Considerations on WLL / AOBS system

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Ralf Palmisano Ralf Palmisano
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Considerations on WLL / AOBS system

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Dear community,

I am kindly asking for feedback and sharing of expertise.

  * What is the difference comparing a white light laser & AOBS system
    to a classical gas laser & AOBS set-up in maintenance and service costs.
  * I would love to get feedback on experience about the reliability of
    a WLL against a classical approach in a multi-user high performance
    environment.
  * Any knowledge of a true average life-time in the wild of a WLL (~
    2.000 usage hours / year).
  * Is there any particular fact one should be aware of?
  * The system would mainly be used with fixed samples (2D & 3D) and
    normally 3 to 4 channels.
  * Has anyone already experience with far red / near IR dyes on such
    system?

You can also send me a pm. Thank you for your time and eventual support.

Best Ralph

--
Ralph Palmisano
Head - Optical Imaging Centre Erlangen

Fellow Royal Microscopical Society
Member Royal Society of Medicine

Speaker Scientific Advisory Board "German BioImaging"
Board of Directors Core Technologies for Life Science

Cauerstr. 3
91058 Erlangen, Germany

+49-9131-85-70320 (Office)
+49-9131-85-70321 (Secretary)

www.oice.uni-erlangen.de
Jonkman, James Jonkman, James
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Re: [External] Considerations on WLL / AOBS system

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Hi, Ralph.  We got two Leica SP8s at the same time about 3 years ago: one that's more basic with CW lasers, and the other with WLL, STED, and FLIM.  I don't yet know the difference in maintenance costs as they are all still under warranty.  So far the WLL has been very reliable, but it's main advantage for us is that it allows for FLIM and gated STED.

Personally, I wouldn't get it just for regular confocal microscopy for several reasons:
- You still need a 405nm laser, so probably 405nm laser + WLL is more expensive than 4 CW lasers (though I'm not certain).
- I share your concern about the cost of service contract or repair when it comes to that: I hope somebody else can reply with knowledge of this.
- The average power of the WLL at 488nm is about 350uW, compared to 5mW for the 488 solid state laser on our other SP8.  This makes it pretty poor for FRAP, and even slow for acceptor photobleaching to confirm FRET.
- The AOBS isn't perfect.  You have to move the spectral gate quite far away from the laser line to avoid capturing reflected light, which lowers your efficiency for light collection.  Unless you choose one of the special laser lines that has an associated laser line filter (but then you are back to using discrete wavelengths); or you use gating to throw away the very fastest signal (but then you need special detectors).  I would be curious to see the overall efficiency of the AOBS: I know that AOTFs are not 100% transmissive, but with excitation it isn't really a problem to just throw a fair bit of it away.
- I like the flexibility of the WLL, but in our hands it has not proven to be crucial for new probes or anything like that.  There is not really an advantage to hitting the excitation wavelength exactly on the peak.  It's critical to capture as much emission as possible, which usually means you should excite a little to the "left" of the peak.  The photons that don't absorb don't really do any harm - in thinner samples especially, they either absorb or they pass through the sample without interacting and therefore they do no harm.  (I suppose in thicker samples they could also scatter, and then they may absorb elsewhere).

The WLL works well for far red dyes (AF647 for example).

Finally, I saw with envy that the specs on the WLL with their new Stellaris model go from 440nm to 850nm (whereas mine is 470 to 670nm).  This is a huge improvement!  We have to use a separate pulsed 440nm laser for CFP on our instrument (still used by some people for FLIM), which is not terribly well integrated (manual power dial for example), but the new WLL will eliminate that and give you more options of probes in the NIR as well (when combined with their new HyD detectors).  I'm not sure if this exactly counts "Confocal Re-imagined " as their recent slick marketing video claims, but it is definitely a welcome improvement!

Cheers,
James
-----------------------------------------------
   James Jonkman, Staff Scientist
   Advanced Optical Microscopy Facility (AOMF)
   and Wright Cell Imaging Facility (WCIF)
   University Health Network
   MaRS, PMCRT tower, 101 College St., Room 15-305
   Toronto, ON, CANADA    M5G 1L7
  [hidden email]  Tel: 416-581-8593
   www.aomf.ca




-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Ralf Palmisano
Sent: Wednesday, April 22, 2020 12:06 PM
To: [hidden email]
Subject: [External] Considerations on WLL / AOBS system

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

Dear community,

I am kindly asking for feedback and sharing of expertise.

  * What is the difference comparing a white light laser & AOBS system
    to a classical gas laser & AOBS set-up in maintenance and service costs.
  * I would love to get feedback on experience about the reliability of
    a WLL against a classical approach in a multi-user high performance
    environment.
  * Any knowledge of a true average life-time in the wild of a WLL (~
    2.000 usage hours / year).
  * Is there any particular fact one should be aware of?
  * The system would mainly be used with fixed samples (2D & 3D) and
    normally 3 to 4 channels.
  * Has anyone already experience with far red / near IR dyes on such
    system?

You can also send me a pm. Thank you for your time and eventual support.

Best Ralph

--
Ralph Palmisano
Head - Optical Imaging Centre Erlangen

Fellow Royal Microscopical Society
Member Royal Society of Medicine

Speaker Scientific Advisory Board "German BioImaging"
Board of Directors Core Technologies for Life Science

Cauerstr. 3
91058 Erlangen, Germany

+49-9131-85-70320 (Office)
+49-9131-85-70321 (Secretary)

www.oice.uni-erlangen.de


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Zdenek Svindrych-2 Zdenek Svindrych-2
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Re: [External] Considerations on WLL / AOBS system

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

Hi James,

I will just comment to the AOBS efficiency: without any electrical signal
the AOBS behaves just like two "glass" crystals (AR coated, of course) for
the returning fluorescence signal, so very little of the precious signal is
lost. With electrical-piezo excitation only smart part of the emission
spectrum is lost, it's exactly the few nanometers that correspond to the
excitation wavelength +/- bandwidth. So to make the AOTF analogy, the laser
light is diffracted into the excitation path with less-than-ideal
efficiency (and polarization sensitive as well), but the emission
throughput is essentially perfect.

Also, Leica uses (or at least used to use) the same principle to combine
discrete lasers with the WLL inside the WLL box. This is the Leica combiner
inside one of the early generation NKT WLLs...
https://drive.google.com/file/d/0B5vWyBYrDvcJWmZTSkpCZ0xqVnM/view?usp=sharing

A side note, I also responded to Ralph's questions, but by mistake it went
out as a personal message and not to the confolist. I won't copy it here
again, as my comments were identical to those of James.

Best, zdenek

On Thu, Apr 23, 2020 at 3:03 PM Jonkman, James <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi, Ralph.  We got two Leica SP8s at the same time about 3 years ago: one
> that's more basic with CW lasers, and the other with WLL, STED, and FLIM.
> I don't yet know the difference in maintenance costs as they are all still
> under warranty.  So far the WLL has been very reliable, but it's main
> advantage for us is that it allows for FLIM and gated STED.
>
> Personally, I wouldn't get it just for regular confocal microscopy for
> several reasons:
> - You still need a 405nm laser, so probably 405nm laser + WLL is more
> expensive than 4 CW lasers (though I'm not certain).
> - I share your concern about the cost of service contract or repair when
> it comes to that: I hope somebody else can reply with knowledge of this.
> - The average power of the WLL at 488nm is about 350uW, compared to 5mW
> for the 488 solid state laser on our other SP8.  This makes it pretty poor
> for FRAP, and even slow for acceptor photobleaching to confirm FRET.
> - The AOBS isn't perfect.  You have to move the spectral gate quite far
> away from the laser line to avoid capturing reflected light, which lowers
> your efficiency for light collection.  Unless you choose one of the special
> laser lines that has an associated laser line filter (but then you are back
> to using discrete wavelengths); or you use gating to throw away the very
> fastest signal (but then you need special detectors).  I would be curious
> to see the overall efficiency of the AOBS: I know that AOTFs are not 100%
> transmissive, but with excitation it isn't really a problem to just throw a
> fair bit of it away.
> - I like the flexibility of the WLL, but in our hands it has not proven to
> be crucial for new probes or anything like that.  There is not really an
> advantage to hitting the excitation wavelength exactly on the peak.  It's
> critical to capture as much emission as possible, which usually means you
> should excite a little to the "left" of the peak.  The photons that don't
> absorb don't really do any harm - in thinner samples especially, they
> either absorb or they pass through the sample without interacting and
> therefore they do no harm.  (I suppose in thicker samples they could also
> scatter, and then they may absorb elsewhere).
>
> The WLL works well for far red dyes (AF647 for example).
>
> Finally, I saw with envy that the specs on the WLL with their new
> Stellaris model go from 440nm to 850nm (whereas mine is 470 to 670nm).
> This is a huge improvement!  We have to use a separate pulsed 440nm laser
> for CFP on our instrument (still used by some people for FLIM), which is
> not terribly well integrated (manual power dial for example), but the new
> WLL will eliminate that and give you more options of probes in the NIR as
> well (when combined with their new HyD detectors).  I'm not sure if this
> exactly counts "Confocal Re-imagined " as their recent slick marketing
> video claims, but it is definitely a welcome improvement!
>
> Cheers,
> James
> -----------------------------------------------
>    James Jonkman, Staff Scientist
>    Advanced Optical Microscopy Facility (AOMF)
>    and Wright Cell Imaging Facility (WCIF)
>    University Health Network
>    MaRS, PMCRT tower, 101 College St., Room 15-305
>    Toronto, ON, CANADA    M5G 1L7
>   [hidden email]  Tel: 416-581-8593
>    www.aomf.ca
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Ralf Palmisano
> Sent: Wednesday, April 22, 2020 12:06 PM
> To: [hidden email]
> Subject: [External] Considerations on WLL / AOBS system
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear community,
>
> I am kindly asking for feedback and sharing of expertise.
>
>   * What is the difference comparing a white light laser & AOBS system
>     to a classical gas laser & AOBS set-up in maintenance and service
> costs.
>   * I would love to get feedback on experience about the reliability of
>     a WLL against a classical approach in a multi-user high performance
>     environment.
>   * Any knowledge of a true average life-time in the wild of a WLL (~
>     2.000 usage hours / year).
>   * Is there any particular fact one should be aware of?
>   * The system would mainly be used with fixed samples (2D & 3D) and
>     normally 3 to 4 channels.
>   * Has anyone already experience with far red / near IR dyes on such
>     system?
>
> You can also send me a pm. Thank you for your time and eventual support.
>
> Best Ralph
>
> --
> Ralph Palmisano
> Head - Optical Imaging Centre Erlangen
>
> Fellow Royal Microscopical Society
> Member Royal Society of Medicine
>
> Speaker Scientific Advisory Board "German BioImaging"
> Board of Directors Core Technologies for Life Science
>
> Cauerstr. 3
> 91058 Erlangen, Germany
>
> +49-9131-85-70320 (Office)
> +49-9131-85-70321 (Secretary)
>
> www.oice.uni-erlangen.de
>
>
> This e-mail may contain confidential and/or privileged information for the
> sole use of the intended recipient.
> Any review or distribution by anyone other than the person for whom it was
> originally intended is strictly prohibited.
> If you have received this e-mail in error, please contact the sender and
> delete all copies.
> Opinions, conclusions or other information contained in this e-mail may
> not be that of the organization.
>
> If you feel you have received an email from UHN of a commercial nature and
> would like to be removed from the sender's mailing list please do one of
> the following:
> (1) Follow any unsubscribe process the sender has included in their email
> (2) Where no unsubscribe process has been included, reply to the sender
> and type "unsubscribe" in the subject line. If you require additional
> information please go to our UHN Newsletters and Mailing Lists page.
> Please note that we are unable to automatically unsubscribe individuals
> from all UHN mailing lists.
>


--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
Arvydas Matiukas Arvydas Matiukas
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Re: [External] Considerations on WLL / AOBS system

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

Nobody mentioned yet that WLL is an ultrashort pulse laser required for FLIM and other advanced methods while the gas laser typically is a CW laser at fixed narrowband wavelength. The latter makes a difference for the metrology applications while for fluorescence imaging it can be safely replaced by a cheaper and simpler laser diode.

Best regards,
Arvydas



>>> Zdenek Svindrych <[hidden email]> 04/23/20 5:18 PM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5WeKnwHEA$ 
Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5XCvEeSUw$  and include the link in your posting.
*****

Hi James,

I will just comment to the AOBS efficiency: without any electrical signal
the AOBS behaves just like two "glass" crystals (AR coated, of course) for
the returning fluorescence signal, so very little of the precious signal is
lost. With electrical-piezo excitation only smart part of the emission
spectrum is lost, it's exactly the few nanometers that correspond to the
excitation wavelength +/- bandwidth. So to make the AOTF analogy, the laser
light is diffracted into the excitation path with less-than-ideal
efficiency (and polarization sensitive as well), but the emission
throughput is essentially perfect.

Also, Leica uses (or at least used to use) the same principle to combine
discrete lasers with the WLL inside the WLL box. This is the Leica combiner
inside one of the early generation NKT WLLs...
https://urldefense.com/v3/__https://drive.google.com/file/d/0B5vWyBYrDvcJWmZTSkpCZ0xqVnM/view?usp=sharing__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5XtKMCuJw$ 

A side note, I also responded to Ralph's questions, but by mistake it went
out as a personal message and not to the confolist. I won't copy it here
again, as my comments were identical to those of James.

Best, zdenek

On Thu, Apr 23, 2020 at 3:03 PM Jonkman, James <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5WeKnwHEA$ 
> Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5XCvEeSUw$  and include the link in your posting.
> *****
>
> Hi, Ralph.  We got two Leica SP8s at the same time about 3 years ago: one
> that's more basic with CW lasers, and the other with WLL, STED, and FLIM.
> I don't yet know the difference in maintenance costs as they are all still
> under warranty.  So far the WLL has been very reliable, but it's main
> advantage for us is that it allows for FLIM and gated STED.
>
> Personally, I wouldn't get it just for regular confocal microscopy for
> several reasons:
> - You still need a 405nm laser, so probably 405nm laser + WLL is more
> expensive than 4 CW lasers (though I'm not certain).
> - I share your concern about the cost of service contract or repair when
> it comes to that: I hope somebody else can reply with knowledge of this.
> - The average power of the WLL at 488nm is about 350uW, compared to 5mW
> for the 488 solid state laser on our other SP8.  This makes it pretty poor
> for FRAP, and even slow for acceptor photobleaching to confirm FRET.
> - The AOBS isn't perfect.  You have to move the spectral gate quite far
> away from the laser line to avoid capturing reflected light, which lowers
> your efficiency for light collection.  Unless you choose one of the special
> laser lines that has an associated laser line filter (but then you are back
> to using discrete wavelengths); or you use gating to throw away the very
> fastest signal (but then you need special detectors).  I would be curious
> to see the overall efficiency of the AOBS: I know that AOTFs are not 100%
> transmissive, but with excitation it isn't really a problem to just throw a
> fair bit of it away.
> - I like the flexibility of the WLL, but in our hands it has not proven to
> be crucial for new probes or anything like that.  There is not really an
> advantage to hitting the excitation wavelength exactly on the peak.  It's
> critical to capture as much emission as possible, which usually means you
> should excite a little to the "left" of the peak.  The photons that don't
> absorb don't really do any harm - in thinner samples especially, they
> either absorb or they pass through the sample without interacting and
> therefore they do no harm.  (I suppose in thicker samples they could also
> scatter, and then they may absorb elsewhere).
>
> The WLL works well for far red dyes (AF647 for example).
>
> Finally, I saw with envy that the specs on the WLL with their new
> Stellaris model go from 440nm to 850nm (whereas mine is 470 to 670nm).
> This is a huge improvement!  We have to use a separate pulsed 440nm laser
> for CFP on our instrument (still used by some people for FLIM), which is
> not terribly well integrated (manual power dial for example), but the new
> WLL will eliminate that and give you more options of probes in the NIR as
> well (when combined with their new HyD detectors).  I'm not sure if this
> exactly counts "Confocal Re-imagined " as their recent slick marketing
> video claims, but it is definitely a welcome improvement!
>
> Cheers,
> James
> -----------------------------------------------
>    James Jonkman, Staff Scientist
>    Advanced Optical Microscopy Facility (AOMF)
>    and Wright Cell Imaging Facility (WCIF)
>    University Health Network
>    MaRS, PMCRT tower, 101 College St., Room 15-305
>    Toronto, ON, CANADA    M5G 1L7
>   [hidden email]  Tel: 416-581-8593
>    https://urldefense.com/v3/__http://www.aomf.ca__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5XL4mAarA$ 
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Ralf Palmisano
> Sent: Wednesday, April 22, 2020 12:06 PM
> To: [hidden email]
> Subject: [External] Considerations on WLL / AOBS system
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5WeKnwHEA$ 
> Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5XCvEeSUw$  and include the link in your posting.
> *****
>
> Dear community,
>
> I am kindly asking for feedback and sharing of expertise.
>
>   * What is the difference comparing a white light laser & AOBS system
>     to a classical gas laser & AOBS set-up in maintenance and service
> costs.
>   * I would love to get feedback on experience about the reliability of
>     a WLL against a classical approach in a multi-user high performance
>     environment.
>   * Any knowledge of a true average life-time in the wild of a WLL (~
>     2.000 usage hours / year).
>   * Is there any particular fact one should be aware of?
>   * The system would mainly be used with fixed samples (2D & 3D) and
>     normally 3 to 4 channels.
>   * Has anyone already experience with far red / near IR dyes on such
>     system?
>
> You can also send me a pm. Thank you for your time and eventual support.
>
> Best Ralph
>
> --
> Ralph Palmisano
> Head - Optical Imaging Centre Erlangen
>
> Fellow Royal Microscopical Society
> Member Royal Society of Medicine
>
> Speaker Scientific Advisory Board "German BioImaging"
> Board of Directors Core Technologies for Life Science
>
> Cauerstr. 3
> 91058 Erlangen, Germany
>
> +49-9131-85-70320 (Office)
> +49-9131-85-70321 (Secretary)
>
> https://urldefense.com/v3/__http://www.oice.uni-erlangen.de__;!!GobTDDpD7A!dijTjmjHGm5bswEVnuHWy8pl-HMxvU1pMNtWJ-jW7rF3-OAooKmOqbKNb5WMkB1JAA$ 
>
>
> This e-mail may contain confidential and/or privileged information for the
> sole use of the intended recipient.
> Any review or distribution by anyone other than the person for whom it was
> originally intended is strictly prohibited.
> If you have received this e-mail in error, please contact the sender and
> delete all copies.
> Opinions, conclusions or other information contained in this e-mail may
> not be that of the organization.
>
> If you feel you have received an email from UHN of a commercial nature and
> would like to be removed from the sender's mailing list please do one of
> the following:
> (1) Follow any unsubscribe process the sender has included in their email
> (2) Where no unsubscribe process has been included, reply to the sender
> and type "unsubscribe" in the subject line. If you require additional
> information please go to our UHN Newsletters and Mailing Lists page.
> Please note that we are unable to automatically unsubscribe individuals
> from all UHN mailing lists.
>


--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
Glyn Nelson Glyn Nelson
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Re: [External] Considerations on WLL / AOBS system

In reply to this post by Ralf Palmisano
*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

I can say that James' comments all match our experience (we also have two SP8, one WLL and one CW).  I would add that the WLL is very expensive and consequently makes a more expensive maintenance contract. I would argue it is worth paying- ours is almost 4 years old and we have had the WLL blow up (well, okay, it set on fire, not quite that bad, but pretty bad!!).  In that respect, it is a case of all eggs in one basket, and left the system completely unusable until we got a replacement, which took a bit longer than we would have liked.   The newer version going down to 440 would be of benefit for quite a few FPs, eg our WLL+405 can't image CFP or mKeima.  I do like the standard CW version though- very few reflection problems, the software seems to run faster than the STED version and it has been a good workhorse.

Glyn.
Christian Kukat Christian Kukat
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Re: Considerations on WLL / AOBS system

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

Hi Ralf,

We have 3 SP8 systems and all of them have a WLL. Many aspects were already discussed in previous emails.

The main reason, why we got our first WLL, was the flexibility that we can excite most fluorophores at the excitation maximum. Because in a core facility/SRL you never know, what kind of samples the user bring. A WLL in combination with a spectral detector gives a lot of flexibility. One can combine 8 laser lines with the other lasers in the system, e.g. 405 nm or Argon laser lines.

The lifetime was not a big issue in our hands, but as any other laser, they break after some time. You can get an extended warranty or a maintenace contract, if this is possible.
The problem is, if the WLL breaks, one is very much limited in the choice of fluorophores, until it is repaired or replaced.

Our users like the features of exciting at excitation maxima. For some dyes, one can reduce the laser power a lot to get the same brightness, compared to exciting not close to the maximum. It makes us less worry about combinations of fluorescent dyes in comparison to other microscopes with fixed laser lines and onyl specific dichroic combinations.
Some users are scanning their samples with an excitation lambda scan to find autofluorescence and avoid then later colors in that spectral range. Some are scanning their samples, because they forgot which fluorophores they have used. ;-)

In some cases, we look at strange or new dyes and created our own excitation and emission spectra for them.

Notch filters have limited usage on a WLL system, but we use the gating mode instead, to exclude reflection or back-scattered light. This works efficiently.
As mentioned before, the WLL has less laser power, than for example an Argon laser. This can be an issue if you would like to perform FRAP experiments. For this reason, we kept Argon lasers at our systems.

Leica has the possibilty to use a PMT for DIC or brightfield. Here, we have observed, that sometimes the classical lasers (405 nm or Argon) create a better looking image, but this is only an asthetic aspect.

We have used WLLs for many years and the microscopes have been good workhorses in our core facility.

Best wishes,

Christian

_______________________________________________________________________

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

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

> On 22. Apr 2020, at 18:06, Ralf Palmisano <[hidden email]> wrote:
>
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>
> Dear community,
>
> I am kindly asking for feedback and sharing of expertise.
>
> * What is the difference comparing a white light laser & AOBS system
>   to a classical gas laser & AOBS set-up in maintenance and service costs.
> * I would love to get feedback on experience about the reliability of
>   a WLL against a classical approach in a multi-user high performance
>   environment.
> * Any knowledge of a true average life-time in the wild of a WLL (~
>   2.000 usage hours / year).
> * Is there any particular fact one should be aware of?
> * The system would mainly be used with fixed samples (2D & 3D) and
>   normally 3 to 4 channels.
> * Has anyone already experience with far red / near IR dyes on such
>   system?
>
> You can also send me a pm. Thank you for your time and eventual support.
>
> Best Ralph
>
> --
> Ralph Palmisano
> Head - Optical Imaging Centre Erlangen
>
> Fellow Royal Microscopical Society
> Member Royal Society of Medicine
>
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> Board of Directors Core Technologies for Life Science
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