*****
To join or leave the confocal microscopy listserv or to change your email address, go to:
https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
Post images on http://www.imgur.com and include the link in your posting.
*****

You can detect 2P saturation by ramping the power up and down and
monitoring the resulting signal. Ideally if incident power is doubled, the
generated signal will be quadrupled. If it is not quadrupled then you are
in the realm of saturation. In reality there are many things that influence
your sample and will change your results from the theoretical square
relationship, but strong deviations away from the relationship are a sign
that you have saturated your nonlinear process.

Craig

On Sat, Jun 3, 2023 at 12:37 PM Rowlands, Christopher J <
[log in to unmask]> wrote:

> *****
> To join or leave the confocal microscopy listserv or to change your email
> address, go to:
> https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Nino,
>
> Sorry, should have been more clear - I don't think there is ever a point
> where you formally "start" saturating. There's always a small probability
> that you'll get two excitation events occurring quasi-simultaneously (so
> the second event does not produce a fluorescence photon); the key question
> is how much saturation is OK. If you were worried about it I'd say you
> could scan some beads (or other uniform-intensity sample) at steadily
> increasing power and plot the deviation of the bead's fluorescence from an
> I^2 dependence (1 - measured brightness / predicted brightness) - this
> deviation should asymptotically approach 1 at infinite power, then you can
> select your scanning power based on how much saturation you're prepared to
> tolerate. I was always taught to aim for ~10% of saturation as a rule of
> thumb; if you want a more rigorous justification, the fact that a Strehl
> ratio of 0.8 is considered "diffraction limited" implies that anything up
> to 20% of saturation is negligible to optical engineers.
>
> Hope that helps,
>
> Chris
>
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]> On
> Behalf Of Sebastian 'Nino' Karpf
> Sent: Saturday, June 3, 2023 9:12 AM
> To: [log in to unmask]
> Subject: Re: Power-dependent PSF in multiphoton microscopy
>
> *****
> To join or leave the confocal microscopy listserv or to change your email
> address, go to:
> https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Chris,
>
> thank you, this paper is indeed very helpful. To add to the discussion:
> How will I notice that I start saturating in the focus? In a typical
> Imaging situation, I imagine the user will just set the power based on the
> imaging results (i.e. turning up the laser power - percentage slider in the
> software - until the image looks nice). However, bright spots in the image
> are only avoided for the detector to not saturate, not for a PSF-elongating
> saturation effect. Or am I wrong and users do actually monitor the power
> dependent PSF on a daily basis?
>
> I guess this is even more of a problem in higher than quadratic
> dependencies, as Craig mentioned. In machining, the effect will be highly
> nonlinear (plasma, avalanche effects, free-electrons), so I guess if you
> have a order >3 process going on, your typical Gaussian curve will get
> heavily skewed, so the argument that FWHM is always the same as it is a
> relative measure fails here (although it was a very good explanation to my
> original question, thank you all for that!).
>
> My take: PSF and thus resolution will be power dependent, but it is an
> overseen effect. Especially in three-photon imaging, saturation might be
> easily reached in the focus and thus resolution decreased in bright areas.
>
> Best,
> Nino
>
> > Am 31.05.2023 um 12:29 schrieb Rowlands, Christopher J <
> [log in to unmask]>:
> >
> > *****
> > To join or leave the confocal microscopy listserv or to change your
> email address, go to:
> > https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > I've always found this paper very helpful:
> >
> > Cianci GC, Wu J, Berland KM. Saturation modified point spread functions
> in two-photon microscopy. Microsc Res Tech. 2004 Jun 1;64(2):135-41. doi:
> 10.1002/jemt.20071. PMID: 15352084.
> >
> > The key isn't increasing laser power (in the same way that decreasing
> your power in confocal doesn't improve your PSF) rather it is when you
> start saturating.
> >
> > Chris
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <[log in to unmask]> On
> Behalf Of Sebastian 'Nino' Karpf
> > Sent: Wednesday, May 31, 2023 10:27 AM
> > To: [log in to unmask]
> > Subject: Power-dependent PSF in multiphoton microscopy
> >
> >
> > *******************
> > This email originates from outside Imperial. Do not click on links and
> attachments unless you recognise the sender.
> > If you trust the sender, add them to your safe senders list
> https://spam.ic.ac.uk/SpamConsole/Senders.aspx to disable email stamping
> for this address.
> > *******************
> > *****
> > To join or leave the confocal microscopy listserv or to change your
> email address, go to:
> > https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear list members,
> >
> > do you know of any report on power-dependent point-spread function (PSF)
> in multiphoton microscopy? There is a common concept of using higher peak
> power for more signal, thus leveraging the quadratic power dependence (or
> cubic in THG etc.). But if I increase power, for instance by using shorter
> pulses (e.g. 50fs instead of 140fs), then the excitation volume should also
> increase, correct? This would lead to out-of-focus regions being excited
> and thus an increased 3D PSF (vice-versa a reduced three-dimensional
> resolution).
> >
> > Do you know of any discussion in literature on this? Or do you have any
> experience/thoughts on this?
> >
> >
> > Another rephrasing of this would be: if I increase the power levels of
> my Gaussian excitation beam, then the region, or 3D excitation volume,
> where a two-photon absorption can occur/is probable should also increase in
> size. The effect would be analogous to opening the detection pinhole in
> confocal microscopy - yes there will be more signal reaching the detector,
> but the 3D PSF will get worse.
> >
> >
> > As a background: we are working on an adaptive approach of generating
> more two-photon signal per pixel and came across a problem that SNR
> increase is not the best measure, because images looked worse due to
> out-of-focus light being generated.
> >
> > Hope that my multiphoton question is still fitting for this list.
> Looking forward to an interesting discussion.
> >
> > Best,
> > Nino
>