Improving two-photon excitation efficiency

[Steven Hou]

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Hi everyone, one of our two photon microscopes currently doesn't seem to perform as well as some others in our lab. On this microscope, when imaging the same sample, we need to increase the laser power to higher levels than on other microscopes to achieve a similar signal level which results in bleaching. In my mind the two main things to confirm is that 1) the excitation laser beam is going parallel/centered through the objective (while overfilling the back aperture), 2) the detection path is properly aligned so that we are capturing all of the fluorescence (since, we are imaging cells in a plate, I'm not even considering the scattered fluorescence light). Since I have confirmed both of these points, I'm considering other factors that may be causing this poor performance. One thing I'm wondering about is the laser itself which is an old Coherent Chameleon from more than 20 years ago. When I look at the beam on a piece of paper I see a bright spot overlayed with some "background" haze. I don't see this "background" haze when looking at other Ti-Sapphs in our lab. Also, there is a strange thing I noticed where even when I set the laser wavelength to 800 nm, I can actually "see" the laser spot more than I expected. When I put a 700 nm dichroic in the path, I found with a spectrometer that there is a ~600 nm component combined with the 800 nm output. For now, I am just filtering out this 600 nm component with a dichroic but I wonder if this is indicative of something wrong with the laser.

To deal with the poor spatial beam profile of the laser, I was thinking of trying to set up a spatial filter (pinhole) system to try to recover a Gaussian beam. I'm wondering if anyone has any idea whether this is worth trying and whether improving a laser's beam profile like this can noticeably improve two photon excitation efficiency.

Thanks for any feedback,
Steve

[Stpehen Ausden]

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Hi Steve, Are you able to say what mak/model the system is please? Thanks Steve

[Mark Cannell-2]

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Try testing out of mode lock in CW. The extra light could be generated by something fluorescing in the beam path? Haze may be dirt collected on the optics and/or degradation of the ultrafast mirrors...

HTH

Mark B. Cannell. Ph.D. FRSNZ FISHR
Department of Physiology, Pharmacology & Neuroscience
School of Medical Sciences
University Walk
Bristol BS8 1TD
 
[hidden email]
 
 

On 3/12/20, 12:48 AM, "Confocal Microscopy List on behalf of Steven Hou" <[hidden email] on behalf of [hidden email]> wrote:

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    Hi everyone, one of our two photon microscopes currently doesn't seem to perform as well as some others in our lab. On this microscope, when imaging the same sample, we need to increase the laser power to higher levels than on other microscopes to achieve a similar signal level which results in bleaching. In my mind the two main things to confirm is that 1) the excitation laser beam is going parallel/centered through the objective (while overfilling the back aperture), 2) the detection path is properly aligned so that we are capturing all of the fluorescence (since, we are imaging cells in a plate, I'm not even considering the scattered fluorescence light). Since I have confirmed both of these points, I'm considering other factors that may be causing this poor performance. One thing I'm wondering about is the laser itself which is an old Coherent Chameleon from more than 20 years ago. When I look at the beam on a piece of paper I see a bright spot overlayed with some "background" haze. I don't see this "background" haze when looking at other Ti-Sapphs in our lab. Also, there is a strange thing I noticed where even when I set the laser wavelength to 800 nm, I can actually "see" the laser spot more than I expected. When I put a 700 nm dichroic in the path, I found with a spectrometer that there is a ~600 nm component combined with the 800 nm output. For now, I am just filtering out this 600 nm component with a dichroic but I wonder if this is indicative of something wrong with the laser.

    To deal with the poor spatial beam profile of the laser, I was thinking of trying to set up a spatial filter (pinhole) system to try to recover a Gaussian beam. I'm wondering if anyone has any idea whether this is worth trying and whether improving a laser's beam profile like this can noticeably improve two photon excitation efficiency.

    Thanks for any feedback,
    Steve

[Michael Giacomelli-2]

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Photobleaching in the NIR tends to be a two photon process, meaning that
you are probably exciting your sample efficiently but something is wrong
with the detection.  If there was a problem with the laser (poor mode
locking, etc.) usually you see no signal at all or a noisy signal but don't
get too much photobleaching since not much of the NIR light is being
absorbed.

Since you checked alignment and found no issues, have you tried swapping
PMTs?  They have a finite life (especially for GaAsP) and gradually lose
sensitivity, often so slowly you don't notice it.  If your PMTs are past
their lifespan, then you will find that you need to use a lot more power to
get the same signal.

Mike



On Wed, Dec 2, 2020 at 8:12 PM Mark Cannell <[hidden email]>
wrote:

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> and include the link in your posting.
> *****
>
> Try testing out of mode lock in CW. The extra light could be generated by
> something fluorescing in the beam path? Haze may be dirt collected on the
> optics and/or degradation of the ultrafast mirrors...
>
> HTH
>
> Mark B. Cannell. Ph.D. FRSNZ FISHR
> Department of Physiology, Pharmacology & Neuroscience
> School of Medical Sciences
> University Walk
> Bristol BS8 1TD
>
> [hidden email]
>
>
>
> On 3/12/20, 12:48 AM, "Confocal Microscopy List on behalf of Steven Hou" <
> [hidden email] on behalf of [hidden email]> wrote:
>
>     *****
>     To join, leave or search the confocal microscopy listserv, go to:
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> and include the link in your posting.
>     *****
>
>     Hi everyone, one of our two photon microscopes currently doesn't seem
> to perform as well as some others in our lab. On this microscope, when
> imaging the same sample, we need to increase the laser power to higher
> levels than on other microscopes to achieve a similar signal level which
> results in bleaching. In my mind the two main things to confirm is that 1)
> the excitation laser beam is going parallel/centered through the objective
> (while overfilling the back aperture), 2) the detection path is properly
> aligned so that we are capturing all of the fluorescence (since, we are
> imaging cells in a plate, I'm not even considering the scattered
> fluorescence light). Since I have confirmed both of these points, I'm
> considering other factors that may be causing this poor performance. One
> thing I'm wondering about is the laser itself which is an old Coherent
> Chameleon from more than 20 years ago. When I look at the beam on a piece
> of paper I see a bright spot overlayed with some "background" haze. I don't
> see this "background" haze when looking at other Ti-Sapphs in our lab.
> Also, there is a strange thing I noticed where even when I set the laser
> wavelength to 800 nm, I can actually "see" the laser spot more than I
> expected. When I put a 700 nm dichroic in the path, I found with a
> spectrometer that there is a ~600 nm component combined with the 800 nm
> output. For now, I am just filtering out this 600 nm component with a
> dichroic but I wonder if this is indicative of something wrong with the
> laser.
>
>     To deal with the poor spatial beam profile of the laser, I was
> thinking of trying to set up a spatial filter (pinhole) system to try to
> recover a Gaussian beam. I'm wondering if anyone has any idea whether this
> is worth trying and whether improving a laser's beam profile like this can
> noticeably improve two photon excitation efficiency.
>
>     Thanks for any feedback,
>     Steve
>
>

[Benjamin Smith]

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Hey Steve,

The leaking 600 nm sounds like something might be off with the aperture in
the prism compressor used to tune the wavelength.  Have you tried re-homing
the aperture?  The older Coherent Chameleon laser interfaces are a lot less
locked down, so I think you should be able to do this via the front panel
options or serial communication (look in the manual for the table of serial
commands).

Another possibility, which may be the eventual death knell for the laser,
is that something got knocked out of alignment in the prism compressor.
This not only could cause a spurious wavelength to leak through, but would
also explain the poor two photon excitation as you would wind up with a bit
of both temporal and spatial dispersion.

That said, I have put a pinhole in a two-photon launch path, in my case to
ensure that four separate laser lines were perfectly coaligned, but as a
secondary effect it did help clean off the faint side modes one often gets
from a Pockels cell, resulting in a beautiful TM00 beam, and it is our
highest resolution two-photon rig (as measured by FFT).  One thing to keep
in mind is that with a 2P laser the power density at the pinhole is quite
high, so if a lot of the beam is in higher order modes, you could wind up
damaging a conventional pinhole.  If this turns out to be an issue, Edmund
Optics makes high power pinholes out of materials like tungsten:
https://www.edmundoptics.com/f/high-power-apertures/12205/

This also brings up another good point: you can readily infer the beam
quality at the sample plane by measuring the resolution of your microscope
using a FFT.  Basically, take a sample with a lot of fine detail (although
a slant edge or slant grid would be best) and image it with pixels that are
<200 nm across.  Then look for the highest frequency modulation you can
find in the FFT of the image, this will give you the effective resolution.
 If the dimmer laser gives a lower resolution than the bright one, that
would point towards a beam quality issue.  If both lasers generate the same
resolution, then you likely have a temporal dispersion problem.  However,
the only way to definitively confirm temporal dispersion would be to get
your hands on an autocorrelator (which aren't cheap).

One final note, I would also try getting in touch with Coherent, in general
I've found their technicians to be quite knowledgeable and more than
willing to openly share that knowledge.

If you find the solution I would love to hear it!

Cheers,
   Ben Smith



On Wed, Dec 2, 2020 at 4:48 PM Steven Hou <[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 everyone, one of our two photon microscopes currently doesn't seem to
> perform as well as some others in our lab. On this microscope, when imaging
> the same sample, we need to increase the laser power to higher levels than
> on other microscopes to achieve a similar signal level which results in
> bleaching. In my mind the two main things to confirm is that 1) the
> excitation laser beam is going parallel/centered through the objective
> (while overfilling the back aperture), 2) the detection path is properly
> aligned so that we are capturing all of the fluorescence (since, we are
> imaging cells in a plate, I'm not even considering the scattered
> fluorescence light). Since I have confirmed both of these points, I'm
> considering other factors that may be causing this poor performance. One
> thing I'm wondering about is the laser itself which is an old Coherent
> Chameleon from more than 20 years ago. When I look at the beam on a piece
> of paper I see a bright spot overlayed with some "background" haze. I don't
> see this "background" haze when looking at other Ti-Sapphs in our lab.
> Also, there is a strange thing I noticed where even when I set the laser
> wavelength to 800 nm, I can actually "see" the laser spot more than I
> expected. When I put a 700 nm dichroic in the path, I found with a
> spectrometer that there is a ~600 nm component combined with the 800 nm
> output. For now, I am just filtering out this 600 nm component with a
> dichroic but I wonder if this is indicative of something wrong with the
> laser.
>
> To deal with the poor spatial beam profile of the laser, I was thinking of
> trying to set up a spatial filter (pinhole) system to try to recover a
> Gaussian beam. I'm wondering if anyone has any idea whether this is worth
> trying and whether improving a laser's beam profile like this can
> noticeably improve two photon excitation efficiency.
>
> Thanks for any feedback,
> Steve
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

[Paolo Bianchini]

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Dear Steve,
In my experience, a slight misalignment could require a much higher power without compromising the image quality. It could be a slight tilt of the PSF along the optical axis, but most of that can be seen looking at the whole field of view (FOV). Maximising the intensity in the centre of the FOV in an iterative way, while imaging a uniform fluorescence sample (a drop of fluorescein or Chroma slide) could strongly help.
Another issue, as you suggested, can be a strong CW component in the beam. This component should visible looking with the spectrometer at the pulse. A tiny peak over the main Gaussian pulse shape could be a sign. If it happens, the laser has a problem or is a back reflection in the cavity, due to some optics in the beam path.
I noticed the component at 600-700 nm only once working very closed to the laser source. The explanation I found was the fluorescence of the Ti:Sapphire crystal. It is CW and not so collimated, thus it should not reach the sample over a long beam path, although removing as you do could be important.

I hope it can help
Good luck!
Paolo


--
Paolo Bianchini, PhD
Scientist
Optical Nanoscopy, Nanophysics
ISTITUTO ITALIANO DI TECNOLOGIA

Via Enrico Melen 83, 16152 Genova, Italy
Tel: [phone]
skype: bianchinipaolo
www.iit.it <http://www.iit.it/>

> On 3 Dec 2020, at 05:29, Benjamin Smith <[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.
> *****
>
> Hey Steve,
>
> The leaking 600 nm sounds like something might be off with the aperture in
> the prism compressor used to tune the wavelength.  Have you tried re-homing
> the aperture?  The older Coherent Chameleon laser interfaces are a lot less
> locked down, so I think you should be able to do this via the front panel
> options or serial communication (look in the manual for the table of serial
> commands).
>
> Another possibility, which may be the eventual death knell for the laser,
> is that something got knocked out of alignment in the prism compressor.
> This not only could cause a spurious wavelength to leak through, but would
> also explain the poor two photon excitation as you would wind up with a bit
> of both temporal and spatial dispersion.
>
> That said, I have put a pinhole in a two-photon launch path, in my case to
> ensure that four separate laser lines were perfectly coaligned, but as a
> secondary effect it did help clean off the faint side modes one often gets
> from a Pockels cell, resulting in a beautiful TM00 beam, and it is our
> highest resolution two-photon rig (as measured by FFT).  One thing to keep
> in mind is that with a 2P laser the power density at the pinhole is quite
> high, so if a lot of the beam is in higher order modes, you could wind up
> damaging a conventional pinhole.  If this turns out to be an issue, Edmund
> Optics makes high power pinholes out of materials like tungsten:
> https://www.edmundoptics.com/f/high-power-apertures/12205/
>
> This also brings up another good point: you can readily infer the beam
> quality at the sample plane by measuring the resolution of your microscope
> using a FFT.  Basically, take a sample with a lot of fine detail (although
> a slant edge or slant grid would be best) and image it with pixels that are
> <200 nm across.  Then look for the highest frequency modulation you can
> find in the FFT of the image, this will give you the effective resolution.
> If the dimmer laser gives a lower resolution than the bright one, that
> would point towards a beam quality issue.  If both lasers generate the same
> resolution, then you likely have a temporal dispersion problem.  However,
> the only way to definitively confirm temporal dispersion would be to get
> your hands on an autocorrelator (which aren't cheap).
>
> One final note, I would also try getting in touch with Coherent, in general
> I've found their technicians to be quite knowledgeable and more than
> willing to openly share that knowledge.
>
> If you find the solution I would love to hear it!
>
> Cheers,
>   Ben Smith
>
>
>
> On Wed, Dec 2, 2020 at 4:48 PM Steven Hou <[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 everyone, one of our two photon microscopes currently doesn't seem to
>> perform as well as some others in our lab. On this microscope, when imaging
>> the same sample, we need to increase the laser power to higher levels than
>> on other microscopes to achieve a similar signal level which results in
>> bleaching. In my mind the two main things to confirm is that 1) the
>> excitation laser beam is going parallel/centered through the objective
>> (while overfilling the back aperture), 2) the detection path is properly
>> aligned so that we are capturing all of the fluorescence (since, we are
>> imaging cells in a plate, I'm not even considering the scattered
>> fluorescence light). Since I have confirmed both of these points, I'm
>> considering other factors that may be causing this poor performance. One
>> thing I'm wondering about is the laser itself which is an old Coherent
>> Chameleon from more than 20 years ago. When I look at the beam on a piece
>> of paper I see a bright spot overlayed with some "background" haze. I don't
>> see this "background" haze when looking at other Ti-Sapphs in our lab.
>> Also, there is a strange thing I noticed where even when I set the laser
>> wavelength to 800 nm, I can actually "see" the laser spot more than I
>> expected. When I put a 700 nm dichroic in the path, I found with a
>> spectrometer that there is a ~600 nm component combined with the 800 nm
>> output. For now, I am just filtering out this 600 nm component with a
>> dichroic but I wonder if this is indicative of something wrong with the
>> laser.
>>
>> To deal with the poor spatial beam profile of the laser, I was thinking of
>> trying to set up a spatial filter (pinhole) system to try to recover a
>> Gaussian beam. I'm wondering if anyone has any idea whether this is worth
>> trying and whether improving a laser's beam profile like this can
>> noticeably improve two photon excitation efficiency.
>>
>> Thanks for any feedback,
>> Steve
>>
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

[Steven Hou]

*****
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Thank you everyone for the great feedback and suggestions. I will update further if I gain more insights into our issue.

Best,
Steve