http://confocal-microscopy-list.275.s1.nabble.com/405-laser-intensity-at-the-objective-is-0-15-of-actually-intensity-is-this-normal-tp7587264p7587280.html
traversing the multimode fiber. The wavelength is short enough that you
on how the fiber is bent. It's remotely possible that this is causing some
through the fiber start to bleed out through the cladding. I would call
> *****
> 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
>