405 laser intensity at the objective is 0.15% of actually intensity ... is this normal?

[Emmanuel Levy]

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Dear All,

We are experiencing problems to detect fluorescent proteins, in particular
with blue fluorescence. BFPs are generally weak and so I initially thought
it was normal, but today we measured the laser intensities at the fiber
exit or "entrance to the microscope" as well as at the objective and we
loose a lot of intensity on the path.

We have three lasers:
405 - 120mW
488 - 100mW
561 - 75mW

The lasers are combined inside a combiner, enter a multi-mode fiber that is
attached to a spinning disk microscope (Yokogawa W1), itself coupled to an
Olympus X83.

The intensities measured at the fiber exit are:
405 - 21mW
488 - 34.5 mW
561 - 32.4mW

The intensities measured at the objective (measured after removing the
objective) are:

405 - 0.18 mW
488 - 1.18 mW
561 - 2.16 mW

Does this look reasonable to you? I guess that the 488 and 561 lines are
somewhat OK, but for the 405 laser, the intensity at the objective is ~700x
lower compared to the original intensity.

I would be keen to hear about typical values you are used to see with
spinning disk systems.

Thank you for your feedback,
Best wishes,

Emmanuel

[Peter Brunt AVR]

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*commercial (non-commercial) post*

Hi Emmanuel
Are you running your lasers at full power?  What combiner are you using?  I can't comment so much on the power you have at your objective but on your combiner side the values look low.  Going into a multi-mode fibre should be like a barn door for your lasers so if we imagine a ~5% (x2) loss due to combining optics and an ~80% coupling efficiency, you should still be getting a ~72% average coupling efficiency into the fibre.  It sounds to me like the setup may need to be realigned. If you still don't see an improvement then one other possibility is that the 405nm laser has degraded your fibre due to solarisation and the fibre needs replacing.  

I hope this helps.


Kind Regards

Pete Brunt

[Emmanuel Levy]

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Dear Peter,

Thank you for this information.

Are you running your lasers at full power?


Yes we are.


> What combiner are you using?


It is a custom made combiner, from the company that assembled the
microscope.


> I can't comment so much on the power you have at your objective but on
> your combiner side the values look low.  Going into a multi-mode fibre
> should be like a barn door for your lasers so if we imagine a ~5% (x2) loss
> due to combining optics and an ~80% coupling efficiency, you should still
> be getting a ~72% average coupling efficiency into the fibre.  It sounds to
> me like the setup may need to be realigned. If you still don't see an
> improvement then one other possibility is that the 405nm laser has degraded
> your fibre due to solarisation and the fibre needs replacing.
>

Thanks a lot for this info, I'll discuss it with the company.

If there are other opinions, in particular regarding the loss between te
fiber-output and the objective, I'd be glad to hear them.

Best wishes,

Emmanuel





>
> I hope this helps.
>
>
> Kind Regards
>
> Pete Brunt
>

[Jana Nieder]

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Dear all,

we are organising an event on single quantum emitters - including single emitters in the context of super resolution microscopy!

Hope to see many of you there!

Registration deadline is already in three days (9.9.2017)!

Best wishes,

Jana



--
Jana B. Nieder
Dr. rer. nat.
Head of Nanophotonics Department/Group Leader
Ultrafast Bio- and Nanophotonics Group
Av. Mestre José Veiga s/n
4715-330 Braga – Portugal
T: + 351 253 140 112 – (2254)





****************************************************************************************

TOPICAL MEETING ON SINGLE QUANTUM EMITTERS (SQE2017)

4.- 6. October 2017
INL-International Iberian Nanotechnology Laboratory, Braga, Portugal


!!!! Deadline for active participation poster / talk is already the 9.9. 2017!!!!


DESCRIPTION
The topical meeting SQE2017 shall bring together international scientists from in- and outside the COST Action MP1302 Nanospectroscopy to discuss novel technologies and findings in the context of Single Quantum Emitters which form the foundation of the emerging second quantum revolution.

The topics covered include but are not restricted to:
□ Impurities in 2D materials and crystals
□ Single emitters and interactions with cavities, plasmonics, surface waves, meta-materials etc.
□ Integration of single emitters in quantum information devices
□ Single emitter sources and interdisciplinary applications e.g. in super-resolution microscopy for life science applications, etc.
The Topical Meeting is organized by INL - International Iberian Nanotechnology Laboratory and with the support of the COST Action MP 1302 Nanospectroscopy.
The event follows the final management meeting of the COST Action being held on 3./4. October 2017 at INL.


Confirmed invited speakers at the Topical meeting on Single Quantum Emitters (so far):

Cristina Flors, IMDEA nanoscience, Madrid, Spain
Ronen Rapaport, The Hebrew University of Jerusalem, Israel
Ruth Oulton, University of Bristol, UK
Alper Kiraz, Koç University, Istanbul, Turkey
Julien Houel, U Lyon, France
John Rarity, Bristol, UK
Alfred Meixner, Tuebingen, Germany
Andreas Schell, ICFO, Spain
Paulo Ferreira, INL, Portugal



Registration via:

https://www.eventbrite.co.uk/e/topical-meeting-on-single-quantum-emitters-sqe2017-registration-36063759676

Abstract templates available under:

http://www.cost-nanospectroscopy.eu/final-meeting.html


************************************************************************************************************************************

[John Oreopoulos]

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Emmanuel,

Check and make sure you don't have a filter inserted into the optical pathway that blocks the 405nm wavelength, and then check that all the other filters (dichroic and emission) actually are what you think they are. Make sure the filter wheels and linear motor positions are indexed and homing properly. Check your microscope filter turret as well.

John Oreopoulos

> On Sep 6, 2017, at 3:28 PM, Emmanuel Levy <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Peter,
>
> Thank you for this information.
>
> Are you running your lasers at full power?
>
>
> Yes we are.
>
>
>> What combiner are you using?
>
>
> It is a custom made combiner, from the company that assembled the
> microscope.
>
>
>> I can't comment so much on the power you have at your objective but on
>> your combiner side the values look low.  Going into a multi-mode fibre
>> should be like a barn door for your lasers so if we imagine a ~5% (x2) loss
>> due to combining optics and an ~80% coupling efficiency, you should still
>> be getting a ~72% average coupling efficiency into the fibre.  It sounds to
>> me like the setup may need to be realigned. If you still don't see an
>> improvement then one other possibility is that the 405nm laser has degraded
>> your fibre due to solarisation and the fibre needs replacing.
>
> Thanks a lot for this info, I'll discuss it with the company.
>
> If there are other opinions, in particular regarding the loss between te
> fiber-output and the objective, I'd be glad to hear them.
>
> Best wishes,
>
> Emmanuel
>
>
>
>
>
>>
>> I hope this helps.
>>
>>
>> Kind Regards
>>
>> Pete Brunt
>>

[Craig Brideau]

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*****

Try measuring the power of all three lines using a different objective
lens. Many objectives are not designed to work below 450nm and so they
absorb considerably for lasers shorter than the blue. Also, remove the
objective lens and try measuring the power of the beam. You will probably
overfill your power meter, but you will be able to compare the relative
power between the three lines from what light does hit your sensor.

Craig

On Wed, Sep 6, 2017 at 1:28 PM, Emmanuel Levy <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Peter,
>
> Thank you for this information.
>
> Are you running your lasers at full power?
>
>
> Yes we are.
>
>
> > What combiner are you using?
>
>
> It is a custom made combiner, from the company that assembled the
> microscope.
>
>
> > I can't comment so much on the power you have at your objective but on
> > your combiner side the values look low.  Going into a multi-mode fibre
> > should be like a barn door for your lasers so if we imagine a ~5% (x2)
> loss
> > due to combining optics and an ~80% coupling efficiency, you should still
> > be getting a ~72% average coupling efficiency into the fibre.  It sounds
> to
> > me like the setup may need to be realigned. If you still don't see an
> > improvement then one other possibility is that the 405nm laser has
> degraded
> > your fibre due to solarisation and the fibre needs replacing.
> >
>
> Thanks a lot for this info, I'll discuss it with the company.
>
> If there are other opinions, in particular regarding the loss between te
> fiber-output and the objective, I'd be glad to hear them.
>
> Best wishes,
>
> Emmanuel
>
>
>
>
>
> >
> > I hope this helps.
> >
> >
> > Kind Regards
> >
> > Pete Brunt
> >
>

[Emmanuel Levy]

*****
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*****

Dear Craig,

The numbers i gave initially do correspond to a scenario where I removed the objectives before measuring the power of each line.

Best wishes,

Emmanuel

> Try measuring the power of all three lines using a different objective
> lens. Many objectives are not designed to work below 450nm and so they
> absorb considerably for lasers shorter than the blue. Also, remove the
> objective lens and try measuring the power of the beam. You will probably
> overfill your power meter, but you will be able to compare the relative
> power between the three lines from what light does hit your sensor.
>
> Craig
>
> On Wed, Sep 6, 2017 at 1:28 PM, Emmanuel Levy <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Dear Peter,
>>
>> Thank you for this information.
>>
>> Are you running your lasers at full power?
>>
>>
>> Yes we are.
>>
>>
>>> What combiner are you using?
>>
>>
>> It is a custom made combiner, from the company that assembled the
>> microscope.
>>
>>
>>> I can't comment so much on the power you have at your objective but on
>>> your combiner side the values look low.  Going into a multi-mode fibre
>>> should be like a barn door for your lasers so if we imagine a ~5% (x2)
>> loss
>>> due to combining optics and an ~80% coupling efficiency, you should still
>>> be getting a ~72% average coupling efficiency into the fibre.  It sounds
>> to
>>> me like the setup may need to be realigned. If you still don't see an
>>> improvement then one other possibility is that the 405nm laser has
>> degraded
>>> your fibre due to solarisation and the fibre needs replacing.
>>>
>>
>> Thanks a lot for this info, I'll discuss it with the company.
>>
>> If there are other opinions, in particular regarding the loss between te
>> fiber-output and the objective, I'd be glad to hear them.
>>
>> Best wishes,
>>
>> Emmanuel
>>
>>
>>
>>
>>
>>>
>>> I hope this helps.
>>>
>>>
>>> Kind Regards
>>>
>>> Pete Brunt
>>>
>>

[John Oreopoulos]

*****
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*****

I took a closer look at your reported power levels along the optical train. First, recognize that most silicon-based power meters are not accurate at 405nm. There could be an error there as high as 20%. Also, as previously mentioned, these sensors are angle dependent and you might not be capturing all the light because of beam size (at the back of the objective perhaps, but probably fine at laser and fiber tip). But assuming you've taken those points into consideration and you're confident in the powers you've indicated, there's something a bit more troubling here.

You mentioned this system uses a multi-mode fiber delivery method, but the incurred losses are much higher from laser to fiber tip than I would expect, and from fiber to objective, even more so. This is a very inefficient illumination setup - at all wavelengths.

Disclaimer - I work for Andor. Andor (Spectral Applied Research) in the past manufactured the Borealis multi-mode fibre illumination upgrade for Yokogawa CSUs, and so I have some familiarity with what efficiency levels should be achievable with a multi-mode fiber delivery scheme. Here, it seems it's not much better than a single-mode fiber approach, so if I were you, I'd get this checked out with the manufacturer.

John Oreopoulos

> On Sep 6, 2017, at 3:54 PM, John Oreopoulos <[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.
> *****
>
> Emmanuel,
>
> Check and make sure you don't have a filter inserted into the optical pathway that blocks the 405nm wavelength, and then check that all the other filters (dichroic and emission) actually are what you think they are. Make sure the filter wheels and linear motor positions are indexed and homing properly. Check your microscope filter turret as well.
>
> John Oreopoulos
>
>> On Sep 6, 2017, at 3:28 PM, Emmanuel Levy <[hidden email]> wrote:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Dear Peter,
>>
>> Thank you for this information.
>>
>> Are you running your lasers at full power?
>>
>>
>> Yes we are.
>>
>>
>>> What combiner are you using?
>>
>>
>> It is a custom made combiner, from the company that assembled the
>> microscope.
>>
>>
>>> I can't comment so much on the power you have at your objective but on
>>> your combiner side the values look low.  Going into a multi-mode fibre
>>> should be like a barn door for your lasers so if we imagine a ~5% (x2) loss
>>> due to combining optics and an ~80% coupling efficiency, you should still
>>> be getting a ~72% average coupling efficiency into the fibre.  It sounds to
>>> me like the setup may need to be realigned. If you still don't see an
>>> improvement then one other possibility is that the 405nm laser has degraded
>>> your fibre due to solarisation and the fibre needs replacing.
>>
>> Thanks a lot for this info, I'll discuss it with the company.
>>
>> If there are other opinions, in particular regarding the loss between te
>> fiber-output and the objective, I'd be glad to hear them.
>>
>> Best wishes,
>>
>> Emmanuel
>>
>>
>>
>>
>>
>>>
>>> I hope this helps.
>>>
>>>
>>> Kind Regards
>>>
>>> Pete Brunt
>>>

[Gary Laevsky]

*****
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*****

Hi Emmanuel,

Also, don't forget there is an expected huge excitation loss through the
disc.  If I recall, it was "increased" to something like 14% with the X1,
not sure where it is with the W1.  Maybe a bad dichroic in the disc? And,
mind which disc is in the path, if the lower mag optimized disc, I believe
the pinholes are half as big (25 um) and spaced twice as far apart to
maximize confocality and minimize bleedthrough from adjacent pinholes.
This is all for naught if you are using the bypass mode.

As others have mentioned, using a multi-mode should make the coupling
really easy.  I used to work for Andor and with a single-mode I remember
upwards of 75% using 488 (on great days:)).

Good luck!

On Wed, Sep 6, 2017 at 10:23 PM, John Oreopoulos <
[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.
> *****
>
> I took a closer look at your reported power levels along the optical
> train. First, recognize that most silicon-based power meters are not
> accurate at 405nm. There could be an error there as high as 20%. Also, as
> previously mentioned, these sensors are angle dependent and you might not
> be capturing all the light because of beam size (at the back of the
> objective perhaps, but probably fine at laser and fiber tip). But assuming
> you've taken those points into consideration and you're confident in the
> powers you've indicated, there's something a bit more troubling here.
>
> You mentioned this system uses a multi-mode fiber delivery method, but the
> incurred losses are much higher from laser to fiber tip than I would
> expect, and from fiber to objective, even more so. This is a very
> inefficient illumination setup - at all wavelengths.
>
> Disclaimer - I work for Andor. Andor (Spectral Applied Research) in the
> past manufactured the Borealis multi-mode fibre illumination upgrade for
> Yokogawa CSUs, and so I have some familiarity with what efficiency levels
> should be achievable with a multi-mode fiber delivery scheme. Here, it
> seems it's not much better than a single-mode fiber approach, so if I were
> you, I'd get this checked out with the manufacturer.
>
> John Oreopoulos
>
> > On Sep 6, 2017, at 3:54 PM, John Oreopoulos <[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.
> > *****
> >
> > Emmanuel,
> >
> > Check and make sure you don't have a filter inserted into the optical
> pathway that blocks the 405nm wavelength, and then check that all the other
> filters (dichroic and emission) actually are what you think they are. Make
> sure the filter wheels and linear motor positions are indexed and homing
> properly. Check your microscope filter turret as well.
> >
> > John Oreopoulos
> >
> >> On Sep 6, 2017, at 3:28 PM, Emmanuel Levy <[hidden email]>
> wrote:
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your
> posting.
> >> *****
> >>
> >> Dear Peter,
> >>
> >> Thank you for this information.
> >>
> >> Are you running your lasers at full power?
> >>
> >>
> >> Yes we are.
> >>
> >>
> >>> What combiner are you using?
> >>
> >>
> >> It is a custom made combiner, from the company that assembled the
> >> microscope.
> >>
> >>
> >>> I can't comment so much on the power you have at your objective but on
> >>> your combiner side the values look low.  Going into a multi-mode fibre
> >>> should be like a barn door for your lasers so if we imagine a ~5% (x2)
> loss
> >>> due to combining optics and an ~80% coupling efficiency, you should
> still
> >>> be getting a ~72% average coupling efficiency into the fibre.  It
> sounds to
> >>> me like the setup may need to be realigned. If you still don't see an
> >>> improvement then one other possibility is that the 405nm laser has
> degraded
> >>> your fibre due to solarisation and the fibre needs replacing.
> >>
> >> Thanks a lot for this info, I'll discuss it with the company.
> >>
> >> If there are other opinions, in particular regarding the loss between te
> >> fiber-output and the objective, I'd be glad to hear them.
> >>
> >> Best wishes,
> >>
> >> Emmanuel
> >>
> >>
> >>
> >>
> >>
> >>>
> >>> I hope this helps.
> >>>
> >>>
> >>> Kind Regards
> >>>
> >>> Pete Brunt
> >>>
>

--
Best,

Gary Laevsky, Ph.D.
Director, Confocal Imaging Facility
Nikon Center of Excellence
Dept. of Molecular Biology
Washington Rd.
Princeton University
Princeton, New Jersey, 08544-1014
(O) 609 258 5432
(C) 508 507 1310

[Craig Brideau]

*****
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*****

Actually I wonder if the mode from the 405 is still adequate after
traversing the multimode fiber. The wavelength is short enough that you
could possibly have some very unusual profile exiting the fiber dependent
on how the fiber is bent. It's remotely possible that this is causing some
of your loss, especially if some of the modes from the 405 propagating
through the fiber start to bleed out through the cladding. I would call
this a long shot though, as it is more likely the fiber is solarizing or
your disk is having issues with the 405nm light.

Craig

On Thu, Sep 7, 2017 at 4:14 AM, Gary Laevsky <[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 Emmanuel,
>
> Also, don't forget there is an expected huge excitation loss through the
> disc.  If I recall, it was "increased" to something like 14% with the X1,
> not sure where it is with the W1.  Maybe a bad dichroic in the disc? And,
> mind which disc is in the path, if the lower mag optimized disc, I believe
> the pinholes are half as big (25 um) and spaced twice as far apart to
> maximize confocality and minimize bleedthrough from adjacent pinholes.
> This is all for naught if you are using the bypass mode.
>
> As others have mentioned, using a multi-mode should make the coupling
> really easy.  I used to work for Andor and with a single-mode I remember
> upwards of 75% using 488 (on great days:)).
>
> Good luck!
>
> On Wed, Sep 6, 2017 at 10:23 PM, John Oreopoulos <
> [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.
> > *****
> >
> > I took a closer look at your reported power levels along the optical
> > train. First, recognize that most silicon-based power meters are not
> > accurate at 405nm. There could be an error there as high as 20%. Also, as
> > previously mentioned, these sensors are angle dependent and you might not
> > be capturing all the light because of beam size (at the back of the
> > objective perhaps, but probably fine at laser and fiber tip). But
> assuming
> > you've taken those points into consideration and you're confident in the
> > powers you've indicated, there's something a bit more troubling here.
> >
> > You mentioned this system uses a multi-mode fiber delivery method, but
> the
> > incurred losses are much higher from laser to fiber tip than I would
> > expect, and from fiber to objective, even more so. This is a very
> > inefficient illumination setup - at all wavelengths.
> >
> > Disclaimer - I work for Andor. Andor (Spectral Applied Research) in the
> > past manufactured the Borealis multi-mode fibre illumination upgrade for
> > Yokogawa CSUs, and so I have some familiarity with what efficiency levels
> > should be achievable with a multi-mode fiber delivery scheme. Here, it
> > seems it's not much better than a single-mode fiber approach, so if I
> were
> > you, I'd get this checked out with the manufacturer.
> >
> > John Oreopoulos
> >
> > > On Sep 6, 2017, at 3:54 PM, John Oreopoulos <
> [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.
> > > *****
> > >
> > > Emmanuel,
> > >
> > > Check and make sure you don't have a filter inserted into the optical
> > pathway that blocks the 405nm wavelength, and then check that all the
> other
> > filters (dichroic and emission) actually are what you think they are.
> Make
> > sure the filter wheels and linear motor positions are indexed and homing
> > properly. Check your microscope filter turret as well.
> > >
> > > John Oreopoulos
> > >
> > >> On Sep 6, 2017, at 3:28 PM, Emmanuel Levy <[hidden email]>
> > wrote:
> > >>
> > >> *****
> > >> To join, leave or search the confocal microscopy listserv, go to:
> > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> Post images on http://www.imgur.com and include the link in your
> > posting.
> > >> *****
> > >>
> > >> Dear Peter,
> > >>
> > >> Thank you for this information.
> > >>
> > >> Are you running your lasers at full power?
> > >>
> > >>
> > >> Yes we are.
> > >>
> > >>
> > >>> What combiner are you using?
> > >>
> > >>
> > >> It is a custom made combiner, from the company that assembled the
> > >> microscope.
> > >>
> > >>
> > >>> I can't comment so much on the power you have at your objective but
> on
> > >>> your combiner side the values look low.  Going into a multi-mode
> fibre
> > >>> should be like a barn door for your lasers so if we imagine a ~5%
> (x2)
> > loss
> > >>> due to combining optics and an ~80% coupling efficiency, you should
> > still
> > >>> be getting a ~72% average coupling efficiency into the fibre.  It
> > sounds to
> > >>> me like the setup may need to be realigned. If you still don't see an
> > >>> improvement then one other possibility is that the 405nm laser has
> > degraded
> > >>> your fibre due to solarisation and the fibre needs replacing.
> > >>
> > >> Thanks a lot for this info, I'll discuss it with the company.
> > >>
> > >> If there are other opinions, in particular regarding the loss between
> te
> > >> fiber-output and the objective, I'd be glad to hear them.
> > >>
> > >> Best wishes,
> > >>
> > >> Emmanuel
> > >>
> > >>
> > >>
> > >>
> > >>
> > >>>
> > >>> I hope this helps.
> > >>>
> > >>>
> > >>> Kind Regards
> > >>>
> > >>> Pete Brunt
> > >>>
> >
>
>
>
> --
> Best,
>
> Gary Laevsky, Ph.D.
> Director, Confocal Imaging Facility
> Nikon Center of Excellence
> Dept. of Molecular Biology
> Washington Rd.
> Princeton University
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
> (C) 508 507 1310
>

[Emmanuel Levy]

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Dear Pete,

I was wrong, we actually have a single mode and not a multi-mode fiber. May
I ask what is to be expected in terms of coupling efficiency for single
mode fibers?

Thank you for your input,

All the best,

Emmanuel



On 6 September 2017 at 22:15, Peter Brunt <[hidden email]>
wrote:

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> *****
>
> *commercial (non-commercial) post*
>
> Hi Emmanuel
> Are you running your lasers at full power?  What combiner are you using?
> I can't comment so much on the power you have at your objective but on your
> combiner side the values look low.  Going into a multi-mode fibre should be
> like a barn door for your lasers so if we imagine a ~5% (x2) loss due to
> combining optics and an ~80% coupling efficiency, you should still be
> getting a ~72% average coupling efficiency into the fibre.  It sounds to me
> like the setup may need to be realigned. If you still don't see an
> improvement then one other possibility is that the 405nm laser has degraded
> your fibre due to solarisation and the fibre needs replacing.
>
> I hope this helps.
>
>
> Kind Regards
>
> Pete Brunt
>

[Emmanuel Levy]

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Dear Gary and John,

My apologies for the confusion, but we actually have a single-mode fiber.
I'm not sure if this means that coupling is as expected on the combiner
end? Are 20% / 30% / 50% efficiency for 405/488/561nm in the normal range?

Thanks for your feedback,

Best wishes,

Emmanuel

PS: On the microscope end we do have a specific device that "widens" the
beam in order to get more homogeneous illumination, which appears to be the
cause for the more pronounced loss in the 405nm wavelength. Otherwise,
there are no unnecessary filters on the optical path.



On 7 September 2017 at 13:14, Gary Laevsky <[hidden email]> wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Emmanuel,
>
> Also, don't forget there is an expected huge excitation loss through the
> disc.  If I recall, it was "increased" to something like 14% with the X1,
> not sure where it is with the W1.  Maybe a bad dichroic in the disc? And,
> mind which disc is in the path, if the lower mag optimized disc, I believe
> the pinholes are half as big (25 um) and spaced twice as far apart to
> maximize confocality and minimize bleedthrough from adjacent pinholes.
> This is all for naught if you are using the bypass mode.
>
> As others have mentioned, using a multi-mode should make the coupling
> really easy.  I used to work for Andor and with a single-mode I remember
> upwards of 75% using 488 (on great days:)).
>
> Good luck!
>
> On Wed, Sep 6, 2017 at 10:23 PM, John Oreopoulos <
> [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.
> > *****
> >
> > I took a closer look at your reported power levels along the optical
> > train. First, recognize that most silicon-based power meters are not
> > accurate at 405nm. There could be an error there as high as 20%. Also, as
> > previously mentioned, these sensors are angle dependent and you might not
> > be capturing all the light because of beam size (at the back of the
> > objective perhaps, but probably fine at laser and fiber tip). But
> assuming
> > you've taken those points into consideration and you're confident in the
> > powers you've indicated, there's something a bit more troubling here.
> >
> > You mentioned this system uses a multi-mode fiber delivery method, but
> the
> > incurred losses are much higher from laser to fiber tip than I would
> > expect, and from fiber to objective, even more so. This is a very
> > inefficient illumination setup - at all wavelengths.
> >
> > Disclaimer - I work for Andor. Andor (Spectral Applied Research) in the
> > past manufactured the Borealis multi-mode fibre illumination upgrade for
> > Yokogawa CSUs, and so I have some familiarity with what efficiency levels
> > should be achievable with a multi-mode fiber delivery scheme. Here, it
> > seems it's not much better than a single-mode fiber approach, so if I
> were
> > you, I'd get this checked out with the manufacturer.
> >
> > John Oreopoulos
> >
> > > On Sep 6, 2017, at 3:54 PM, John Oreopoulos <
> [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.
> > > *****
> > >
> > > Emmanuel,
> > >
> > > Check and make sure you don't have a filter inserted into the optical
> > pathway that blocks the 405nm wavelength, and then check that all the
> other
> > filters (dichroic and emission) actually are what you think they are.
> Make
> > sure the filter wheels and linear motor positions are indexed and homing
> > properly. Check your microscope filter turret as well.
> > >
> > > John Oreopoulos
> > >
> > >> On Sep 6, 2017, at 3:28 PM, Emmanuel Levy <[hidden email]>
> > wrote:
> > >>
> > >> *****
> > >> To join, leave or search the confocal microscopy listserv, go to:
> > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> Post images on http://www.imgur.com and include the link in your
> > posting.
> > >> *****
> > >>
> > >> Dear Peter,
> > >>
> > >> Thank you for this information.
> > >>
> > >> Are you running your lasers at full power?
> > >>
> > >>
> > >> Yes we are.
> > >>
> > >>
> > >>> What combiner are you using?
> > >>
> > >>
> > >> It is a custom made combiner, from the company that assembled the
> > >> microscope.
> > >>
> > >>
> > >>> I can't comment so much on the power you have at your objective but
> on
> > >>> your combiner side the values look low.  Going into a multi-mode
> fibre
> > >>> should be like a barn door for your lasers so if we imagine a ~5%
> (x2)
> > loss
> > >>> due to combining optics and an ~80% coupling efficiency, you should
> > still
> > >>> be getting a ~72% average coupling efficiency into the fibre.  It
> > sounds to
> > >>> me like the setup may need to be realigned. If you still don't see an
> > >>> improvement then one other possibility is that the 405nm laser has
> > degraded
> > >>> your fibre due to solarisation and the fibre needs replacing.
> > >>
> > >> Thanks a lot for this info, I'll discuss it with the company.
> > >>
> > >> If there are other opinions, in particular regarding the loss between
> te
> > >> fiber-output and the objective, I'd be glad to hear them.
> > >>
> > >> Best wishes,
> > >>
> > >> Emmanuel
> > >>
> > >>
> > >>
> > >>
> > >>
> > >>>
> > >>> I hope this helps.
> > >>>
> > >>>
> > >>> Kind Regards
> > >>>
> > >>> Pete Brunt
> > >>>
> >
>
>
>
> --
> Best,
>
> Gary Laevsky, Ph.D.
> Director, Confocal Imaging Facility
> Nikon Center of Excellence
> Dept. of Molecular Biology
> Washington Rd.
> Princeton University
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
> (C) 508 507 1310
>

[Peter Brunt AVR]

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Hi Emmanuel,
For singlemode fibre coupling across multiple wavelengths ranging from 405-642nm I'd typically expect >60% coupling efficiency for all lines in the worst case.  For 405nm, 488nm and 561nm I'd actually expect closer to ~70% on the basis that most fibre coupling systems (industry wide) aren't entirely achromatic and the closer the wavelengths, the easier it is to adjust the coupling optics to maximise the efficiency.  

Still, if we assume there is still some loss on the dichroics, you should still expect ~54% average power out of the fibre.  I think there is a good chance your lasers / fibre coupler need realigning.  This is somewhat evidence by the fact that your coupling seems to be biased towards 561nm.  

I hope this helps.


Pete Brunt

AVR Optics

[Zdenek Svindrych-2]

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Also, some single mode fibers are actually not single mode at 405 nm. The
higher modes won't be useful for fluorescence excitation in a confocal
microscope... So just check the far filed pattern from your fiber when
aligning the 405 line to make sure you're only coupling to the TEM(0,0) mode
- it should be nice gaussian when you point the bare fiber at a sheet of
common white (fluorescent ) paper.
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
Department of Biology,University of Virginia
409 McCormick Rd, Charlottesville, VA-22904
http://www.kcci.virginia.edu/
tel: [phone]

---------- Původní e-mail ----------
Od: Peter Brunt <[hidden email]>
Komu: [hidden email]
Datum: 11. 9. 2017 10:45:05
Předmět: Re: 405 laser intensity at the objective is 0.15% of actually
intensity ... is this normal?
"*****
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Hi Emmanuel,
For singlemode fibre coupling across multiple wavelengths ranging from 405-
642nm I'd typically expect >60% coupling efficiency for all lines in the
worst case. For 405nm, 488nm and 561nm I'd actually expect closer to ~70% on
the basis that most fibre coupling systems (industry wide) aren't entirely
achromatic and the closer the wavelengths, the easier it is to adjust the
coupling optics to maximise the efficiency.

Still, if we assume there is still some loss on the dichroics, you should
still expect ~54% average power out of the fibre. I think there is a good
chance your lasers / fibre coupler need realigning. This is somewhat
evidence by the fact that your coupling seems to be biased towards 561nm.

I hope this helps.


Pete Brunt

AVR Optics
"

[Craig Brideau]

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Pete's estimates feel optimistic to me when you take into consideration
that multiple wavelengths are typically coupled down one single mode fiber
for microscopy applications. For one-laser-one-fiber applications Pete is
quite correct as the vendor can optimize the fiber and coupling optics for
one wavelength. For multiple lasers down a single fiber, however,
compromises must be made, and coupling efficiency <50% are not uncommon for
diode lasers. As Zdenek says, some fibers are not single mode for 405nm. To
expand on this, single-mode fibers are only single mode to a rated
wavelength known as the cutoff wavelength. Below the cutoff wavelength, the
core of the fiber is actually too wide to act as a single mode waveguide
for a short wavelength. Typically 405nm suffers from this, as again most
systems were initially designed for ~450nm. Zdenek's test of checking the
pattern for each line out of the fiber should give you some idea of how
well it performs for all the laser lines. In my own experience, at least on
slightly outdated coupling systems, 405nm often goes multi-modal in
extended band single mode fibers. You will see two lobes in the output
instead of a single Gaussian spot if this should happen. You may also
notice strange behavior if you bend the fiber in different ways as the
stresses will alter the properties of the fiber, especially impacting
shorter wavelength boundary cases where the wavelength of light is near the
cutoff for single mode behavior in the fiber.

Craig

On Mon, Sep 11, 2017 at 10:16 AM, <[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.
> *****
>
> Also, some single mode fibers are actually not single mode at 405 nm. The
> higher modes won't be useful for fluorescence excitation in a confocal
> microscope... So just check the far filed pattern from your fiber when
> aligning the 405 line to make sure you're only coupling to the TEM(0,0)
> mode
> - it should be nice gaussian when you point the bare fiber at a sheet of
> common white (fluorescent ) paper.
> --
> Zdenek Svindrych, Ph.D.
> W.M. Keck Center for Cellular Imaging (PLSB 003)
> Department of Biology,University of Virginia
> 409 McCormick Rd, Charlottesville, VA-22904
> http://www.kcci.virginia.edu/
> tel: [phone]
>
> ---------- Původní e-mail ----------
> Od: Peter Brunt <[hidden email]>
> Komu: [hidden email]
> Datum: 11. 9. 2017 10:45:05
> Předmět: Re: 405 laser intensity at the objective is 0.15% of actually
> intensity ... is this normal?
> "*****
> 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 Emmanuel,
> For singlemode fibre coupling across multiple wavelengths ranging from 405-
> 642nm I'd typically expect >60% coupling efficiency for all lines in the
> worst case. For 405nm, 488nm and 561nm I'd actually expect closer to ~70%
> on
> the basis that most fibre coupling systems (industry wide) aren't entirely
> achromatic and the closer the wavelengths, the easier it is to adjust the
> coupling optics to maximise the efficiency.
>
> Still, if we assume there is still some loss on the dichroics, you should
> still expect ~54% average power out of the fibre. I think there is a good
> chance your lasers / fibre coupler need realigning. This is somewhat
> evidence by the fact that your coupling seems to be biased towards 561nm.
>
> I hope this helps.
>
>
> Pete Brunt
>
> AVR Optics
> "
>

[lgelman]

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Hi Emmanuel,

These are the values we get at our 10x/0.45 objective mounted on a Zeiss Axioimager equipped with a Yokogawa W1 (Note that laser power is set to 10%):
Laser 488 at 10% with 10x/0.45 = 1.7 mW
Laser 561 at 10% with 10x/0.45 = 0.227 mW
Laser 405 at 10% with 10x/0.45 = 0.201 mW
Our lasers must be rather in the 150-200mW range.
As the others mentioned already, I would try to realign the lasers in the combiner and/or to by a new fiber.
Best regards,
Laurent.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Emmanuel Levy
Sent: Wednesday, September 6, 2017 11:54
To: [hidden email]
Subject: 405 laser intensity at the objective is 0.15% of actually intensity ... is this normal?

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Dear All,

We are experiencing problems to detect fluorescent proteins, in particular with blue fluorescence. BFPs are generally weak and so I initially thought it was normal, but today we measured the laser intensities at the fiber exit or "entrance to the microscope" as well as at the objective and we loose a lot of intensity on the path.

We have three lasers:
405 - 120mW
488 - 100mW
561 - 75mW

The lasers are combined inside a combiner, enter a multi-mode fiber that is attached to a spinning disk microscope (Yokogawa W1), itself coupled to an Olympus X83.

The intensities measured at the fiber exit are:
405 - 21mW
488 - 34.5 mW
561 - 32.4mW

The intensities measured at the objective (measured after removing the
objective) are:

405 - 0.18 mW
488 - 1.18 mW
561 - 2.16 mW

Does this look reasonable to you? I guess that the 488 and 561 lines are somewhat OK, but for the 405 laser, the intensity at the objective is ~700x lower compared to the original intensity.

I would be keen to hear about typical values you are used to see with spinning disk systems.

Thank you for your feedback,
Best wishes,

Emmanuel