bordeaux Imaging Center in Bordeaux, France has a STED and a
>> TIRF/PALM http://www.bic.u-bordeaux2.fr/
>>
>> PALM/STORM setups are becoming more common (I'm aware of 2 setups
>> already running in Marseille), because (at least this is what the
>> people who did it told me) it is quite easy to add to an existing TIRF
>> setup (provided you find software for the detection and localisation
>> of individual fluorophores, but there is now  an available ImageJ plugin for that).
>>
>>
>> --
>> Christophe Leterrier
>> Postdoc
>> INSERM UMR641 // Ionic channels Lab
>> IFR Jean Roche, Mediterranée University Marseille, France
>> [hidden email]
>>
>>
>>
>>
>> On Sat, Jan 29, 2011 at 16:52, G. Esteban Fernandez <
>> [hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> UCLA has a STED
>>>
>>> http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791
>>> <http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791>
>>> http://www.cnsi.ucla.edu/staticpages/core-facilities#alms
>>>
>>>
>>> <http://www.cnsi.ucla.edu/staticpages/core-facilities#alms>
>>>
>>> On Sat, Jan 29, 2011 at 7:32 AM, George McNamara
>>> <[hidden email]>wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear Confocal listserv,
>>>>
>>>> Who has purchased a fluorescence nanoscope? What can you tell me
>>>> about
>>> your
>>>> experiences - either here or privately?
>>>>
>>>> I see 11 OMX labs listed at http://www.api.com/omx-labs.asp (may be
>>>> out
>>> of
>>>> date or some customers may be shy).
>>>>
>>>> I am aware of STED systems in the USA at Yale Univ, NIH and I was
>>>> told
>>> San
>>>> Diego (have not found where), and possibly UC Denver. Paul French
>>> apparently
>>>> did his own upgrade of a Leica SP2 (
>>>> http://www.focusonmicroscopy.org/2008/PDF/070_Auksorius.pdf and a
>>>> Leica Scientific Forum video ... see also related work at
>>>> ftp://ftp.espci.fr/incoming/Gilles/INSERM-STED-x6.pdf ).
>>>>
>>>> I am also aware of one 4pi microscope in the USA.
>>>>
>>>> So, who has N-SIM, N-STORM, SR-SIM, PALm, SR-200, and what can you
>>>> tell
>>> me
>>>> about your experiences with your (purchased) nanoscope(s)?
>>>>
>>>> Sincerely,
>>>>
>>>> George
>>>> p.s. Please no need to clutter up the listserv with other people's
>>>> nanoscope references - I know how to use PubMed. For that matter,
>>>> I've replicated the results of the following two papers on my confocal's:
>>>>
>>>> Subdiffraction fluorescence imaging of biomolecular structure and
>>>> distributions with quantum dots. </pubmed/20600360>
>>>>
>>>> Heidbreder M, Endesfelder U, van de Linde S, Hennig S, Widera D,
>>>> Kaltschmidt B, Kaltschmidt C, Heilemann M.
>>>>
>>>> Biochim Biophys Acta. 2010 Oct;1803(10):1224-9. Epub 2010 Jun
>>>> 23.PMID: 20600360
>>>>
>>>>
>>>> Quantum dot triexciton imaging with three-dimensional subdiffraction
>>>> resolution. </pubmed/19453186>
>>>>
>>>> Hennig S, van de Linde S, Heilemann M, Sauer M.
>>>>
>>>> Nano Lett. 2009 Jun;9(6):2466-70.PMID: 19453186
>>>>
>>>> I have used <1 Airy unit pinhole to improve triexciton resolution
>>> further.
>>>> I have not tried doing 3D deconvolution on the data.
>>>>
>>>>
>>>> --
>>>>
>>>>
>>>> George McNamara, PhD
>>>> Analytical Imaging Core Facility
>>>> University of Miami
>>>>
>>>
>
>
>
> ISTITUTO ITALIANO
> DI TECNOLOGIA
>
> Prof. Alberto Diaspro
> Scientific Head
> Nanophysics
> Via Morego, 30 16163 Genova
> Tel: +39-010.71.781.503
> Fax +39-010-72.03.21
> Mobile +39-3666719968
> www.iit.it
> [hidden email]

________________________________

No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1204 / Virus Database: 1435/3413 - Release Date: 01/30/11
John Oreopoulos John Oreopoulos
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Re: Building a STORM/FPALM

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

Whoops, sorry about that.

John Oreopoulos

On 2011-02-01, at 2:26 AM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Just to make it clear, his name is Ricardo Henriques.  He showed me an early version of his software and I was quite amazed by its speed.
>
>
>
>                                                               Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone +61 2 9351 3176     Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
>      http://www.guycox.net
>
>
> ________________________________
>
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
> Sent: Tuesday, 1 February 2011 3:19 AM
> To: [hidden email]
> Subject: Re: Building a STORM/FPALM
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> David, when it comes to the software side of things for STORM/PALM, you might consider Henriques Ricardo's QuickPALM ImageJ/MicroManager plugin:
>
> Henriques, R., et al., Quickpalm: 3d real-time photoactivation nanoscopy image processing in ImageJ. Nat Meth, 2010. 7(5): p. 339-340.
>
> Henriques has also put together a few youtube videos guiding a user step-by-step on how to put it all together (hardware and software):
>
> http://www.youtube.com/user/paxcal
>
> Cheers!
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> 9078 Leslie Street, Unit 11
> Richmond Hill
> Ontario, Canada
> www.spectral.ca
>
>
> On 2011-01-31, at 10:05 AM, David Burk wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Since the topic of home built super-resolution systems has been brought up I was wondering if anyone had a very simple "Super Res for Dummies" type manual for building such a system.  While I feel quite capable of following directions and assembling components together I have a certain degree of trepidation when it comes to figuring out how to drive all the components software-wise.  I will admit I lack programming experience and limit myself to making ImageJ macros (poorly) or Cell Profiler pipelines - not writing code in Matlab to open shutters and such.  I am aware that many articles describe their setups and give overview diagrams but, from a true schematic standpoint, they lack sufficient detail for someone as rigidly OCD as myself.  Perhaps there is a class or course offered somewhere that covers this "roll your own" approach and I have yet to convince Google to divulge that information.
>>
>> Along the same lines, I am very interested in trying to construct an optical projection tomography system for our facility and, again, while I know many labs have built their own systems and published details of them, some critical details elude me.  Have any listers built their own OPT rig and, if so, could you provide a detailed component list as well as assembly instructions?  Personally, I would be more than willing to come visit a lab that has implemented their own solution to the FPALM/STORM and/or OPT method if they wouldn't mind spending a little time answering what they most likely would think are silly questions (and would allow me to take pictures of their system).
>>
>> David
>>
>> David H. Burk, PhD
>> Cell Biology and Bioimaging Core
>> Pennington Biomedical Research Center
>> Baton Rouge, LA 70808
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alberto Diaspro
>> Sent: Saturday, January 29, 2011 10:47 AM
>> To: [hidden email]
>> Subject: Re: Who has purchased a fluorescence nanoscope?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> At the Italian Institute of Technology, we got a Leica STED-CW and home built an FPALM on an inverted Nikon thanks to software sharing from Sam Hess Lab, University of Maine. We home built a WLL and a CW STED controlled by MPI Nanobiophotonics software. We are currently interested n the Nikon N-STORM.
>> All the best
>> Alby
>> Il giorno 29/gen/2011, alle ore 17.19, Christophe Leterrier ha scritto:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> The Bordeaux Imaging Center in Bordeaux, France has a STED and a
>>> TIRF/PALM http://www.bic.u-bordeaux2.fr/
>>>
>>> PALM/STORM setups are becoming more common (I'm aware of 2 setups
>>> already running in Marseille), because (at least this is what the
>>> people who did it told me) it is quite easy to add to an existing TIRF
>>> setup (provided you find software for the detection and localisation
>>> of individual fluorophores, but there is now  an available ImageJ plugin for that).
>>>
>>>
>>> --
>>> Christophe Leterrier
>>> Postdoc
>>> INSERM UMR641 // Ionic channels Lab
>>> IFR Jean Roche, Mediterranée University Marseille, France
>>> [hidden email]
>>>
>>>
>>>
>>>
>>> On Sat, Jan 29, 2011 at 16:52, G. Esteban Fernandez <
>>> [hidden email]> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> UCLA has a STED
>>>>
>>>> http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791
>>>> <http://clms.cnsi.ucla.edu/cnsi/clms/equipment-list?search_lab=6791>
>>>> http://www.cnsi.ucla.edu/staticpages/core-facilities#alms
>>>>
>>>>
>>>> <http://www.cnsi.ucla.edu/staticpages/core-facilities#alms>
>>>>
>>>> On Sat, Jan 29, 2011 at 7:32 AM, George McNamara
>>>> <[hidden email]>wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Dear Confocal listserv,
>>>>>
>>>>> Who has purchased a fluorescence nanoscope? What can you tell me
>>>>> about
>>>> your
>>>>> experiences - either here or privately?
>>>>>
>>>>> I see 11 OMX labs listed at http://www.api.com/omx-labs.asp (may be
>>>>> out
>>>> of
>>>>> date or some customers may be shy).
>>>>>
>>>>> I am aware of STED systems in the USA at Yale Univ, NIH and I was
>>>>> told
>>>> San
>>>>> Diego (have not found where), and possibly UC Denver. Paul French
>>>> apparently
>>>>> did his own upgrade of a Leica SP2 (
>>>>> http://www.focusonmicroscopy.org/2008/PDF/070_Auksorius.pdf and a
>>>>> Leica Scientific Forum video ... see also related work at
>>>>> ftp://ftp.espci.fr/incoming/Gilles/INSERM-STED-x6.pdf ).
>>>>>
>>>>> I am also aware of one 4pi microscope in the USA.
>>>>>
>>>>> So, who has N-SIM, N-STORM, SR-SIM, PALm, SR-200, and what can you
>>>>> tell
>>>> me
>>>>> about your experiences with your (purchased) nanoscope(s)?
>>>>>
>>>>> Sincerely,
>>>>>
>>>>> George
>>>>> p.s. Please no need to clutter up the listserv with other people's
>>>>> nanoscope references - I know how to use PubMed. For that matter,
>>>>> I've replicated the results of the following two papers on my confocal's:
>>>>>
>>>>> Subdiffraction fluorescence imaging of biomolecular structure and
>>>>> distributions with quantum dots. </pubmed/20600360>
>>>>>
>>>>> Heidbreder M, Endesfelder U, van de Linde S, Hennig S, Widera D,
>>>>> Kaltschmidt B, Kaltschmidt C, Heilemann M.
>>>>>
>>>>> Biochim Biophys Acta. 2010 Oct;1803(10):1224-9. Epub 2010 Jun
>>>>> 23.PMID: 20600360
>>>>>
>>>>>
>>>>> Quantum dot triexciton imaging with three-dimensional subdiffraction
>>>>> resolution. </pubmed/19453186>
>>>>>
>>>>> Hennig S, van de Linde S, Heilemann M, Sauer M.
>>>>>
>>>>> Nano Lett. 2009 Jun;9(6):2466-70.PMID: 19453186
>>>>>
>>>>> I have used <1 Airy unit pinhole to improve triexciton resolution
>>>> further.
>>>>> I have not tried doing 3D deconvolution on the data.
>>>>>
>>>>>
>>>>> --
>>>>>
>>>>>
>>>>> George McNamara, PhD
>>>>> Analytical Imaging Core Facility
>>>>> University of Miami
>>>>>
>>>>
>>
>>
>>
>> ISTITUTO ITALIANO
>> DI TECNOLOGIA
>>
>> Prof. Alberto Diaspro
>> Scientific Head
>> Nanophysics
>> Via Morego, 30 16163 Genova
>> Tel: +39-010.71.781.503
>> Fax +39-010-72.03.21
>> Mobile +39-3666719968
>> www.iit.it
>> [hidden email]
>
> ________________________________
>
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1204 / Virus Database: 1435/3413 - Release Date: 01/30/11
Christian-103 Christian-103
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a couple Arabidopsis questions

In reply to this post by David Burk
I have two problems, both in Arabidopsis.

 

First, has an
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I have two problems, both in Arabidopsis.

 

First, has anyone on this list done FRET in live plant
cells?  If so, may I contact you off list
for a few pointers?  The entire
cytoplasmic streaming and movement of organelles bit has me a bit stumped.

 

Secondly, we have GFP which is targeting two organelles.   The question is if the amounts going to each
organelle can be quantified.  The issue
being one is roughly 10x as bright.  I am
unsure if any of the various settings on a Nikon A1 are linear, as it is
impossible to image the two organelles in the same cell and have them be on the
same intensity scale. 

 

Any pointers or ideas are greatly appreciated.

 

Christian