Leica SP5 behaviour using 405 nm laser
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Dear All,
We have a fairly new Leica SP5 system on a DM6000 scope, equipped with AOBS/AOTF, 405 nm diode, argon, HeNe and 561 nm DPSS lasers. When imaging a monolayer of cells, in this case the epithelial cell layer of a fly embryo, using the 405 nm laser (Hoechst labelled) we get two in-focus images, appr. 10 microns apart. One of these is the "real" image, corresponding to the top of the embryo, whereas the second ("ghost") image appears deep inside the embryo. It is physically/biologically impossible to have a second layer of labelled epithelial cells inside the embryo, so it must be an optical effect. I tested the system with a double coverslip sample with a thin layer of air between the two coverslips (using two pieces of tape as spacers) in reflection mode, which revealed a very strong second maximum (optical intererence at the glass surface) with the 405 nm laser, but not with the other three lasers. The 405 nm lightpath avoids the AOBS, so I'm wondering if that holds the key to this problem. Leica is also trying to figure this out. Has anyone come across this weird behaviour?? Any help or advice will be much appreciated!
Thanks,
Zoltan
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Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
 
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Chere Petty |
Re: Leica SP5 behaviour using 405 nm laser
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Are you imaging line by line, frame by frame , or stack by stack? We had this same problem and our Leica rep (Linda Brown) suggested we do either frame by frame or stack by stack scanning. This solved our problem of what appeared to be bleed through but is really a very energetic and long life time signal from the UV laser. Chere Petty M.S. Manager of UMBC Keith R. Porter Core Imaging Facility Department of Biological Sciences University of Maryland Baltimore County (UMBC) 1000 Hilltop Circle Baltimore Maryland 21250 301-367-8408 [hidden email] emumbc.com On Mar 10, 2008, at 9:19 AM, Zoltan Cseresnyes wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Dear All, > > We have a fairly new Leica SP5 system on a DM6000 scope, equipped > with AOBS/AOTF, 405 nm diode, argon, HeNe and 561 nm DPSS lasers. > When imaging a monolayer of cells, in this case the epithelial cell > layer of a fly embryo, using the 405 nm laser (Hoechst labelled) we > get two in-focus images, appr. 10 microns apart. One of these is > the "real" image, corresponding to the top of the embryo, whereas > the second ("ghost") image appears deep inside the embryo. It is > physically/biologically impossible to have a second layer of > labelled epithelial cells inside the embryo, so it must be an > optical effect. I tested the system with a double coverslip sample > with a thin layer of air between the two coverslips (using two > pieces of tape as spacers) in reflection mode, which revealed a > very strong second maximum (optical intererence at the glass > surface) with the 405 nm laser, but not with the other three > lasers. The 405 nm lightpath avoids the AOBS, so I'm wondering if > that holds the key to this problem. Leica is also trying to figure > this out. Has anyone come across this weird behaviour?? Any help > or advice will be much appreciated! > Thanks, > > Zoltan > > > > -- > Zoltan Cseresnyes > Facility manager, Imaging Suite > Dept. of Zoology University of Cambridge > Downing Street, Cambridge > CB2 3EJ UK > > Tel.: (++44) (0)1223 769282 > Fax.: (++44) (0)1223 336676 |
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Jacqueline Ross |
Re: Leica SP5 behaviour using 405 nm laser
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In reply to this post by Zoltan
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Dear Zoltan,
I don't know if this will help you at all since you have a
newer system and a different laser.
We had an odd reflection artifact with our TCS SP2 some
time ago when using our UV laser (Coherent Enterprise II). Our system also does
not have AOBS and we use neutral density filters to alter the laser
power.
We originally noticed a problem because the UV images
appeared to be rather fuzzy compared to the visible ones. I tested the system
using multicoloured microspheres and discovered that where there should
have been one bead (as in the visible), there were actually 2 in the UV
image, one of which was slightly weaker.
It turned out to be due to a tiny washer that was sitting
inside the neutral density assembly at the head of the laser. Once the washer
was removed, everything was fine but we still don't know where the washer came
from!
Kind regards,
Jacqui
Jacqueline Ross
Biomedical Imaging
Microscopist From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zoltan Cseresnyes Sent: Tuesday, 11 March 2008 2:19 a.m. To: [hidden email] Subject: Leica SP5 behaviour using 405 nm laser Dear All,
We have a fairly new Leica SP5 system on a DM6000 scope, equipped with
AOBS/AOTF, 405 nm diode, argon, HeNe and 561 nm DPSS lasers. When imaging
a monolayer of cells, in this case the epithelial cell layer of a fly
embryo, using the 405 nm laser (Hoechst labelled) we get two in-focus images,
appr. 10 microns apart. One of these is the "real" image,
corresponding to the top of the embryo, whereas the second
("ghost") image appears deep inside the embryo. It is
physically/biologically impossible to have a second layer of labelled
epithelial cells inside the embryo, so it must be an optical effect. I
tested the system with a double coverslip sample with a thin layer of air
between the two coverslips (using two pieces of tape as spacers) in
reflection mode, which revealed a very strong second maximum (optical
intererence at the glass surface) with the 405 nm laser, but not with the other
three lasers. The 405 nm lightpath avoids the AOBS, so I'm wondering if
that holds the key to this problem. Leica is also trying to figure this
out. Has anyone come across this weird behaviour?? Any help or
advice will be much appreciated!
Thanks,
Zoltan
--
Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear All,
I very much appreciate the replies I've received so far!! The problem may indeed be caused by a misaligned UV pinhole lens, which seems even likelier in light of today's tests where I examined the same sample with 4 different objectives (our system has separate UV pinhole lenses for the 20x, 40x and 63x objectives, and no lens for the 10x). The results showed that the 10x objective produced ghost images throughout the entire sample, whereas the 40x and 63x objectives produced no ghost image at all. I have a Leica engineer come in on Wednesday to check and possibly re-align the system. This will also give us a chance to look inside for loose reflective items.
Thanks very much again,
Zoltan On 3/10/08, Jacqui Ross <[hidden email]> wrote:
Search the CONFOCAL archive at <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- -- Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
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S. Pagakis (IIBEAA) |
Re: Leica SP5 behaviour using 405 nm laser
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Zoltan we had a similar problem and was fixed by pinhole lens alignment. So this should do it for you. However, since sequential +405 imaging was mentioned in this thread, I would like to report a problem we discovered on an SP5 recently. (LAS AF version 1.8.2) When someone is doing "by frame" or "by stack" sequential imaging, the software allows the selection of a different hardware settings per scan method. This is OK so far. However it also allows the selection of a different 405 pinhole lens per scan method, even if the objective lens is not changed between scans. Therefore it is possible to have a "wrong" 405 pinhole lens when a "non--405" scan has been defined. Obviously this does not affect the images, because the wrong 405 pinhole lens is introduced when a different colour is acquired. The first problem this creates is a long delay switching between scan because it, unnecessarily, switches the 405 pinhole lens between scans. The BIGGEST problem however is when someone switches to "Line by line" sequential imaging, while a scan method with the wrong 405 pinhole lens is active. Then, because during "Line by Line' sequential scanning hardware changes are NOT allowed, the wrong 405-pinhole lens is used for ALL scan methods, even for the one which collects the 405 sugnal. Then we have serious 405 image misalignment. It is, therefore, also possible that you are seeing these double images not because of misalignment of your corresponding pinhole lens but because the wrong 405-pinhole lens is used with the objective, EVEN IF YOU SELECTED THE CORRECT ONE WHEN STARTING THE EXPERIMENT. It is obviously a software "logic" bug and we will report it to Leica as well. Regards ********************************* Stamatis Pagakis Ph.D. Biological Imaging Unit Biomedical Research Foundation, Academy of Athens Soranou Efessiou 4, Athens 115 27 - Greece M: +306946644955 W: +302106597481 FAX: +302106597545 [hidden email] On 11 Mar 2008, at 03:09, Zoltan Cseresnyes wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Dear All, > > I very much appreciate the replies I've received so far!! The problem > may indeed be caused by a misaligned UV pinhole lens, which seems even > likelier in light of today's tests where I examined the same sample > with 4 different objectives (our system has separate UV pinhole lenses > for the 20x, 40x and 63x objectives, and no lens for the 10x). The > results showed that the 10x objective produced ghost images throughout > the entire sample, whereas the 40x and 63x objectives produced no > ghost image at all. I have a Leica engineer come in on Wednesday to > check and possibly re-align the system. This will also give us a > chance to look inside for loose reflective items. > Thanks very much again, > > Zoltan > > > > |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thank you all very much for the extremely useful replies! The 405 nm laser-produced ghost image was indeed the result of a misaligned pinhole lens, especially the one for the 20X multi-immersion objective, although all others (for 40X oil, 63X oil and 63X water) had to be re-aligned as well. The system is now good as new. Thanks again; this is indeed a wonderful forum! Zoltan On 3/12/08, S. Pagakis (IIBEAA) <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello Zoltan > > we had a similar problem and was fixed by pinhole lens alignment. So > this should do it for you. > However, since sequential +405 imaging was mentioned in this thread, I > would like to report a problem we discovered on an SP5 recently. (LAS > AF version 1.8.2) > > When someone is doing "by frame" or "by stack" sequential imaging, the > software allows the selection of a different hardware settings per scan > method. This is OK so far. However it also allows the selection of a > different 405 pinhole lens per scan method, even if the objective lens > is not changed between scans. > > Therefore it is possible to have a "wrong" 405 pinhole lens when a > "non--405" scan has been defined. Obviously this does not affect the > images, because the wrong 405 pinhole lens is introduced when a > different colour is acquired. > > The first problem this creates is a long delay switching between scan > because it, unnecessarily, switches the 405 pinhole lens between scans. > > The BIGGEST problem however is when someone switches to "Line by line" > sequential imaging, while a scan method with the wrong 405 pinhole lens > is active. > > Then, because during "Line by Line' sequential scanning hardware > changes are NOT allowed, the wrong 405-pinhole lens is used for ALL > scan methods, even for the one which collects the 405 sugnal. Then we > have serious 405 image misalignment. > > It is, therefore, also possible that you are seeing these double images > not because of misalignment of your corresponding pinhole lens but > because the wrong 405-pinhole lens is used with the objective, EVEN IF > YOU SELECTED THE CORRECT ONE WHEN STARTING THE EXPERIMENT. > > It is obviously a software "logic" bug and we will report it to Leica > as well. > > Regards > > > ********************************* > Stamatis Pagakis Ph.D. > Biological Imaging Unit > Biomedical Research Foundation, Academy of Athens > Soranou Efessiou 4, Athens 115 27 - Greece > M: +306946644955 W: +302106597481 > FAX: +302106597545 [hidden email] > > > On 11 Mar 2008, at 03:09, Zoltan Cseresnyes wrote: > > > Search the CONFOCAL archive at > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear All, > > > > I very much appreciate the replies I've received so far!! The problem > > may indeed be caused by a misaligned UV pinhole lens, which seems even > > likelier in light of today's tests where I examined the same sample > > with 4 different objectives (our system has separate UV pinhole lenses > > for the 20x, 40x and 63x objectives, and no lens for the 10x). The > > results showed that the 10x objective produced ghost images throughout > > the entire sample, whereas the 40x and 63x objectives produced no > > ghost image at all. I have a Leica engineer come in on Wednesday to > > check and possibly re-align the system. This will also give us a > > chance to look inside for loose reflective items. > > Thanks very much again, > > > > Zoltan > > > > > > > > > -- -- Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
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