http://confocal-microscopy-list.275.s1.nabble.com/405-laser-intensity-at-the-objective-is-0-15-of-actually-intensity-is-this-normal-tp7587264p7587287.html
cause for the more pronounced loss in the 405nm wavelength. Otherwise,
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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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
>