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

Chirped mirrors are a quick way to get a bit of pulse compression with
minimal effort (and cost) in a situation like this.
As for video-rate and 2-photon imaging, I'll stick to resonant scanners
before trying to do it with spinning disk.

Craig


On Mon, Feb 25, 2013 at 3:23 AM, Zdenek Svindrych <[log in to unmask]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks to the List I am aware of this paper.
>
> I have spent some time doing 2PE and SHG on a scanning confocal. Though our
> setup was not tuned optimally (a prechirp would really help), we were often
> limited by the absorbed heat. So increasing the power to get larger FOV  of
> the 2P spinning disc might be problematic.
>
> The work reported in the paper is great, no doubt (I'd also like to design
> my own disc :-). But I think it will be only useful with new effective up-
> converting dyes or something the like.
>
> Cheers,
> zdenek
> P.S.:
> I didn't mean unsuitable, i just thing it is not the preferred combination,
> at least for now.
>
>
> ---------- Původní zpráva ----------
> Od: George McNamara <[log in to unmask]>
> Datum: 25. 2. 2013
> Předmět: Re: multiphoton spinning disc
>
> "*****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Zdenek: "it is a simple matter, multiphoton is not suitable for spinning
> disc as it's not suitable for widefield. "
>
> Shimozawa et al: "Second, the generation of out-of-focus light is
> prevented by two-photon excitation that achieves selective-plane
> illumination. </pubmed/23401517>"
>
> *Shimozawa* T, Yamagata K, Kondo T, Hayashi S, Shitamukai A, Konno D,
> Matsuzaki F, Takayama J, Onami S, Nakayama H, Kosugi Y, Watanabe TM,
> Fujita K, Mimori-Kiyosue Y. Improving spinning disk confocal microscopy
> by preventing pinhole cross-talk for intravital imaging.
> </pubmed/23401517>* *Proc Natl Acad Sci U S A. 2013 Feb 11. PMID: 23401517
>
> A recent key requirement in life sciences is the observation of
> biological processes in their natural in vivo context. However, imaging
> techniques that allow fast imaging with higher resolution in 3D thick
> specimens are still limited. Spinning disk confocal microscopy using a
> Yokogawa Confocal Scanner Unit, which offers high-speed multipoint
> confocal live imaging, has been found to have wide utility among cell
> biologists. A conventional Confocal Scanner Unit configuration, however,
> is not optimized for thick specimens, for which the background noise
> attributed to "pinhole cross-talk," which is unintended pinhole
> transmission of out-of-focus light, limits overall performance in focal
> discrimination and reduces confocal capability. Here, we improve
> spinning disk confocal microscopy by eliminating pinhole cross-talk.
> First, the amount of pinhole cross-talk is reduced by increasing the
> interpinhole distance. Second, the generation of out-of-focus light is
> prevented by two-photon excitation that achieves selective-plane
> illumination. We evaluate the effect of these modifications and test the
> applicability to the live imaging of green fluorescent
> protein-expressing model animals. As demonstrated by visualizing the
> fine details of the 3D cell shape and submicron-size cytoskeletal
> structures inside animals, these strategies dramatically improve
> higher-resolution intravital imaging. </pubmed/23401517>*
> *
>
> On 2/16/2013 2:04 AM, Zdenek Svindrych wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi guys,
> >
> > it is a simple matter, multiphoton is not suitable for spinning disc as
> it's not suitable for widefield.
> > It's a nonlinear process and with the same average intensity of
> illumination the signal will be the higher the higher is the peak
> intensity.
> To get intense peak illumination you can focus it spatially (to a
> diffraction-limited spot) or 'temporally' (with fs pulses); one has to use
> both approaches to get appreciable results with common fluorescent dyes...
> >
> > Shortening the pulses is vital, but you cannot go to 1fs - the spectrum
> would be too broad. I don't think the microlenses could stretch the pulses,
> because they are fairly thin. The do destroy the wavefronts a bit (coz the
> are so small...), but this is cleaned up by the pinholes...
> >
> > Simply put, in a regular confocal you are creating one
> diffraction-limited
> spot, in a spinning disc you work with thousand spots. So you would need
> thousand times more power (assuming the same pulse duration). Bu then the
> thermal effects (assuming the contribution of the multiphoton processes is
> negligible) also increase thousand times...
> >
> > And now the solutions:
> > 1) use less pinholes - you hardly can modify your disc, so you decrease
> FOV instead, like in that paper.
> > 2) use better dyes - there are special up-conversion dyes that work even
> in widefield and without femtoseconds, so some could be found that would
> work here.
> > 3) more powerful pulses - not necessarily shorter, if I got the money I
> would find someone to build a pulse picker and an amplifier for me. I think
> 80 kHz repetition with thousand times the peak power could work and the
> scanning artifacts wouldn't be too severe (you may imagine this can't work
> with regular confocal...)
> >
> > Sorry for the lengthy post, bu I didn't want to disclose my brightest
> ideas on the first few lines :-).
> >
> > Cheers,
> >
> > zdenek
> >
> > ---------- Původní zpráva ----------
> > Od: Unruh, Jay
> > Datum: 15. 2. 2013
> > Předmět: Re: multiphoton spinning disc
> >
> > I would imagine that quite a bit could be gained by going to a shorter
> pulsed laser and performing dispersion compensation. I would guess that the
> micro lens array introduces a significant amount of dispersion. Of course
> many of the shorter pulsed lasers aren't tunable either. Large field of
> view
> applications with longer pulsed lasers may result in an undesired amount of
> heating anyway. Del Mar ventures (no commercial interest) actually sells a
> DIY kit for a<20 fs laser for much less than a single box tunable system.
> >
> > Jay
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Craig Brideau
> > Sent: Friday, February 15, 2013 11:46 AM
> > To: [log in to unmask]
> > Subject: Re: multiphoton spinning disc
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > If you could sacrifice tunability, fiber lasers are pretty much there, at
> least for 1040nm. Time-bandwidth-products (the company, not the unit of
> merit for pulsed lasers) makes a death ray called the Fortis that outputs
> 50
> W at 50 MHz...
> >
> > Craig
> >
> >
> > On Fri, Feb 15, 2013 at 9:37 AM, Tim Feinstein wrote:
> >
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hello all,
> >>
> >> There is an interesting demonstration of multiphoton spinning disc
> >> confocal imaging in the latest PNAS by Yuko Mimori-Kosue from RIKEN.
> >> It shows the expected Z-resolution, depth and s/n improvements (maybe
> >> a little better than due to pinhole refinement), but their last
> paragraph
> is a doozy.
> >>
> >> "In putting the system to practical use, the available output laser
> >> power is the limiting factor for the illumination area and the maximum
> >> imaging depth. Even with the two-photon lasers having the highest
> >> levels of output power currently available, we could trigger
> >> two-photon absorption for only ∼40-μm-diameter areas of GFP-expressing
> >> animals (less than 10% of the effective frame size with a 60×
> >> objective; Fig. S1B). Lasers with roughly
> >> 5–10 times the power would be required. The higher laser power is also
> >> required to excite red fluorescent proteins, which can be excited at
> >> wavelengths between 1,000 and 1,200 nm, at which the output power of
> >> the existing mode-locked laser decreases to ∼15% of peak power. "
> >>
> >> Is there any chance of seeing a ti-sapphire with 5 to 10-fold more
> power?
> >> Would it cost 5-10 times as much? It sounds like a nice approach but
> >> maybe not so much if you need a military LASER to use it. On the
> >> other hand maybe resonant two-photon (or slit-scanning, with
> >> sacrifices) can already deliver similar performance. Thoughts
> appreciated.
> >>
> >> All the best,
> >>
> >>
> >> TF
> >>
> >> Timothy Feinstein, PhD
> >> Visiting Research Associate
> >> Laboratory for GPCR Biology
> >> Dept. of Pharmacology& Chemical Biology University of Pittsburgh,
> >> School of Medicine BST W1301, 200 Lothrop St.
> >> Pittsburgh, PA 15261
> >>
> >>"
>