Ideal wavelength for bleaching step of FRAP

[Ben Abrams]

Hi All,

I'd love to hear what the consensus is on this.  If I was able to choose between different lasers with equal power for FRAP, would it be better to bleach with a wavelength that is ideal for excitation of the fluorophore being bleached, or is it better to bleach with a shorter more intense wavelength?  I can imagine the former exciting molecules more efficiently and the latter doing more damage to the fluorophore.

Thanks!
Ben

[WILDE Geraint]

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Dear Ben,

We deal with FRAPPA devices.  I will be interested in the broad experience of the listserv on this, but my opinion would be that it is better to bleach at the wavelength you would optimally excite at.  Bleaching is all about exciting as optimally as possible, and so it makes sense to try and hit the absorption peak.

Sometimes this is not possible if your bleaching device is using a different optical path to your imaging device.  For example a point-scanner or camera on a side port, and bleaching device on the fluorescence axis, when you want simultaneous bleaching and imaging.  In this instance you will use a shorter wavelength to your imaging lines, so that your dichroic reflecting your short bleaching wavelength, can then pass your longer imaging excitation and emission wavelengths.

Short wavelengths down towards UV are also of course increasingly detrimental to cell health.

Best

Geraint

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ben Abrams
Sent: 08 May 2012 21:42
To: [hidden email]
Subject: [CONFOCALMICROSCOPY] Ideal wavelength for bleaching step of FRAP

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Hi All,

I'd love to hear what the consensus is on this.  If I was able to choose between different lasers with equal power for FRAP, would it be better to bleach with a wavelength that is ideal for excitation of the fluorophore being bleached, or is it better to bleach with a shorter more intense wavelength?  I can imagine the former exciting molecules more efficiently and the latter doing more damage to the fluorophore.

Thanks!
Ben

--
View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRAP-tp7540631.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.


[http://www.andor.com/newsletter/footer/sig.jpg]<http://www.andor.com/newsletter/footer>

[Tim Feinstein-2]

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Hi Ben,

In my experience you have two basic choices for a FRAP wavelength.  One, you can use a 405 nm laser or similar wavelength of transmitted light and bleach everything as long as you use enough light.  Or, one can bleach at or about the optimal excitation wavelength for a fluorophore and get a little more specificity with what you bleach.  In terms of raw effectiveness it is quite easy to get enough 405nm light to nuke an ROI whereas a 442 or 561 nm laser may take more power or more time.  

The selective approach is a bit trickier also because some fluorophores bleach more readily than others.  CFP, for example, can take quite a lot of light before it gives up the ghost.  However, we do this whenever we can because it is extremely useful to have a 'control' fluorophore in the experiment to track the movement of bleached objects (e.g., an endosome) in the X, Y and Z planes.  In our experience bleaching a given fluorophore (e.g., GFP) tends to also bleach every fluorescent protein to the red of it (e.g., dTomato or mCherry) whereas you can bleach red proteins without any detectable effect on proteins excited by shorter wavelength light.  Whether this is due to redder proteins being inherently more sensitive to bleaching I cannot say.  

cheers,


TF


Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On May 8, 2012, at 4:42 PM, Ben Abrams wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi All,
>
> I'd love to hear what the consensus is on this.  If I was able to choose
> between different lasers with equal power for FRAP, would it be better to
> bleach with a wavelength that is ideal for excitation of the fluorophore
> being bleached, or is it better to bleach with a shorter more intense
> wavelength?  I can imagine the former exciting molecules more efficiently
> and the latter doing more damage to the fluorophore.
>
> Thanks!
> Ben
>
> --
> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRAP-tp7540631.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[Timothy Gould]

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Hello,
      Does anyone know how to align the laser on our Noran odyssey XL .
Guidance notes would help at least !
Regards, Tim

[Stanislav Vitha]

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Ben,
bleachin at wavelength of maximum absorption of the fluorophore will give you
the best damage to the fluorohore (that is what you want).
However, for essentially all fluorophores I can bleach  with the 405 nm laser  
on our Olympus FV1000  (either using the main scanner or the SIM scanner).

Nevertheless, I think it is best to limit your bleaching to the fluorophore of
interest. The 405 nm laser is likely to damage/bleach/photoconvert other
cellular constituents besides bleaching your GFP/YFP/mCherry.

With some specimens, like plant chloroplasts, the 405 nm laser will induce
strong autofluorescence, so instead of nice dark spot where the GFP was
bleached, I get a giant bright spot.  That is not ideal for FRAP :(
 
Stan Vitha
Microscopy and Imaging Center
Texas A&M Universuty
 
On Tue, 8 May 2012 13:42:28 -0700, Ben Abrams <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi All,
>
>I'd love to hear what the consensus is on this.  If I was able to choose
>between different lasers with equal power for FRAP, would it be better to
>bleach with a wavelength that is ideal for excitation of the fluorophore
>being bleached, or is it better to bleach with a shorter more intense
>wavelength?  I can imagine the former exciting molecules more efficiently
>and the latter doing more damage to the fluorophore.
>
>Thanks!
>Ben
>
>--
>View this message in context: http://confocal-microscopy-

list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRAP-
tp7540631.html
>Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[Todd Adams Clason]

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We have a Noran OZ that is still running. We recently put a Coherent  
488 nm Sapphire on it to replace the Melles Ar, so I may be able to  
help you. You can also contact Prairie Instruments. Depending on how  
swamped they are, they might be able to offer some moral support.

Todd

-----------------------------------
Todd Clason
Manager, Neuroscience COBRE Imaging and Physiology Core Department of  
Anatomy and Neurobiology College of Medicine, University of Vermont
E015 Given
89 Beaumont Ave.
Burlington, VT 05405
p. 802.656.0413
[hidden email]



-----Original Message-----
From: Confocal Microscopy List  
[mailto:[hidden email]] On Behalf Of Timothy Gould
Sent: May 09, 2012 11:16 AM
To: [hidden email]
Subject: Re: Noran Odyssey xl laser alignment

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Hello,
       Does anyone know how to align the laser on our Noran odyssey XL .
Guidance notes would help at least !
Regards, Tim


-----------------------------------
Todd Clason
Director, COBRE Imaging and Physiology Core
Department of Anatomy and Neurobiology
College of Medicine, University of Vermont
E015 Given
89 Beaumont Ave.
Burlington, VT 05405
p. 802.656.0413
[hidden email]

[Gregg Jarvis]

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http://books.google.com/books?id=16Ft5k8RC-AC&pg=PA476&lpg=PA476&dq=Noran+odyssey+XL+laser&source=bl&ots=5XFABquCUt&sig=fu9L0Kuf5OxpblfIowX6VGSY8EQ&hl=en&sa=X&ei=Zo6qT_iGF4X6ggfQhZmqAQ&ved=0CE0Q6AEwAg#v=onepage&q=Noran%20odyssey%20XL%20laser&f=false
http://www.lplarson.com/Laser%20Alignment%20Services.html

looks like a service call unless you have the Big book of laser alignment
atyour disposal.
Why would it be out of alignment?
wear your safety glasses



On Wed, May 9, 2012 at 11:26 AM, <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> We have a Noran OZ that is still running. We recently put a Coherent 488
> nm Sapphire on it to replace the Melles Ar, so I may be able to help you.
> You can also contact Prairie Instruments. Depending on how swamped they
> are, they might be able to offer some moral support.
>
> Todd
>
> ------------------------------**-----
> Todd Clason
> Manager, Neuroscience COBRE Imaging and Physiology Core Department of
> Anatomy and Neurobiology College of Medicine, University of Vermont
> E015 Given
> 89 Beaumont Ave.
> Burlington, VT 05405
> p. 802.656.0413
> [hidden email]
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU<[hidden email]>]
> On Behalf Of Timothy Gould
> Sent: May 09, 2012 11:16 AM
> To: [hidden email].**EDU <[hidden email]>
> Subject: Re: Noran Odyssey xl laser alignment
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Hello,
>      Does anyone know how to align the laser on our Noran odyssey XL .
> Guidance notes would help at least !
> Regards, Tim
>
>
> ------------------------------**-----
> Todd Clason
> Director, COBRE Imaging and Physiology Core
> Department of Anatomy and Neurobiology
> College of Medicine, University of Vermont
> E015 Given
> 89 Beaumont Ave.
> Burlington, VT 05405
> p. 802.656.0413
> [hidden email]
>

--
Gregg Jarvis
Advanced Products Group
Omega Optical Inc.
24 Omega Drive
Brattleboro, VT 05301
[hidden email]
1-802-251-7316

[Kurt Thorn]

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It's not always the case that the peak excitation wavelength gives the
fastest bleaching.  I did some tests a number of years ago where we saw
that we could get faster bleaching of YFP when excited with a CFP
excitation filter than with a YFP excitation filter.  That said, I don't
know how common this is, and I've not seen any systematic comparison of
bleaching rates for different dyes at different wavelengths, so you'd
probably have to test it yourself.

Kurt

On 5/9/2012 8:16 AM, Stanislav Vitha wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Ben,
> bleachin at wavelength of maximum absorption of the fluorophore will give you
> the best damage to the fluorohore (that is what you want).
> However, for essentially all fluorophores I can bleach  with the 405 nm laser
> on our Olympus FV1000  (either using the main scanner or the SIM scanner).
>
> Nevertheless, I think it is best to limit your bleaching to the fluorophore of
> interest. The 405 nm laser is likely to damage/bleach/photoconvert other
> cellular constituents besides bleaching your GFP/YFP/mCherry.
>
> With some specimens, like plant chloroplasts, the 405 nm laser will induce
> strong autofluorescence, so instead of nice dark spot where the GFP was
> bleached, I get a giant bright spot.  That is not ideal for FRAP :(
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M Universuty
>
> On Tue, 8 May 2012 13:42:28 -0700, Ben Abrams<[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi All,
>>
>> I'd love to hear what the consensus is on this.  If I was able to choose
>> between different lasers with equal power for FRAP, would it be better to
>> bleach with a wavelength that is ideal for excitation of the fluorophore
>> being bleached, or is it better to bleach with a shorter more intense
>> wavelength?  I can imagine the former exciting molecules more efficiently
>> and the latter doing more damage to the fluorophore.
>>
>> Thanks!
>> Ben
>>
>> --
>> View this message in context: http://confocal-microscopy-
> list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRAP-
> tp7540631.html
>> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>

[James Pawley]

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Hi Folks,

I know that FRAP is very popular and possibly
even useful. But the story I am hearing here
seems to be ignoring some important aspects of
photo-bleaching.

If you are planning to spend a lot of "academic
capital" on experiments that depend on FRAP I do
recommend you take the time to read chapters 38,
CELL DAMAGE DURING MULTI-PHOTON MICROSCOPY and 39
PHOTOBLEACHING.

The short version is that although some bleaching
reactions may involve the absorption of a second
(or third) photon by an already excited molecule,
leading to its demise, (i.e., a simple
two-component system where a photon or 3 are
absorbed by a single molecule and the molecule
ceases to be fluorescent) this is believed to be
a rare event. More common is the situation where
the (singlet- or triplet-) excited dye molecule
passes its excess energy on to (usually) O2,
creating a variety of reactive oxygen species
(ROS). Reactions between this ROS and the dye
create a modified dye molecule that is no longer
fluorescent (or is not fluorescent at different
wavelengths etc.)

The problem with this is that the ROS can move
some distance (0.1-1 µm?) between the time it is
created and the time (ms?) that it reacts, and in
addition, there is no reason to assume that this
second reaction will involve a dye molecule.
Once the ROS is formed, it can damage many of the
molecules within its diffusion radius.

Well, this is getting a bit long but it is
important to remember that the details of the
bleaching event can depend strongly on the exact
chemistry of the dye, and the probability of it
occurring may not be proportional to dose
(intensity:  in photons/square µm x exposure
time) but may also vary non-linearly with
intensity.

Cell type may be important, because some cells
are better provided with molecules designed to
mop up ROS (which are also produced by
mitochondria), as will the stain concentration:
no stain implies only autofluorescence and so
probably only very weak excitation and little
bleaching, (unless there are a lot of pigments or
naturally-fluorescent compounds (as are
particularly common in plants).

But please remember

1. That you cannot assume that a fluorophor will
be bleached ONLY because you hit it with light at
it's excitation peak. The absorption of your
"bleaching" light may bleach any dye in the
vicinity at a rate depending on the propensity of
the dye you do excite to make ROS and the
sensitivity of any other dye to ROS attack.

2. The cell may not like the ROS any more than
the dyes do and is likely to respond to this
insult. Some folks say that if a cells bleaches,
it must be damaged.

If this seems unnecessarily cautious, please have
a look at the text surrounding Figs 49-12 and
49-13.

Sorry to complicate your story.

Cheers,

Jim Pawley

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>It's not always the case that the peak
>excitation wavelength gives the fastest
>bleaching.  I did some tests a number of years
>ago where we saw that we could get faster
>bleaching of YFP when excited with a CFP
>excitation filter than with a YFP excitation
>filter.  That said, I don't know how common this
>is, and I've not seen any systematic comparison
>of bleaching rates for different dyes at
>different wavelengths, so you'd probably have to
>test it yourself.
>
>Kurt
>
>On 5/9/2012 8:16 AM, Stanislav Vitha wrote:
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>Ben,
>>bleachin at wavelength of maximum absorption of the fluorophore will give you
>>the best damage to the fluorohore (that is what you want).
>>However, for essentially all fluorophores I can bleach  with the 405 nm laser
>>on our Olympus FV1000  (either using the main scanner or the SIM scanner).
>>
>>Nevertheless, I think it is best to limit your
>>bleaching to the fluorophore of
>>interest. The 405 nm laser is likely to damage/bleach/photoconvert other
>>cellular constituents besides bleaching your GFP/YFP/mCherry.
>>
>>With some specimens, like plant chloroplasts, the 405 nm laser will induce
>>strong autofluorescence, so instead of nice dark spot where the GFP was
>>bleached, I get a giant bright spot.  That is not ideal for FRAP :(
>>
>>Stan Vitha
>>Microscopy and Imaging Center
>>Texas A&M Universuty
>>
>>On Tue, 8 May 2012 13:42:28 -0700, Ben Abrams<[hidden email]>
>>wrote:
>>
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>Hi All,
>>>
>>>I'd love to hear what the consensus is on this.  If I was able to choose
>>>between different lasers with equal power for FRAP, would it be better to
>>>bleach with a wavelength that is ideal for excitation of the fluorophore
>>>being bleached, or is it better to bleach with a shorter more intense
>>>wavelength?  I can imagine the former exciting molecules more efficiently
>>>and the latter doing more damage to the fluorophore.
>>>
>>>Thanks!
>>>Ben
>>>
>>>--
>>>View this message in context: http://confocal-microscopy-
>>list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRAP-
>>tp7540631.html
>>>Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

--
James and Christine Pawley, 21 N. Prospect Ave.
Madison, WI, 53726   Phone: [phone]
<[hidden email]>

[Timothy Gould]

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Hi Todd,
    Thanks for reply. We sent our Odyssey to VisiTech Int. after it kept
failing during acquisition ( it kept re-initialising  the Odyssey scan
module) . They couldn't fix it so I put it back together and now the
software/ hardware seems to stay functioning without falling over? Being
quite pleased with myself I decided to try realigning the laser, I'm a
complete novice with the inside of the odyssey and after getting a very
weak Blue 488 line coming out of the objective I thought my luck was in,
until my colleague pointed out that what I'm seeing might be the 0 or
first order beam ?
I'm a little perplexed now. It might be that I need an engineer to at
least go through the alignment once with me ?
Regarding you're Prairie idea, maybe you're right, because  having spent a
fortune on a Prairie 2-photon, perhaps they'll help a little. I was told
that some of  Norans' engineers went to Priarie ?
Kind regards, Tim



From:   [hidden email]
To:     [hidden email]
Date:   09/05/2012 16:27
Subject:        Re: Noran Odyssey xl laser alignment
Sent by:        Confocal Microscopy List
<[hidden email]>



*****
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*****

We have a Noran OZ that is still running. We recently put a Coherent
488 nm Sapphire on it to replace the Melles Ar, so I may be able to
help you. You can also contact Prairie Instruments. Depending on how
swamped they are, they might be able to offer some moral support.

Todd

-----------------------------------
Todd Clason
Manager, Neuroscience COBRE Imaging and Physiology Core Department of
Anatomy and Neurobiology College of Medicine, University of Vermont
E015 Given
89 Beaumont Ave.
Burlington, VT 05405
p. 802.656.0413
[hidden email]



-----Original Message-----
From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Timothy Gould
Sent: May 09, 2012 11:16 AM
To: [hidden email]
Subject: Re: Noran Odyssey xl laser alignment

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

Hello,
       Does anyone know how to align the laser on our Noran odyssey XL .
Guidance notes would help at least !
Regards, Tim


-----------------------------------
Todd Clason
Director, COBRE Imaging and Physiology Core
Department of Anatomy and Neurobiology
College of Medicine, University of Vermont
E015 Given
89 Beaumont Ave.
Burlington, VT 05405
p. 802.656.0413
[hidden email]

[Guy Cox-2]

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I've crossed swords with Jim on this several times, and it's clear that there have been FRAP experiments in which no collateral damage occurred, though I also agree that this has not been established in all published experiments.  But there's no need for this debate any more!   There's no need for FRAP any more!  If you work with photo-switchable proteins all these problems disappear.  No ROS, no damage.  End of story.

                                                   Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Thursday, 10 May 2012 1:22 PM
To: [hidden email]
Subject: Re: Ideal wavelength for bleaching step of FRAP

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

Hi Folks,

I know that FRAP is very popular and possibly even useful. But the story I am hearing here seems to be ignoring some important aspects of photo-bleaching.

If you are planning to spend a lot of "academic capital" on experiments that depend on FRAP I do recommend you take the time to read chapters 38, CELL DAMAGE DURING MULTI-PHOTON MICROSCOPY and 39 PHOTOBLEACHING.

The short version is that although some bleaching reactions may involve the absorption of a second (or third) photon by an already excited molecule, leading to its demise, (i.e., a simple two-component system where a photon or 3 are absorbed by a single molecule and the molecule ceases to be fluorescent) this is believed to be a rare event. More common is the situation where the (singlet- or triplet-) excited dye molecule passes its excess energy on to (usually) O2, creating a variety of reactive oxygen species (ROS). Reactions between this ROS and the dye create a modified dye molecule that is no longer fluorescent (or is not fluorescent at different wavelengths etc.)

The problem with this is that the ROS can move some distance (0.1-1 µm?) between the time it is created and the time (ms?) that it reacts, and in addition, there is no reason to assume that this second reaction will involve a dye molecule.
Once the ROS is formed, it can damage many of the molecules within its diffusion radius.

Well, this is getting a bit long but it is important to remember that the details of the bleaching event can depend strongly on the exact chemistry of the dye, and the probability of it occurring may not be proportional to dose
(intensity:  in photons/square µm x exposure
time) but may also vary non-linearly with intensity.

Cell type may be important, because some cells are better provided with molecules designed to mop up ROS (which are also produced by mitochondria), as will the stain concentration:
no stain implies only autofluorescence and so probably only very weak excitation and little bleaching, (unless there are a lot of pigments or naturally-fluorescent compounds (as are particularly common in plants).

But please remember

1. That you cannot assume that a fluorophor will be bleached ONLY because you hit it with light at it's excitation peak. The absorption of your "bleaching" light may bleach any dye in the vicinity at a rate depending on the propensity of the dye you do excite to make ROS and the sensitivity of any other dye to ROS attack.

2. The cell may not like the ROS any more than the dyes do and is likely to respond to this insult. Some folks say that if a cells bleaches, it must be damaged.

If this seems unnecessarily cautious, please have a look at the text surrounding Figs 49-12 and 49-13.

Sorry to complicate your story.

Cheers,

Jim Pawley

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>It's not always the case that the peak excitation wavelength gives the
>fastest bleaching.  I did some tests a number of years ago where we saw
>that we could get faster bleaching of YFP when excited with a CFP
>excitation filter than with a YFP excitation filter.  That said, I
>don't know how common this is, and I've not seen any systematic
>comparison of bleaching rates for different dyes at different
>wavelengths, so you'd probably have to test it yourself.
>
>Kurt
>
>On 5/9/2012 8:16 AM, Stanislav Vitha wrote:
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>Ben,
>>bleachin at wavelength of maximum absorption of the fluorophore will
>>give you the best damage to the fluorohore (that is what you want).
>>However, for essentially all fluorophores I can bleach  with the 405
>>nm laser on our Olympus FV1000  (either using the main scanner or the SIM scanner).
>>
>>Nevertheless, I think it is best to limit your bleaching to the
>>fluorophore of interest. The 405 nm laser is likely to
>>damage/bleach/photoconvert other cellular constituents besides
>>bleaching your GFP/YFP/mCherry.
>>
>>With some specimens, like plant chloroplasts, the 405 nm laser will
>>induce strong autofluorescence, so instead of nice dark spot where the
>>GFP was bleached, I get a giant bright spot.  That is not ideal for
>>FRAP :(
>>
>>Stan Vitha
>>Microscopy and Imaging Center
>>Texas A&M Universuty
>>
>>On Tue, 8 May 2012 13:42:28 -0700, Ben Abrams<[hidden email]>
>>wrote:
>>
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>Hi All,
>>>
>>>I'd love to hear what the consensus is on this.  If I was able to
>>>choose between different lasers with equal power for FRAP, would it
>>>be better to bleach with a wavelength that is ideal for excitation of
>>>the fluorophore being bleached, or is it better to bleach with a
>>>shorter more intense wavelength?  I can imagine the former exciting
>>>molecules more efficiently and the latter doing more damage to the fluorophore.
>>>
>>>Thanks!
>>>Ben
>>>
>>>--
>>>View this message in context: http://confocal-microscopy-
>>list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRAP-
>>tp7540631.html
>>>Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

--
James and Christine Pawley, 21 N. Prospect Ave.
Madison, WI, 53726   Phone: [phone]
<[hidden email]>

[Mark Cannell]

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

Assuming of course the expressed protein does not do anything to the cell and behaves exactly like the moiety of interest ... (and that's a huge skeleton in the closet)

Cheers.



On 10/05/2012, at 12:26 PM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I've crossed swords with Jim on this several times, and it's clear that there have been FRAP experiments in which no collateral damage occurred, though I also agree that this has not been established in all published experiments.  But there's no need for this debate any more!   There's no need for FRAP any more!  If you work with photo-switchable proteins all these problems disappear.  No ROS, no damage.  End of story.
>
>                                                   Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
> Sent: Thursday, 10 May 2012 1:22 PM
> To: [hidden email]
> Subject: Re: Ideal wavelength for bleaching step of FRAP
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Folks,
>
> I know that FRAP is very popular and possibly even useful. But the story I am hearing here seems to be ignoring some important aspects of photo-bleaching.
>
> If you are planning to spend a lot of "academic capital" on experiments that depend on FRAP I do recommend you take the time to read chapters 38, CELL DAMAGE DURING MULTI-PHOTON MICROSCOPY and 39 PHOTOBLEACHING.
>
> The short version is that although some bleaching reactions may involve the absorption of a second (or third) photon by an already excited molecule, leading to its demise, (i.e., a simple two-component system where a photon or 3 are absorbed by a single molecule and the molecule ceases to be fluorescent) this is believed to be a rare event. More common is the situation where the (singlet- or triplet-) excited dye molecule passes its excess energy on to (usually) O2, creating a variety of reactive oxygen species (ROS). Reactions between this ROS and the dye create a modified dye molecule that is no longer fluorescent (or is not fluorescent at different wavelengths etc.)
>
> The problem with this is that the ROS can move some distance (0.1-1 µm?) between the time it is created and the time (ms?) that it reacts, and in addition, there is no reason to assume that this second reaction will involve a dye molecule.
> Once the ROS is formed, it can damage many of the molecules within its diffusion radius.
>
> Well, this is getting a bit long but it is important to remember that the details of the bleaching event can depend strongly on the exact chemistry of the dye, and the probability of it occurring may not be proportional to dose
> (intensity:  in photons/square µm x exposure
> time) but may also vary non-linearly with intensity.
>
> Cell type may be important, because some cells are better provided with molecules designed to mop up ROS (which are also produced by mitochondria), as will the stain concentration:
> no stain implies only autofluorescence and so probably only very weak excitation and little bleaching, (unless there are a lot of pigments or naturally-fluorescent compounds (as are particularly common in plants).
>
> But please remember
>
> 1. That you cannot assume that a fluorophor will be bleached ONLY because you hit it with light at it's excitation peak. The absorption of your "bleaching" light may bleach any dye in the vicinity at a rate depending on the propensity of the dye you do excite to make ROS and the sensitivity of any other dye to ROS attack.
>
> 2. The cell may not like the ROS any more than the dyes do and is likely to respond to this insult. Some folks say that if a cells bleaches, it must be damaged.
>
> If this seems unnecessarily cautious, please have a look at the text surrounding Figs 49-12 and 49-13.
>
> Sorry to complicate your story.
>
> Cheers,
>
> Jim Pawley
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> It's not always the case that the peak excitation wavelength gives the
>> fastest bleaching.  I did some tests a number of years ago where we saw
>> that we could get faster bleaching of YFP when excited with a CFP
>> excitation filter than with a YFP excitation filter.  That said, I
>> don't know how common this is, and I've not seen any systematic
>> comparison of bleaching rates for different dyes at different
>> wavelengths, so you'd probably have to test it yourself.
>>
>> Kurt
>>
>> On 5/9/2012 8:16 AM, Stanislav Vitha wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Ben,
>>> bleachin at wavelength of maximum absorption of the fluorophore will
>>> give you the best damage to the fluorohore (that is what you want).
>>> However, for essentially all fluorophores I can bleach  with the 405
>>> nm laser on our Olympus FV1000  (either using the main scanner or the SIM scanner).
>>>
>>> Nevertheless, I think it is best to limit your bleaching to the
>>> fluorophore of interest. The 405 nm laser is likely to
>>> damage/bleach/photoconvert other cellular constituents besides
>>> bleaching your GFP/YFP/mCherry.
>>>
>>> With some specimens, like plant chloroplasts, the 405 nm laser will
>>> induce strong autofluorescence, so instead of nice dark spot where the
>>> GFP was bleached, I get a giant bright spot.  That is not ideal for
>>> FRAP :(
>>>
>>> Stan Vitha
>>> Microscopy and Imaging Center
>>> Texas A&M Universuty
>>>
>>> On Tue, 8 May 2012 13:42:28 -0700, Ben Abrams<[hidden email]>
>>> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi All,
>>>>
>>>> I'd love to hear what the consensus is on this.  If I was able to
>>>> choose between different lasers with equal power for FRAP, would it
>>>> be better to bleach with a wavelength that is ideal for excitation of
>>>> the fluorophore being bleached, or is it better to bleach with a
>>>> shorter more intense wavelength?  I can imagine the former exciting
>>>> molecules more efficiently and the latter doing more damage to the fluorophore.
>>>>
>>>> Thanks!
>>>> Ben
>>>>
>>>> --
>>>> View this message in context: http://confocal-microscopy-
>>> list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRAP-
>>> tp7540631.html
>>>> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
> --
> James and Christine Pawley, 21 N. Prospect Ave.
> Madison, WI, 53726   Phone: [phone]
> <[hidden email]>

[Timothy Gould]

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

HI Gregg,
          If you have the big book of Laser alignment and its foolproof
can you tell me how ( unless you're joking of course) I can get my hands
on one.  The Odyssey had an intermittent fault which prevented me doing a
Time series. Being so old It was going to be
too expensive to repair, so we got it back from Visitech ( who are very
friendly and helpful). After putting it
back  together we find, as if by magic, the software now runs okay, but of

course we need to align the laser. Funds are tight so we are trying to do
it ourselves.
Regards, Tim


Tim Gould
V.Crunelli Lab
Life Sciences Building
Bioscience 3
Cardiff University
Museum Avenue
Cardiff.
CF10 3AT
email: [hidden email]
Tel:[phone]



From:   Gregg Jarvis <[hidden email]>
To:     [hidden email]
Date:   09/05/2012 16:49
Subject:        Re: Noran Odyssey xl laser alignment
Sent by:        Confocal Microscopy List
<[hidden email]>



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

http://books.google.com/books?id=16Ft5k8RC-AC&pg=PA476&lpg=PA476&dq=Noran+odyssey+XL+laser&source=bl&ots=5XFABquCUt&sig=fu9L0Kuf5OxpblfIowX6VGSY8EQ&hl=en&sa=X&ei=Zo6qT_iGF4X6ggfQhZmqAQ&ved=0CE0Q6AEwAg#v=onepage&q=Noran%20odyssey%20XL%20laser&f=false

http://www.lplarson.com/Laser%20Alignment%20Services.html

looks like a service call unless you have the Big book of laser alignment
atyour disposal.
Why would it be out of alignment?
wear your safety glasses



On Wed, May 9, 2012 at 11:26 AM, <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> We have a Noran OZ that is still running. We recently put a Coherent 488
> nm Sapphire on it to replace the Melles Ar, so I may be able to help
you.

> You can also contact Prairie Instruments. Depending on how swamped they
> are, they might be able to offer some moral support.
>
> Todd
>
> ------------------------------**-----
> Todd Clason
> Manager, Neuroscience COBRE Imaging and Physiology Core Department of
> Anatomy and Neurobiology College of Medicine, University of Vermont
> E015 Given
> 89 Beaumont Ave.
> Burlington, VT 05405
> p. 802.656.0413
> [hidden email]
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [

mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU
<[hidden email]>]
> On Behalf Of Timothy Gould
> Sent: May 09, 2012 11:16 AM
> To: [hidden email].**EDU
<[hidden email]>
> Subject: Re: Noran Odyssey xl laser alignment
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>

> *****
>
> Hello,
>      Does anyone know how to align the laser on our Noran odyssey XL .
> Guidance notes would help at least !
> Regards, Tim
>
>
> ------------------------------**-----
> Todd Clason
> Director, COBRE Imaging and Physiology Core
> Department of Anatomy and Neurobiology
> College of Medicine, University of Vermont
> E015 Given
> 89 Beaumont Ave.
> Burlington, VT 05405
> p. 802.656.0413
> [hidden email]
>

--
Gregg Jarvis
Advanced Products Group
Omega Optical Inc.
24 Omega Drive
Brattleboro, VT 05301
[hidden email]
1-802-251-7316

[Gregg Jarvis]

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

Tim,
Can you get the cover off the instrument?
As long as you have power to the laser there should be some adjusment
screws that
will allow alignment. I don't understand how it could be so far out of
alignment unless it was dropped or something.
Is this Ebay ad what your laser looks like?

http://www.ebay.com/itm/Noran-Confocal-Laser-Scanning-Microscope-Odyssey-XL-/170664960822

Gregg
On Thu, May 10, 2012 at 10:51 AM, Timothy Gould <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> HI Gregg,
>          If you have the big book of Laser alignment and its foolproof
> can you tell me how ( unless you're joking of course) I can get my hands
> on one.  The Odyssey had an intermittent fault which prevented me doing a
> Time series. Being so old It was going to be
> too expensive to repair, so we got it back from Visitech ( who are very
> friendly and helpful). After putting it
> back  together we find, as if by magic, the software now runs okay, but of
>
> course we need to align the laser. Funds are tight so we are trying to do
> it ourselves.
> Regards, Tim
>
>
> Tim Gould
> V.Crunelli Lab
> Life Sciences Building
> Bioscience 3
> Cardiff University
> Museum Avenue
> Cardiff.
> CF10 3AT
> email: [hidden email]
> Tel:[phone]
>
>
>
> From:   Gregg Jarvis <[hidden email]>
> To:     [hidden email]
> Date:   09/05/2012 16:49
> Subject:        Re: Noran Odyssey xl laser alignment
> Sent by:        Confocal Microscopy List
> <[hidden email]>
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> http://books.google.com/books?id=16Ft5k8RC-AC&pg=PA476&lpg=PA476&dq=Noran+odyssey+XL+laser&source=bl&ots=5XFABquCUt&sig=fu9L0Kuf5OxpblfIowX6VGSY8EQ&hl=en&sa=X&ei=Zo6qT_iGF4X6ggfQhZmqAQ&ved=0CE0Q6AEwAg#v=onepage&q=Noran%20odyssey%20XL%20laser&f=false
>
> http://www.lplarson.com/Laser%20Alignment%20Services.html
>
> looks like a service call unless you have the Big book of laser alignment
> atyour disposal.
> Why would it be out of alignment?
> wear your safety glasses
>
>
>
> On Wed, May 9, 2012 at 11:26 AM, <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > *****
> >
> > We have a Noran OZ that is still running. We recently put a Coherent 488
> > nm Sapphire on it to replace the Melles Ar, so I may be able to help
> you.
> > You can also contact Prairie Instruments. Depending on how swamped they
> > are, they might be able to offer some moral support.
> >
> > Todd
> >
> > ------------------------------**-----
> > Todd Clason
> > Manager, Neuroscience COBRE Imaging and Physiology Core Department of
> > Anatomy and Neurobiology College of Medicine, University of Vermont
> > E015 Given
> > 89 Beaumont Ave.
> > Burlington, VT 05405
> > p. 802.656.0413
> > [hidden email]
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [
> mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU <http://lists.umn.edu/>
> <[hidden email]>]
> > On Behalf Of Timothy Gould
> > Sent: May 09, 2012 11:16 AM
> > To: [hidden email].**EDU
> <[hidden email]>
> > Subject: Re: Noran Odyssey xl laser alignment
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > *****
> >
> > Hello,
> >      Does anyone know how to align the laser on our Noran odyssey XL .
> > Guidance notes would help at least !
> > Regards, Tim
> >
> >
> > ------------------------------**-----
> > Todd Clason
> > Director, COBRE Imaging and Physiology Core
> > Department of Anatomy and Neurobiology
> > College of Medicine, University of Vermont
> > E015 Given
> > 89 Beaumont Ave.
> > Burlington, VT 05405
> > p. 802.656.0413
> > [hidden email]
> >
>
>
>
> --
> Gregg Jarvis
> Advanced Products Group
> Omega Optical Inc.
> 24 Omega Drive
> Brattleboro, VT 05301
> [hidden email]
> 1-802-251-7316
>

--
Gregg Jarvis
Advanced Products Group
Omega Optical Inc.
24 Omega Drive
Brattleboro, VT 05301
[hidden email]
1-802-251-7316

[Guy Cox-2]

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

Yes, of course and it's obviously not, in the end, true.  There's always going to be some interference.  But then people inject phalloidin into living cells and expect to see normal actin behaviour!   In the case of FRAP vs PC that aspect is unchanged, but the chemical environment is much gentler.  One is at least minimising the interference.  

                                                                           Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell
Sent: Thursday, 10 May 2012 11:55 PM
To: [hidden email]
Subject: Re: Ideal wavelength for bleaching step of FRAP

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

Assuming of course the expressed protein does not do anything to the cell and behaves exactly like the moiety of interest ... (and that's a huge skeleton in the closet)

Cheers.



On 10/05/2012, at 12:26 PM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I've crossed swords with Jim on this several times, and it's clear that there have been FRAP experiments in which no collateral damage occurred, though I also agree that this has not been established in all published experiments.  But there's no need for this debate any more!   There's no need for FRAP any more!  If you work with photo-switchable proteins all these problems disappear.  No ROS, no damage.  End of story.
>
>                                                   Guy
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of James Pawley
> Sent: Thursday, 10 May 2012 1:22 PM
> To: [hidden email]
> Subject: Re: Ideal wavelength for bleaching step of FRAP
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Folks,
>
> I know that FRAP is very popular and possibly even useful. But the story I am hearing here seems to be ignoring some important aspects of photo-bleaching.
>
> If you are planning to spend a lot of "academic capital" on experiments that depend on FRAP I do recommend you take the time to read chapters 38, CELL DAMAGE DURING MULTI-PHOTON MICROSCOPY and 39 PHOTOBLEACHING.
>
> The short version is that although some bleaching reactions may
> involve the absorption of a second (or third) photon by an already
> excited molecule, leading to its demise, (i.e., a simple two-component
> system where a photon or 3 are absorbed by a single molecule and the
> molecule ceases to be fluorescent) this is believed to be a rare
> event. More common is the situation where the (singlet- or triplet-)
> excited dye molecule passes its excess energy on to (usually) O2,
> creating a variety of reactive oxygen species (ROS). Reactions between
> this ROS and the dye create a modified dye molecule that is no longer
> fluorescent (or is not fluorescent at different wavelengths etc.)
>
> The problem with this is that the ROS can move some distance (0.1-1 µm?) between the time it is created and the time (ms?) that it reacts, and in addition, there is no reason to assume that this second reaction will involve a dye molecule.
> Once the ROS is formed, it can damage many of the molecules within its diffusion radius.
>
> Well, this is getting a bit long but it is important to remember that
> the details of the bleaching event can depend strongly on the exact
> chemistry of the dye, and the probability of it occurring may not be
> proportional to dose
> (intensity:  in photons/square µm x exposure
> time) but may also vary non-linearly with intensity.
>
> Cell type may be important, because some cells are better provided with molecules designed to mop up ROS (which are also produced by mitochondria), as will the stain concentration:
> no stain implies only autofluorescence and so probably only very weak excitation and little bleaching, (unless there are a lot of pigments or naturally-fluorescent compounds (as are particularly common in plants).
>
> But please remember
>
> 1. That you cannot assume that a fluorophor will be bleached ONLY because you hit it with light at it's excitation peak. The absorption of your "bleaching" light may bleach any dye in the vicinity at a rate depending on the propensity of the dye you do excite to make ROS and the sensitivity of any other dye to ROS attack.
>
> 2. The cell may not like the ROS any more than the dyes do and is likely to respond to this insult. Some folks say that if a cells bleaches, it must be damaged.
>
> If this seems unnecessarily cautious, please have a look at the text surrounding Figs 49-12 and 49-13.
>
> Sorry to complicate your story.
>
> Cheers,
>
> Jim Pawley
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> It's not always the case that the peak excitation wavelength gives
>> the fastest bleaching.  I did some tests a number of years ago where
>> we saw that we could get faster bleaching of YFP when excited with a
>> CFP excitation filter than with a YFP excitation filter.  That said,
>> I don't know how common this is, and I've not seen any systematic
>> comparison of bleaching rates for different dyes at different
>> wavelengths, so you'd probably have to test it yourself.
>>
>> Kurt
>>
>> On 5/9/2012 8:16 AM, Stanislav Vitha wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Ben,
>>> bleachin at wavelength of maximum absorption of the fluorophore will
>>> give you the best damage to the fluorohore (that is what you want).
>>> However, for essentially all fluorophores I can bleach  with the 405
>>> nm laser on our Olympus FV1000  (either using the main scanner or the SIM scanner).
>>>
>>> Nevertheless, I think it is best to limit your bleaching to the
>>> fluorophore of interest. The 405 nm laser is likely to
>>> damage/bleach/photoconvert other cellular constituents besides
>>> bleaching your GFP/YFP/mCherry.
>>>
>>> With some specimens, like plant chloroplasts, the 405 nm laser will
>>> induce strong autofluorescence, so instead of nice dark spot where
>>> the GFP was bleached, I get a giant bright spot.  That is not ideal
>>> for FRAP :(
>>>
>>> Stan Vitha
>>> Microscopy and Imaging Center
>>> Texas A&M Universuty
>>>
>>> On Tue, 8 May 2012 13:42:28 -0700, Ben Abrams<[hidden email]>
>>> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi All,
>>>>
>>>> I'd love to hear what the consensus is on this.  If I was able to
>>>> choose between different lasers with equal power for FRAP, would it
>>>> be better to bleach with a wavelength that is ideal for excitation
>>>> of the fluorophore being bleached, or is it better to bleach with a
>>>> shorter more intense wavelength?  I can imagine the former exciting
>>>> molecules more efficiently and the latter doing more damage to the fluorophore.
>>>>
>>>> Thanks!
>>>> Ben
>>>>
>>>> --
>>>> View this message in context: http://confocal-microscopy-
>>> list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRA
>>> P-
>>> tp7540631.html
>>>> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
> --
> James and Christine Pawley, 21 N. Prospect Ave.
> Madison, WI, 53726   Phone: [phone]
> <[hidden email]>

[Timothy Gould]

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

Hi Gregg,
        Yes our machine is the same as the ebay one and yes I'm tweaking
the laser inside the scan module and now have a faint blue spot exiting
the objective. I've still some way to go and presently concerned about
diffractions. As it's my first time I'm a little in the dark so to speak,
but slowly getting there. The laser misalignment is due to the kit being
dismantled and the scan head being completely separated from it's original
position. It seems that minor movements get exaggerated when dealing with
light .
Regards, Tim


Tim Gould
V.Crunelli Lab
Life Sciences Building
Bioscience 3
Cardiff University
Museum Avenue
Cardiff.
CF10 3AT
email: [hidden email]
Tel:[phone]



From:   Gregg Jarvis <[hidden email]>
To:     [hidden email]
Date:   10/05/2012 17:23
Subject:        Re: Noran Odyssey xl laser alignment
Sent by:        Confocal Microscopy List
<[hidden email]>



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

Tim,
Can you get the cover off the instrument?
As long as you have power to the laser there should be some adjusment
screws that
will allow alignment. I don't understand how it could be so far out of
alignment unless it was dropped or something.
Is this Ebay ad what your laser looks like?

http://www.ebay.com/itm/Noran-Confocal-Laser-Scanning-Microscope-Odyssey-XL-/170664960822


Gregg
On Thu, May 10, 2012 at 10:51 AM, Timothy Gould
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> HI Gregg,
>          If you have the big book of Laser alignment and its foolproof
> can you tell me how ( unless you're joking of course) I can get my hands
> on one.  The Odyssey had an intermittent fault which prevented me doing
a
> Time series. Being so old It was going to be
> too expensive to repair, so we got it back from Visitech ( who are very
> friendly and helpful). After putting it
> back  together we find, as if by magic, the software now runs okay, but
of
>
> course we need to align the laser. Funds are tight so we are trying to
do

> it ourselves.
> Regards, Tim
>
>
> Tim Gould
> V.Crunelli Lab
> Life Sciences Building
> Bioscience 3
> Cardiff University
> Museum Avenue
> Cardiff.
> CF10 3AT
> email: [hidden email]
> Tel:[phone]
>
>
>
> From:   Gregg Jarvis <[hidden email]>
> To:     [hidden email]
> Date:   09/05/2012 16:49
> Subject:        Re: Noran Odyssey xl laser alignment
> Sent by:        Confocal Microscopy List
> <[hidden email]>
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
>

http://books.google.com/books?id=16Ft5k8RC-AC&pg=PA476&lpg=PA476&dq=Noran+odyssey+XL+laser&source=bl&ots=5XFABquCUt&sig=fu9L0Kuf5OxpblfIowX6VGSY8EQ&hl=en&sa=X&ei=Zo6qT_iGF4X6ggfQhZmqAQ&ved=0CE0Q6AEwAg#v=onepage&q=Noran%20odyssey%20XL%20laser&f=false

>
> http://www.lplarson.com/Laser%20Alignment%20Services.html
>
> looks like a service call unless you have the Big book of laser
alignment

> atyour disposal.
> Why would it be out of alignment?
> wear your safety glasses
>
>
>
> On Wed, May 9, 2012 at 11:26 AM, <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > *****
> >
> > We have a Noran OZ that is still running. We recently put a Coherent

488
> > nm Sapphire on it to replace the Melles Ar, so I may be able to help
> you.
> > You can also contact Prairie Instruments. Depending on how swamped
they

> > are, they might be able to offer some moral support.
> >
> > Todd
> >
> > ------------------------------**-----
> > Todd Clason
> > Manager, Neuroscience COBRE Imaging and Physiology Core Department of
> > Anatomy and Neurobiology College of Medicine, University of Vermont
> > E015 Given
> > 89 Beaumont Ave.
> > Burlington, VT 05405
> > p. 802.656.0413
> > [hidden email]
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [
> mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU <http://lists.umn.edu/>
> <[hidden email]>]
> > On Behalf Of Timothy Gould
> > Sent: May 09, 2012 11:16 AM
> > To: [hidden email].**EDU
> <[hidden email]>
> > Subject: Re: Noran Odyssey xl laser alignment
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > *****
> >
> > Hello,
> >      Does anyone know how to align the laser on our Noran odyssey XL .
> > Guidance notes would help at least !
> > Regards, Tim
> >
> >
> > ------------------------------**-----
> > Todd Clason
> > Director, COBRE Imaging and Physiology Core
> > Department of Anatomy and Neurobiology
> > College of Medicine, University of Vermont
> > E015 Given
> > 89 Beaumont Ave.
> > Burlington, VT 05405
> > p. 802.656.0413
> > [hidden email]
> >
>
>
>
> --
> Gregg Jarvis
> Advanced Products Group
> Omega Optical Inc.
> 24 Omega Drive
> Brattleboro, VT 05301
> [hidden email]
> 1-802-251-7316
>

--
Gregg Jarvis
Advanced Products Group
Omega Optical Inc.
24 Omega Drive
Brattleboro, VT 05301
[hidden email]
1-802-251-7316

[Gregg Jarvis]

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

Tim,
keep going!
Is the laser at full power?
Alignment is critical and you are correct that all the reflective surfaces
need to be lined up and focused at the detector end.
Start at one end and follow the path of the laser. Minor adjustments at one
end will have greater effect at the other end depending on how much the
beam may spread out, so go in small steps and just remember how many turns
you go on screws.
I find that putting a piece of tape on the tool you are using, so you can
count revolutions and drawing a diagram as you go will aid in ease of
alignment.
keep me posted.
Focus and patience,
Gregg

On Fri, May 11, 2012 at 9:26 AM, Timothy Gould <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Gregg,
>        Yes our machine is the same as the ebay one and yes I'm tweaking
> the laser inside the scan module and now have a faint blue spot exiting
> the objective. I've still some way to go and presently concerned about
> diffractions. As it's my first time I'm a little in the dark so to speak,
> but slowly getting there. The laser misalignment is due to the kit being
> dismantled and the scan head being completely separated from it's original
> position. It seems that minor movements get exaggerated when dealing with
> light .
> Regards, Tim
>
>
> Tim Gould
> V.Crunelli Lab
> Life Sciences Building
> Bioscience 3
> Cardiff University
> Museum Avenue
> Cardiff.
> CF10 3AT
> email: [hidden email]
> Tel:[phone]
>
>
>
> From:   Gregg Jarvis <[hidden email]>
> To:     [hidden email]
> Date:   10/05/2012 17:23
> Subject:        Re: Noran Odyssey xl laser alignment
> Sent by:        Confocal Microscopy List
> <[hidden email]>
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Tim,
> Can you get the cover off the instrument?
> As long as you have power to the laser there should be some adjusment
> screws that
> will allow alignment. I don't understand how it could be so far out of
> alignment unless it was dropped or something.
> Is this Ebay ad what your laser looks like?
>
>
> http://www.ebay.com/itm/Noran-Confocal-Laser-Scanning-Microscope-Odyssey-XL-/170664960822
>
>
> Gregg
> On Thu, May 10, 2012 at 10:51 AM, Timothy Gould
> <[hidden email]>wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > HI Gregg,
> >          If you have the big book of Laser alignment and its foolproof
> > can you tell me how ( unless you're joking of course) I can get my hands
> > on one.  The Odyssey had an intermittent fault which prevented me doing
> a
> > Time series. Being so old It was going to be
> > too expensive to repair, so we got it back from Visitech ( who are very
> > friendly and helpful). After putting it
> > back  together we find, as if by magic, the software now runs okay, but
> of
> >
> > course we need to align the laser. Funds are tight so we are trying to
> do
> > it ourselves.
> > Regards, Tim
> >
> >
> > Tim Gould
> > V.Crunelli Lab
> > Life Sciences Building
> > Bioscience 3
> > Cardiff University
> > Museum Avenue
> > Cardiff.
> > CF10 3AT
> > email: [hidden email]
> > Tel:[phone]
> >
> >
> >
> > From:   Gregg Jarvis <[hidden email]>
> > To:     [hidden email]
> > Date:   09/05/2012 16:49
> > Subject:        Re: Noran Odyssey xl laser alignment
> > Sent by:        Confocal Microscopy List
> > <[hidden email]>
> >
> >
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> >
> >
>
> http://books.google.com/books?id=16Ft5k8RC-AC&pg=PA476&lpg=PA476&dq=Noran+odyssey+XL+laser&source=bl&ots=5XFABquCUt&sig=fu9L0Kuf5OxpblfIowX6VGSY8EQ&hl=en&sa=X&ei=Zo6qT_iGF4X6ggfQhZmqAQ&ved=0CE0Q6AEwAg#v=onepage&q=Noran%20odyssey%20XL%20laser&f=false
>
> >
> > http://www.lplarson.com/Laser%20Alignment%20Services.html
> >
> > looks like a service call unless you have the Big book of laser
> alignment
> > atyour disposal.
> > Why would it be out of alignment?
> > wear your safety glasses
> >
> >
> >
> > On Wed, May 9, 2012 at 11:26 AM, <[hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > > *****
> > >
> > > We have a Noran OZ that is still running. We recently put a Coherent
> 488
> > > nm Sapphire on it to replace the Melles Ar, so I may be able to help
> > you.
> > > You can also contact Prairie Instruments. Depending on how swamped
> they
> > > are, they might be able to offer some moral support.
> > >
> > > Todd
> > >
> > > ------------------------------**-----
> > > Todd Clason
> > > Manager, Neuroscience COBRE Imaging and Physiology Core Department of
> > > Anatomy and Neurobiology College of Medicine, University of Vermont
> > > E015 Given
> > > 89 Beaumont Ave.
> > > Burlington, VT 05405
> > > p. 802.656.0413
> > > [hidden email]
> > >
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List [
> > mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU <http://lists.umn.edu/> <
> http://lists.umn.edu/>
> > <[hidden email]>]
> > > On Behalf Of Timothy Gould
> > > Sent: May 09, 2012 11:16 AM
> > > To: [hidden email].**EDU
> > <[hidden email]>
> > > Subject: Re: Noran Odyssey xl laser alignment
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > > *****
> > >
> > > Hello,
> > >      Does anyone know how to align the laser on our Noran odyssey XL .
> > > Guidance notes would help at least !
> > > Regards, Tim
> > >
> > >
> > > ------------------------------**-----
> > > Todd Clason
> > > Director, COBRE Imaging and Physiology Core
> > > Department of Anatomy and Neurobiology
> > > College of Medicine, University of Vermont
> > > E015 Given
> > > 89 Beaumont Ave.
> > > Burlington, VT 05405
> > > p. 802.656.0413
> > > [hidden email]
> > >
> >
> >
> >
> > --
> > Gregg Jarvis
> > Advanced Products Group
> > Omega Optical Inc.
> > 24 Omega Drive
> > Brattleboro, VT 05301
> > [hidden email]
> > 1-802-251-7316
> >
>
>
>
> --
> Gregg Jarvis
> Advanced Products Group
> Omega Optical Inc.
> 24 Omega Drive
> Brattleboro, VT 05301
> [hidden email]
> 1-802-251-7316
>

--
Gregg Jarvis
Advanced Products Group
Omega Optical Inc.
24 Omega Drive
Brattleboro, VT 05301
[hidden email]
1-802-251-7316

[Fabiana Caetano]

Sent on the TELUS Mobility network with BlackBerry

-----Original Message-----
From: Guy Cox <[hidden email]>
Sender: Confocal Microscopy List <[hidden email]>
Date:         Fri, 11 May 2012 05:51:20
To: <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Subject: Re: Ideal wavelength for bleaching step of FRAP

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

Yes, of course and it's obviously not, in the end, true.  There's always going to be some interference.  But then people inject phalloidin into living cells and expect to see normal actin behaviour!   In the case of FRAP vs PC that aspect is unchanged, but the chemical environment is much gentler.  One is at least minimising the interference.  

                                                                           Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell
Sent: Thursday, 10 May 2012 11:55 PM
To: [hidden email]
Subject: Re: Ideal wavelength for bleaching step of FRAP

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

Assuming of course the expressed protein does not do anything to the cell and behaves exactly like the moiety of interest ... (and that's a huge skeleton in the closet)

Cheers.



On 10/05/2012, at 12:26 PM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I've crossed swords with Jim on this several times, and it's clear that there have been FRAP experiments in which no collateral damage occurred, though I also agree that this has not been established in all published experiments.  But there's no need for this debate any more!   There's no need for FRAP any more!  If you work with photo-switchable proteins all these problems disappear.  No ROS, no damage.  End of story.
>
>                                                   Guy
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of James Pawley
> Sent: Thursday, 10 May 2012 1:22 PM
> To: [hidden email]
> Subject: Re: Ideal wavelength for bleaching step of FRAP
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Folks,
>
> I know that FRAP is very popular and possibly even useful. But the story I am hearing here seems to be ignoring some important aspects of photo-bleaching.
>
> If you are planning to spend a lot of "academic capital" on experiments that depend on FRAP I do recommend you take the time to read chapters 38, CELL DAMAGE DURING MULTI-PHOTON MICROSCOPY and 39 PHOTOBLEACHING.
>
> The short version is that although some bleaching reactions may
> involve the absorption of a second (or third) photon by an already
> excited molecule, leading to its demise, (i.e., a simple two-component
> system where a photon or 3 are absorbed by a single molecule and the
> molecule ceases to be fluorescent) this is believed to be a rare
> event. More common is the situation where the (singlet- or triplet-)
> excited dye molecule passes its excess energy on to (usually) O2,
> creating a variety of reactive oxygen species (ROS). Reactions between
> this ROS and the dye create a modified dye molecule that is no longer
> fluorescent (or is not fluorescent at different wavelengths etc.)
>
> The problem with this is that the ROS can move some distance (0.1-1 µm?) between the time it is created and the time (ms?) that it reacts, and in addition, there is no reason to assume that this second reaction will involve a dye molecule.
> Once the ROS is formed, it can damage many of the molecules within its diffusion radius.
>
> Well, this is getting a bit long but it is important to remember that
> the details of the bleaching event can depend strongly on the exact
> chemistry of the dye, and the probability of it occurring may not be
> proportional to dose
> (intensity:  in photons/square µm x exposure
> time) but may also vary non-linearly with intensity.
>
> Cell type may be important, because some cells are better provided with molecules designed to mop up ROS (which are also produced by mitochondria), as will the stain concentration:
> no stain implies only autofluorescence and so probably only very weak excitation and little bleaching, (unless there are a lot of pigments or naturally-fluorescent compounds (as are particularly common in plants).
>
> But please remember
>
> 1. That you cannot assume that a fluorophor will be bleached ONLY because you hit it with light at it's excitation peak. The absorption of your "bleaching" light may bleach any dye in the vicinity at a rate depending on the propensity of the dye you do excite to make ROS and the sensitivity of any other dye to ROS attack.
>
> 2. The cell may not like the ROS any more than the dyes do and is likely to respond to this insult. Some folks say that if a cells bleaches, it must be damaged.
>
> If this seems unnecessarily cautious, please have a look at the text surrounding Figs 49-12 and 49-13.
>
> Sorry to complicate your story.
>
> Cheers,
>
> Jim Pawley
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> It's not always the case that the peak excitation wavelength gives
>> the fastest bleaching.  I did some tests a number of years ago where
>> we saw that we could get faster bleaching of YFP when excited with a
>> CFP excitation filter than with a YFP excitation filter.  That said,
>> I don't know how common this is, and I've not seen any systematic
>> comparison of bleaching rates for different dyes at different
>> wavelengths, so you'd probably have to test it yourself.
>>
>> Kurt
>>
>> On 5/9/2012 8:16 AM, Stanislav Vitha wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Ben,
>>> bleachin at wavelength of maximum absorption of the fluorophore will
>>> give you the best damage to the fluorohore (that is what you want).
>>> However, for essentially all fluorophores I can bleach  with the 405
>>> nm laser on our Olympus FV1000  (either using the main scanner or the SIM scanner).
>>>
>>> Nevertheless, I think it is best to limit your bleaching to the
>>> fluorophore of interest. The 405 nm laser is likely to
>>> damage/bleach/photoconvert other cellular constituents besides
>>> bleaching your GFP/YFP/mCherry.
>>>
>>> With some specimens, like plant chloroplasts, the 405 nm laser will
>>> induce strong autofluorescence, so instead of nice dark spot where
>>> the GFP was bleached, I get a giant bright spot.  That is not ideal
>>> for FRAP :(
>>>
>>> Stan Vitha
>>> Microscopy and Imaging Center
>>> Texas A&M Universuty
>>>
>>> On Tue, 8 May 2012 13:42:28 -0700, Ben Abrams<[hidden email]>
>>> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi All,
>>>>
>>>> I'd love to hear what the consensus is on this.  If I was able to
>>>> choose between different lasers with equal power for FRAP, would it
>>>> be better to bleach with a wavelength that is ideal for excitation
>>>> of the fluorophore being bleached, or is it better to bleach with a
>>>> shorter more intense wavelength?  I can imagine the former exciting
>>>> molecules more efficiently and the latter doing more damage to the fluorophore.
>>>>
>>>> Thanks!
>>>> Ben
>>>>
>>>> --
>>>> View this message in context: http://confocal-microscopy-
>>> list.588098.n2.nabble.com/Ideal-wavelength-for-bleaching-step-of-FRA
>>> P-
>>> tp7540631.html
>>>> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
> --
> James and Christine Pawley, 21 N. Prospect Ave.
> Madison, WI, 53726   Phone: [phone]
> <[hidden email]>

[Ben Abrams]

Thank you to everyone who has responded to this post.  Your replies have been informative and I really appreciate them!

-Ben