NDD upgrade on Zeiss LSM510 Meta

[Evangelos Gatzogiannis]

  I have an older Zeiss LSM510 meta in my core facility which serves as
a combination of visible laser scanning confocal, 2-photon, SHG, THG,
and CARS microscope - it's great that it is fairly readily adaptable to
all these.
 
  Has anyone noticed the following: Chroma filters have gotten a lot
better but Semrock ones just don't seem to cut it as much for mp work?  
A Bg39 and 395/100 from Chroma are best for SHG work but my 750SP from
Semrock and 395/100 give me no signal.

I also have some questions with the NDDS on the Zeiss sytems.

(I've put in some UV-sensitive PMTs in place of the standard PMTs in the
NDDs and then some more red-sensitive ones for CARS).  I've heard that
Zeiss has upgraded the NDDs and is offering newer ones to folks like me
with the older ones - any word if it improves things if any of you have
heard of this?  Has anyone on here compared the LSM510 to LSM710 NDDs
(are they the same or different).  Sorry if this post is too
multi-photon and not confocal-only.

      I also use a different condenser as I find the standard Zeiss
0.55NA condenser on the Axiovert a little flimsly and loose - has anyone
else found the standard condenser a little flimsly and what they do
except put their own home-made or external condenser (tightening screws
up helps but isn't enough) - you guys must have noticed the flimsly
condenser, they must have made the thing better on the 710.   I do think
though that the epi-NDD PMT is way too far and can be improved.

     I've found that drilling a hole in a filter turret and mounting a
SP dichroic at 90 degrees to an external PMT is best, but I don't have
the budget or time right now to worry again about pre-amps, high-voltage
boards and synchronizing to software (advice from anyone who has done it
on a Zeiss 510 Meta would be fantastic!, as I've only done it on an FV300).

    In my facility I've tried Olympus, Zeiss, and Leica objectives for
multi-photon work.  Has anyone done multi-photon on Nikon objectives and
what are your comments?  I always trust you guys to have great comments
and sorry for bothering!
 
     I'll be running some special SHG and CARS courses for anyone so if
you're in the area stop by!

Thanks!

Evangelos Gatzogiannis
Advanced Biological Imaging Scientist
Harvard Center for Nanoscale Systems
11 Oxford Street
Cambridge, MA 02138

[Armstrong, Brian]

Regarding the Zeiss NDDs: I did not know Zeiss was giving away PMTs.
We have a 510 with NDDs, and I have tried to figure out why they don't
perform better. I think that the PMTs could be improved, but that this
is not the entire problem. In short, I think that there are many little
problems that affect the overall signal. I also think that Zeiss
listened to user complaints about the system and then tried to offer
solutions to these problems with the new LSM710. I think there is no
question that the LSM710NDD system is better than the LSM510NDD system.
Apparently there are variations in the NDD detectors by Hamamatsu and
that getting "hot" PMTs can help. However, I think that it is really the
sum of all the parts; light path, dichroics, filters, and "selected"
PMTs that make the 710NDD system better, and that changing the PMTs
would only partially address the NDD problems of the LSM510.
I have not noticed a decline in Semrock.
I have no idea what you are talking about regarding the condenser. It's
just a condenser, it sits there, what would you like it to do?

I think there are some inherent problems with using other manufacturer's
objectives on your scope, including filling the back aperture, chromatic
aberration correction, and differential tube lengths. This has already
been addressed here today by Pawley and others, but the back aperture of
these new high NA objectives will create problems with your tube size,
collimating lens, and therefore depth of penetration and flatness of
field. These could be addressed in the MMM and Prairie systems with some
elegant engineering, but perhaps not as easy as some are suggesting here
today.
Just my thoughts, cheers,  

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Evangelos
Sent: Thursday, May 14, 2009 3:20 PM
To: [hidden email]
Subject: NDD upgrade on Zeiss LSM510 Meta


  I have an older Zeiss LSM510 meta in my core facility which serves as
a combination of visible laser scanning confocal, 2-photon, SHG, THG,
and CARS microscope - it's great that it is fairly readily adaptable to
all these.
 
  Has anyone noticed the following: Chroma filters have gotten a lot
better but Semrock ones just don't seem to cut it as much for mp work?  
A Bg39 and 395/100 from Chroma are best for SHG work but my 750SP from
Semrock and 395/100 give me no signal.

I also have some questions with the NDDS on the Zeiss sytems.

(I've put in some UV-sensitive PMTs in place of the standard PMTs in the

NDDs and then some more red-sensitive ones for CARS).  I've heard that
Zeiss has upgraded the NDDs and is offering newer ones to folks like me
with the older ones - any word if it improves things if any of you have
heard of this?  Has anyone on here compared the LSM510 to LSM710 NDDs
(are they the same or different).  Sorry if this post is too
multi-photon and not confocal-only.

      I also use a different condenser as I find the standard Zeiss
0.55NA condenser on the Axiovert a little flimsly and loose - has anyone

else found the standard condenser a little flimsly and what they do
except put their own home-made or external condenser (tightening screws
up helps but isn't enough) - you guys must have noticed the flimsly
condenser, they must have made the thing better on the 710.   I do think

though that the epi-NDD PMT is way too far and can be improved.

     I've found that drilling a hole in a filter turret and mounting a
SP dichroic at 90 degrees to an external PMT is best, but I don't have
the budget or time right now to worry again about pre-amps, high-voltage

boards and synchronizing to software (advice from anyone who has done it

on a Zeiss 510 Meta would be fantastic!, as I've only done it on an
FV300).

    In my facility I've tried Olympus, Zeiss, and Leica objectives for
multi-photon work.  Has anyone done multi-photon on Nikon objectives and

what are your comments?  I always trust you guys to have great comments
and sorry for bothering!
 
     I'll be running some special SHG and CARS courses for anyone so if
you're in the area stop by!

Thanks!

Evangelos Gatzogiannis
Advanced Biological Imaging Scientist
Harvard Center for Nanoscale Systems
11 Oxford Street
Cambridge, MA 02138


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