FW: Cleaning lens.

[Keith Morris]

I think Zeiss’s comment that repeated use of ethanol will damage Zeiss’s lens cement was just that – use it a few times a day and the Zeiss objective will probably fail in 6 months or so [and this might be the case for many solvents, ethanol is simply one more readily at hand*]. Use ethanol every month or so and chances are the objective will last a lot longer [and fail for another reason]. Use ethanol on a very elderly microscope where the lenses are mounted in say gum resin though and you will destroy the objective pretty much instantly [or at least get a nasty smear of gum resin all over the clear bit] - hence some people’s historical aversion is justified. I did mention to a Zeiss rep why did he just use 70% ethanol on our new 100x zillion NA TIRF objective when Zeiss say that repeated use will damage the objective, he said, well repeated use will damage the objective, but once or twice won’t matter [and then I thought ‘well I suppose it’s not actually his £8,000 objective’]. Other manufacturer’s actually recommend ethanol, e.g. Olympus. However immersion oil doesn’t dissolve in ethanol that well, hence another reason for the recommended use of other solvents, e.g. Petroleum ether - see http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379

*from the micro/primer site:  "In the past, solvents have been routinely employed for nearly any cleaning task in microscopy, and particularly for removal of immersion oil. Potential problems associated with solvent cleaning are sufficiently serious that the best current approach in cleaning the microscope is to use solvents only when absolutely necessary, essentially as a last resort rather than a first step. The issue of the use of solvents is further complicated and confused by contradictory recommendations in the scientific literature, as well as by differences in manufacturers' technical publications. Although alcohol and xylene are widely recommended as lens cleaning solvents, they are also named as being harmful to both the mechanical and optical components of many microscopes. Because of the variation in solvent recommendations, and the likelihood that some of the materials used in the instrument components are not known to the user, it is prudent to restrict use of any solvent to an absolute minimum.”

i.e. have a look at: http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html

Most of this discussion of solvents only applies to immersion oil objectives, where the oil isn’t miscible in water. Our inverted microscopes with all air objectives are rarely cleaned, otherwise it’s just a blow with a puffer. On microscopes where oil and air co-exist it’s often immersion oil contamination of the air objective that’s the problem, so it’s solvents again. In the days when only my group operated the microscopes, with all our oil immersion free upright microscopes the objectives almost never needed cleaning.

For other spillages such as culture medium [inverted microscope again] some solvents will probably fix biological muck onto the lens, and there’s the salts content, so the use of water based cleaners has been suggested [e.g. even breathing on the lens and then lens tissue, using optical/glass cleaning solutions]. Water drying onto the lens is a disaster though. Some even recommend things like breaking polystyrene foam [to get a clean surface] and gently rubbing the [oil free] lens with that. Or there’s Sparkle - whatever that is, here in the UK it was a silicon based furniture polish [yuk] not a commercial window glass cleaner. That’s the problem with industrial cleaners, who knows what’s in them or whether the constituents have been modified – you could try it on an inconspicuous area of the objective lens first, I suppose. Presumably optical lens cleaners are glass friendly though, and many use glass cleaning products with no reported problems. All the links in the previous posts [below] give loads of ideas for cleaning objectives [when necessary].

 

http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf

http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html

www.olympus.co.uk/microscopy/images/illum_cleaning.pdf
http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Chris Tully
Sent: 30 March 2009 16:40
To: [hidden email]
Subject: Re: Cleaning lens.

 

Dear all,

While working for a Leica Microsystems dealer the local field service engineer (factor trained) used a sequence of ethanol and heptane to clean truly dirty lenses.  For standard cleaning a lens wipe and Sparkle was his recommendation.  But for dried oil or the like he would graduate to cotton swabs and either ethanol then heptane or a 50:50 mix of the two.

Chris

Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-888-1021
http://www.linkedin.com/in/christully

On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery <[hidden email]> wrote:

Keith,

            Methylated spirit that’s what he said, although I still prefer and use ether when necessary.

Ian.

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: 30 March 2009 14:12

To: [hidden email]

Subject: Re: Cleaning lens.

 

Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’? Methylated spirit is mainly ethanol, and so best avoided - the Axiovert 100 manual says repeated use of 70% ethanol will damage the objectives [but you can use it if you want]. Generally the faster the solvent evaporation from the lens/cement area the better, hence the suggestion of the solvent [pure] diethyl ether by many [and that’s what I use].

 

‘Zeiss cleaning mixture L’, which the engineer’s now use since diethyl ether has been withdrawn from their kit, is 90% by volume ‘benzoline’ [petroleum ether sometimes called medical alcohol] and 10% ‘isopropanol’ [2-proponal, dimethyl carbinol, 2-hydroxyproparne]. The bottle says ‘Clean the optics by moving in circles, slight pressure should be exerted on optics during cleaning’. Petroleum ether or spirit isn’t the same as the diethyl ether solvent/anaesthetic often used to clean objectives, but apparently it does the job for Zeiss optics.  

 

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ian Montgomery
Sent: 30 March 2009 12:28
To: [hidden email]
Subject: Cleaning lens.

 

            In one of our teaching labs many years ago a student complained they were having a problem with the x100 OI objective and sure enough the image was lousy. I cleaned the objective and slide then re-applied a spot of oil and still the image was lousy. I then asked the student how exactly they had set up the microscope. Shock horror, my world collapsed. They had unscrewed the objective, filled it with oil, screwed it back on then put a drop on the slide. After weeks of trying to clean the objective it went into the trash as beyond economic repair.

            Cleaning objectives, I use the fluid recommended by the local Zeiss engineer, 90% methylated spirit and 10% isopropanol.

Ian.   

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

 

 

[Guy Cox]

When I first learnt about microscopes (a very long time ago) I was taught that while dry objectives often had their front element mounted with some adhesive, oil immersion objectives didn't - the element was held by a screw ring.  Hence it was safe to clean them with solvent - we usually used xylene because that was handy - it was also used in mounting specimens (with canada balsam).  Of course this didn't offer any solution to the clumsy clot who gets oil on the x40.  But that wasn't actually quite so common then because oil immersion lenses were longer - ie not parfocal - so if someone swung the turret round the dry lenses would clear the oil.  On the other hand this didn't make using oil lenses too easy - and they didn't have spring noses then either. 

 

Nowadays I'd have thought that if any adhesive is used it would be epoxy which is pretty much immune to solvents. 

 

 

                                                                  Guy

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, 1 April 2009 6:47 PM
To: [hidden email]
Subject: FW: Cleaning lens.

I think Zeiss’s comment that repeated use of ethanol will damage Zeiss’s lens cement was just that – use it a few times a day and the Zeiss objective will probably fail in 6 months or so [and this might be the case for many solvents, ethanol is simply one more readily at hand*]. Use ethanol every month or so and chances are the objective will last a lot longer [and fail for another reason]. Use ethanol on a very elderly microscope where the lenses are mounted in say gum resin though and you will destroy the objective pretty much instantly [or at least get a nasty smear of gum resin all over the clear bit] - hence some people’s historical aversion is justified. I did mention to a Zeiss rep why did he just use 70% ethanol on our new 100x zillion NA TIRF objective when Zeiss say that repeated use will damage the objective, he said, well repeated use will damage the objective, but once or twice won’t matter [and then I thought ‘well I suppose it’s not actually his £8,000 objective’]. Other manufacturer’s actually recommend ethanol, e.g. Olympus. However immersion oil doesn’t dissolve in ethanol that well, hence another reason for the recommended use of other solvents, e.g. Petroleum ether - see http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379

*from the micro/primer site:  "In the past, solvents have been routinely employed for nearly any cleaning task in microscopy, and particularly for removal of immersion oil. Potential problems associated with solvent cleaning are sufficiently serious that the best current approach in cleaning the microscope is to use solvents only when absolutely necessary, essentially as a last resort rather than a first step. The issue of the use of solvents is further complicated and confused by contradictory recommendations in the scientific literature, as well as by differences in manufacturers' technical publications. Although alcohol and xylene are widely recommended as lens cleaning solvents, they are also named as being harmful to both the mechanical and optical components of many microscopes. Because of the variation in solvent recommendations, and the likelihood that some of the materials used in the instrument components are not known to the user, it is prudent to restrict use of any solvent to an absolute minimum.”

i.e. have a look at: http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html

Most of this discussion of solvents only applies to immersion oil objectives, where the oil isn’t miscible in water. Our inverted microscopes with all air objectives are rarely cleaned, otherwise it’s just a blow with a puffer. On microscopes where oil and air co-exist it’s often immersion oil contamination of the air objective that’s the problem, so it’s solvents again. In the days when only my group operated the microscopes, with all our oil immersion free upright microscopes the objectives almost never needed cleaning.

For other spillages such as culture medium [inverted microscope again] some solvents will probably fix biological muck onto the lens, and there’s the salts content, so the use of water based cleaners has been suggested [e.g. even breathing on the lens and then lens tissue, using optical/glass cleaning solutions]. Water drying onto the lens is a disaster though. Some even recommend things like breaking polystyrene foam [to get a clean surface] and gently rubbing the [oil free] lens with that. Or there’s Sparkle - whatever that is, here in the UK it was a silicon based furniture polish [yuk] not a commercial window glass cleaner. That’s the problem with industrial cleaners, who knows what’s in them or whether the constituents have been modified – you could try it on an inconspicuous area of the objective lens first, I suppose. Presumably optical lens cleaners are glass friendly though, and many use glass cleaning products with no reported problems. All the links in the previous posts [below] give loads of ideas for cleaning objectives [when necessary].

 

http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf

http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html

www.olympus.co.uk/microscopy/images/illum_cleaning.pdf
http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Chris Tully
Sent: 30 March 2009 16:40
To: [hidden email]
Subject: Re: Cleaning lens.

 

Dear all,

While working for a Leica Microsystems dealer the local field service engineer (factor trained) used a sequence of ethanol and heptane to clean truly dirty lenses.  For standard cleaning a lens wipe and Sparkle was his recommendation.  But for dried oil or the like he would graduate to cotton swabs and either ethanol then heptane or a 50:50 mix of the two.

Chris

Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-888-1021
http://www.linkedin.com/in/christully

On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery <[hidden email]> wrote:

Keith,

            Methylated spirit that’s what he said, although I still prefer and use ether when necessary.

Ian.

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: 30 March 2009 14:12

To: [hidden email]

Subject: Re: Cleaning lens.

 

Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’? Methylated spirit is mainly ethanol, and so best avoided - the Axiovert 100 manual says repeated use of 70% ethanol will damage the objectives [but you can use it if you want]. Generally the faster the solvent evaporation from the lens/cement area the better, hence the suggestion of the solvent [pure] diethyl ether by many [and that’s what I use].

 

‘Zeiss cleaning mixture L’, which the engineer’s now use since diethyl ether has been withdrawn from their kit, is 90% by volume ‘benzoline’ [petroleum ether sometimes called medical alcohol] and 10% ‘isopropanol’ [2-proponal, dimethyl carbinol, 2-hydroxyproparne]. The bottle says ‘Clean the optics by moving in circles, slight pressure should be exerted on optics during cleaning’. Petroleum ether or spirit isn’t the same as the diethyl ether solvent/anaesthetic often used to clean objectives, but apparently it does the job for Zeiss optics.  

 

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ian Montgomery
Sent: 30 March 2009 12:28
To: [hidden email]
Subject: Cleaning lens.

 

            In one of our teaching labs many years ago a student complained they were having a problem with the x100 OI objective and sure enough the image was lousy. I cleaned the objective and slide then re-applied a spot of oil and still the image was lousy. I then asked the student how exactly they had set up the microscope. Shock horror, my world collapsed. They had unscrewed the objective, filled it with oil, screwed it back on then put a drop on the slide. After weeks of trying to clean the objective it went into the trash as beyond economic repair.

            Cleaning objectives, I use the fluid recommended by the local Zeiss engineer, 90% methylated spirit and 10% isopropanol.

Ian.   

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

 

 

No virus found in this incoming message.
Checked by AVG.
Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

No virus found in this outgoing message.
Checked by AVG.
Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

[Keith Morris]

Well I guess modern epoxy glue is pretty immune to solvents, but it’s how well it sticks to the metal and glass that might be the problem [there is talk about micro-fissures]. All I know is that modern oil objectives have a tendency to let in liquids after a year or two in constant use [or is it abuse] on our inverted microscopes.

 

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: 01 April 2009 11:00
To: [hidden email]
Subject: Re: Cleaning lens.

 

When I first learnt about microscopes (a very long time ago) I was taught that while dry objectives often had their front element mounted with some adhesive, oil immersion objectives didn't - the element was held by a screw ring.  Hence it was safe to clean them with solvent - we usually used xylene because that was handy - it was also used in mounting specimens (with canada balsam).  Of course this didn't offer any solution to the clumsy clot who gets oil on the x40.  But that wasn't actually quite so common then because oil immersion lenses were longer - ie not parfocal - so if someone swung the turret round the dry lenses would clear the oil.  On the other hand this didn't make using oil lenses too easy - and they didn't have spring noses then either. 

 

Nowadays I'd have thought that if any adhesive is used it would be epoxy which is pretty much immune to solvents. 

 

 

                                                                  Guy

 

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net

 

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, 1 April 2009 6:47 PM
To: [hidden email]
Subject: FW: Cleaning lens.

I think Zeiss’s comment that repeated use of ethanol will damage Zeiss’s lens cement was just that – use it a few times a day and the Zeiss objective will probably fail in 6 months or so [and this might be the case for many solvents, ethanol is simply one more readily at hand*]. Use ethanol every month or so and chances are the objective will last a lot longer [and fail for another reason]. Use ethanol on a very elderly microscope where the lenses are mounted in say gum resin though and you will destroy the objective pretty much instantly [or at least get a nasty smear of gum resin all over the clear bit] - hence some people’s historical aversion is justified. I did mention to a Zeiss rep why did he just use 70% ethanol on our new 100x zillion NA TIRF objective when Zeiss say that repeated use will damage the objective, he said, well repeated use will damage the objective, but once or twice won’t matter [and then I thought ‘well I suppose it’s not actually his £8,000 objective’]. Other manufacturer’s actually recommend ethanol, e.g. Olympus. However immersion oil doesn’t dissolve in ethanol that well, hence another reason for the recommended use of other solvents, e.g. Petroleum ether - see http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379

*from the micro/primer site:  "In the past, solvents have been routinely employed for nearly any cleaning task in microscopy, and particularly for removal of immersion oil. Potential problems associated with solvent cleaning are sufficiently serious that the best current approach in cleaning the microscope is to use solvents only when absolutely necessary, essentially as a last resort rather than a first step. The issue of the use of solvents is further complicated and confused by contradictory recommendations in the scientific literature, as well as by differences in manufacturers' technical publications. Although alcohol and xylene are widely recommended as lens cleaning solvents, they are also named as being harmful to both the mechanical and optical components of many microscopes. Because of the variation in solvent recommendations, and the likelihood that some of the materials used in the instrument components are not known to the user, it is prudent to restrict use of any solvent to an absolute minimum.”

i.e. have a look at: http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html

Most of this discussion of solvents only applies to immersion oil objectives, where the oil isn’t miscible in water. Our inverted microscopes with all air objectives are rarely cleaned, otherwise it’s just a blow with a puffer. On microscopes where oil and air co-exist it’s often immersion oil contamination of the air objective that’s the problem, so it’s solvents again. In the days when only my group operated the microscopes, with all our oil immersion free upright microscopes the objectives almost never needed cleaning.

For other spillages such as culture medium [inverted microscope again] some solvents will probably fix biological muck onto the lens, and there’s the salts content, so the use of water based cleaners has been suggested [e.g. even breathing on the lens and then lens tissue, using optical/glass cleaning solutions]. Water drying onto the lens is a disaster though. Some even recommend things like breaking polystyrene foam [to get a clean surface] and gently rubbing the [oil free] lens with that. Or there’s Sparkle - whatever that is, here in the UK it was a silicon based furniture polish [yuk] not a commercial window glass cleaner. That’s the problem with industrial cleaners, who knows what’s in them or whether the constituents have been modified – you could try it on an inconspicuous area of the objective lens first, I suppose. Presumably optical lens cleaners are glass friendly though, and many use glass cleaning products with no reported problems. All the links in the previous posts [below] give loads of ideas for cleaning objectives [when necessary].

 

http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf

http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html

www.olympus.co.uk/microscopy/images/illum_cleaning.pdf
http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Chris Tully
Sent: 30 March 2009 16:40
To: [hidden email]
Subject: Re: Cleaning lens.

 

Dear all,

While working for a Leica Microsystems dealer the local field service engineer (factor trained) used a sequence of ethanol and heptane to clean truly dirty lenses.  For standard cleaning a lens wipe and Sparkle was his recommendation.  But for dried oil or the like he would graduate to cotton swabs and either ethanol then heptane or a 50:50 mix of the two.

Chris

Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-888-1021
http://www.linkedin.com/in/christully

On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery <[hidden email]> wrote:

Keith,

            Methylated spirit that’s what he said, although I still prefer and use ether when necessary.

Ian.

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: 30 March 2009 14:12

To: [hidden email]

Subject: Re: Cleaning lens.

 

Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’? Methylated spirit is mainly ethanol, and so best avoided - the Axiovert 100 manual says repeated use of 70% ethanol will damage the objectives [but you can use it if you want]. Generally the faster the solvent evaporation from the lens/cement area the better, hence the suggestion of the solvent [pure] diethyl ether by many [and that’s what I use].

 

‘Zeiss cleaning mixture L’, which the engineer’s now use since diethyl ether has been withdrawn from their kit, is 90% by volume ‘benzoline’ [petroleum ether sometimes called medical alcohol] and 10% ‘isopropanol’ [2-proponal, dimethyl carbinol, 2-hydroxyproparne]. The bottle says ‘Clean the optics by moving in circles, slight pressure should be exerted on optics during cleaning’. Petroleum ether or spirit isn’t the same as the diethyl ether solvent/anaesthetic often used to clean objectives, but apparently it does the job for Zeiss optics.  

 

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ian Montgomery
Sent: 30 March 2009 12:28
To: [hidden email]
Subject: Cleaning lens.

 

            In one of our teaching labs many years ago a student complained they were having a problem with the x100 OI objective and sure enough the image was lousy. I cleaned the objective and slide then re-applied a spot of oil and still the image was lousy. I then asked the student how exactly they had set up the microscope. Shock horror, my world collapsed. They had unscrewed the objective, filled it with oil, screwed it back on then put a drop on the slide. After weeks of trying to clean the objective it went into the trash as beyond economic repair.

            Cleaning objectives, I use the fluid recommended by the local Zeiss engineer, 90% methylated spirit and 10% isopropanol.

Ian.   

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

 

 

 

No virus found in this incoming message.
Checked by AVG.
Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

No virus found in this outgoing message.
Checked by AVG.
Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

[Leigh Silvester]

Unrelated really but, our biggest problems is undergraduates on the teaching lab microscopes getting DPX on the lenses.

While they are always told how to avoid this, drop the stage before swinging a high magnification lens into place, in the excitement a fair number forget this after having tipped the contents of the DPX bottle onto the slide and floated a cover slip on the top.

 

I have been told that chloroform should get this off, however this has not been particularly successful when the DPX has dried on.

 

Of course I do get the DPX off but I hesitate to mention my technique.

 

Anyone come across a tried and tested method that doesn't involve careful application of a razor blade?

 

Leigh Silvester

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: 01 April 2009 11:00
To: [hidden email]
Subject: Re: Cleaning lens.

When I first learnt about microscopes (a very long time ago) I was taught that while dry objectives often had their front element mounted with some adhesive, oil immersion objectives didn't - the element was held by a screw ring.  Hence it was safe to clean them with solvent - we usually used xylene because that was handy - it was also used in mounting specimens (with canada balsam).  Of course this didn't offer any solution to the clumsy clot who gets oil on the x40.  But that wasn't actually quite so common then because oil immersion lenses were longer - ie not parfocal - so if someone swung the turret round the dry lenses would clear the oil.  On the other hand this didn't make using oil lenses too easy - and they didn't have spring noses then either. 

 

Nowadays I'd have thought that if any adhesive is used it would be epoxy which is pretty much immune to solvents. 

 

 

                                                                  Guy

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, 1 April 2009 6:47 PM
To: [hidden email]
Subject: FW: Cleaning lens.

I think Zeiss’s comment that repeated use of ethanol will damage Zeiss’s lens cement was just that – use it a few times a day and the Zeiss objective will probably fail in 6 months or so [and this might be the case for many solvents, ethanol is simply one more readily at hand*]. Use ethanol every month or so and chances are the objective will last a lot longer [and fail for another reason]. Use ethanol on a very elderly microscope where the lenses are mounted in say gum resin though and you will destroy the objective pretty much instantly [or at least get a nasty smear of gum resin all over the clear bit] - hence some people’s historical aversion is justified. I did mention to a Zeiss rep why did he just use 70% ethanol on our new 100x zillion NA TIRF objective when Zeiss say that repeated use will damage the objective, he said, well repeated use will damage the objective, but once or twice won’t matter [and then I thought ‘well I suppose it’s not actually his £8,000 objective’]. Other manufacturer’s actually recommend ethanol, e.g. Olympus. However immersion oil doesn’t dissolve in ethanol that well, hence another reason for the recommended use of other solvents, e.g. Petroleum ether - see http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379

*from the micro/primer site:  "In the past, solvents have been routinely employed for nearly any cleaning task in microscopy, and particularly for removal of immersion oil. Potential problems associated with solvent cleaning are sufficiently serious that the best current approach in cleaning the microscope is to use solvents only when absolutely necessary, essentially as a last resort rather than a first step. The issue of the use of solvents is further complicated and confused by contradictory recommendations in the scientific literature, as well as by differences in manufacturers' technical publications. Although alcohol and xylene are widely recommended as lens cleaning solvents, they are also named as being harmful to both the mechanical and optical components of many microscopes. Because of the variation in solvent recommendations, and the likelihood that some of the materials used in the instrument components are not known to the user, it is prudent to restrict use of any solvent to an absolute minimum.”

i.e. have a look at: http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html

Most of this discussion of solvents only applies to immersion oil objectives, where the oil isn’t miscible in water. Our inverted microscopes with all air objectives are rarely cleaned, otherwise it’s just a blow with a puffer. On microscopes where oil and air co-exist it’s often immersion oil contamination of the air objective that’s the problem, so it’s solvents again. In the days when only my group operated the microscopes, with all our oil immersion free upright microscopes the objectives almost never needed cleaning.

For other spillages such as culture medium [inverted microscope again] some solvents will probably fix biological muck onto the lens, and there’s the salts content, so the use of water based cleaners has been suggested [e.g. even breathing on the lens and then lens tissue, using optical/glass cleaning solutions]. Water drying onto the lens is a disaster though. Some even recommend things like breaking polystyrene foam [to get a clean surface] and gently rubbing the [oil free] lens with that. Or there’s Sparkle - whatever that is, here in the UK it was a silicon based furniture polish [yuk] not a commercial window glass cleaner. That’s the problem with industrial cleaners, who knows what’s in them or whether the constituents have been modified – you could try it on an inconspicuous area of the objective lens first, I suppose. Presumably optical lens cleaners are glass friendly though, and many use glass cleaning products with no reported problems. All the links in the previous posts [below] give loads of ideas for cleaning objectives [when necessary].

 

http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf

http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html

www.olympus.co.uk/microscopy/images/illum_cleaning.pdf
http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Chris Tully
Sent: 30 March 2009 16:40
To: [hidden email]
Subject: Re: Cleaning lens.

 

Dear all,

While working for a Leica Microsystems dealer the local field service engineer (factor trained) used a sequence of ethanol and heptane to clean truly dirty lenses.  For standard cleaning a lens wipe and Sparkle was his recommendation.  But for dried oil or the like he would graduate to cotton swabs and either ethanol then heptane or a 50:50 mix of the two.

Chris

Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-888-1021
http://www.linkedin.com/in/christully

On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery <[hidden email]> wrote:

Keith,

            Methylated spirit that’s what he said, although I still prefer and use ether when necessary.

Ian.

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: 30 March 2009 14:12

To: [hidden email]

Subject: Re: Cleaning lens.

 

Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’? Methylated spirit is mainly ethanol, and so best avoided - the Axiovert 100 manual says repeated use of 70% ethanol will damage the objectives [but you can use it if you want]. Generally the faster the solvent evaporation from the lens/cement area the better, hence the suggestion of the solvent [pure] diethyl ether by many [and that’s what I use].

 

‘Zeiss cleaning mixture L’, which the engineer’s now use since diethyl ether has been withdrawn from their kit, is 90% by volume ‘benzoline’ [petroleum ether sometimes called medical alcohol] and 10% ‘isopropanol’ [2-proponal, dimethyl carbinol, 2-hydroxyproparne]. The bottle says ‘Clean the optics by moving in circles, slight pressure should be exerted on optics during cleaning’. Petroleum ether or spirit isn’t the same as the diethyl ether solvent/anaesthetic often used to clean objectives, but apparently it does the job for Zeiss optics.  

 

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ian Montgomery
Sent: 30 March 2009 12:28
To: [hidden email]
Subject: Cleaning lens.

 

            In one of our teaching labs many years ago a student complained they were having a problem with the x100 OI objective and sure enough the image was lousy. I cleaned the objective and slide then re-applied a spot of oil and still the image was lousy. I then asked the student how exactly they had set up the microscope. Shock horror, my world collapsed. They had unscrewed the objective, filled it with oil, screwed it back on then put a drop on the slide. After weeks of trying to clean the objective it went into the trash as beyond economic repair.

            Cleaning objectives, I use the fluid recommended by the local Zeiss engineer, 90% methylated spirit and 10% isopropanol.

Ian.   

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

 

 

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[Martin Wessendorf-2]

Leigh Silvester wrote:

> I have been told that chloroform should get this off, however this has
> not been particularly successful when the DPX has dried on.
>  
> Of course I do get the DPX off but I hesitate to mention my technique.
>  
> Anyone come across a tried and tested method that doesn't involve
> careful application of a razor blade?

I think it was our Olympus rep who told us to wait till the DPX dried
hard, and then just chip it off with the broken end of a wooden
applicator.  That seems to have worked beautifully for us the couple of
times that it happened.

Any comments on the wisdom of using wood, from the plant biologists out
there?

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455
**MY E-MAIL ADDRESS HAS CHANGED.  PLEASE USE [hidden email] **

[Dale Callaham]

Hi,

I've tried to follow most of this thread but may have missed some. Seems
that most of the discussion is related to solvents to remove (primarily)
oil. I haven't seem much mention of the aqueous-based cleaners. We have
only used these type of cleaners for decades with no problems. Our
standard procedure for dry lenses is to use a dry cotton swab to mop up
the excess oil in the groove and the surrounding parts that are NOT the
optical surface; then clean those same areas with a swab just moistened
with a single drop of lens cleaner (Kodak lens cleaner, Sparkle glass
cleaner (was recommended by service personnel of two major microscope
companies) or others; Zeiss supplied an aqueous lens cleaner and we
found a commercial product that is reputed to be the same material -
UltraClarity from FilmTools). I think some of these contain a bit of
isopropanol.

Finally, with a cotton swab just moistened and a very light touch, start
at the center of the lens and while spinning the swab between the
fingers also spiral the swab from the center to perimeter; this step may
need to be repeated. There should never be enough lens cleaner that it
sits on the lens - you should see the evaporating film receding
immediately if the cotton swab was just moistened.

In our multiuser facility where we have a range of users from novice to
seasoned professionals and undergrads to advanced postgrads and
professors (note the separation of the categories - they don't
necessarily line up) and we regularly find oil on the dry objectives and
perform this cleaning routine and the lenses do not appear to have
suffered from this method. The use of the objective to crush slides is
another matter; we have deep scoring on the face of the chromed-brass
barrel of a couple of objectives but (amazingly) no damage to the
optical surface which is slightly recessed and concave on the 20x and
40x - the long ones most likely to get oiled or be used for slide
crushers.....

I have always taken the view that oil lenses are designed to be in oil
(and are designed for that) and we just have people use a small square
of Lensx-90 to gently wipe the excess until the tissue "appears" dry -
obviously there will still be a film of oil but the lenses have not
failed in any respect from over 15yr of this protocol. Our microscopes
are used daily and frequently. For the record we use Cargille "DF" oil,
or the Zeiss 518F oil on our Axiovert200.

Once in a while - for mystery materials that range from mounting cement
to nail polish, etc. - we do use xylene sparingly as described for the
aqueous cleaner; first clean up the bulk of the mess away from the
optical surface and always use a just-moistened swab; again the cleaner
should not remain sitting on the lens.

I'm not reporting this as a "you should do it this way" statement; I
just wanted to add to the knowledge base of possible lens care and lens
survivability.

Dale

>     ------------------------------------------------------------------------
>     *From:* Confocal Microscopy List
>     [mailto:[hidden email]] *On Behalf Of *Guy Cox
>     *Sent:* 01 April 2009 11:00
>     *To:* [hidden email]
>     *Subject:* Re: Cleaning lens.
>
>     When I first learnt about microscopes (a very long time ago) I was
>     taught that while dry objectives often had their front element
>     mounted with some adhesive, oil immersion objectives didn't - the
>     element was held by a screw ring.  Hence it was safe to clean them
>     with solvent - we usually used xylene because that was handy - it
>     was also used in mounting specimens (with canada balsam).  Of course
>     this didn't offer any solution to the clumsy clot who gets oil on
>     the x40.  But that wasn't actually quite so common then because oil
>     immersion lenses were longer - ie not parfocal - so if someone swung
>     the turret round the dry lenses would clear the oil.  On the other
>     hand this didn't make using oil lenses too easy - and they didn't
>     have spring noses then either.
>      
>     Nowadays I'd have thought that if any adhesive is used it would be
>     epoxy which is pretty much immune to solvents.
>      
>      
>                                                                       Guy
>      
>
>     Optical Imaging Techniques in Cell Biology
>     by Guy Cox    CRC Press / Taylor & Francis
>         http://www.guycox.com/optical.htm
>     ______________________________________________
>     Associate Professor Guy Cox, MA, DPhil(Oxon)
>     Electron Microscope Unit, Madsen Building F09,
>     University of Sydney, NSW 2006
>     ______________________________________________
>     Phone [phone]     Fax [phone]
>     Mobile [phone]
>     ______________________________________________
>          http://www.guycox.net <http://www.guycox.net/>
>
>      
>
>     ------------------------------------------------------------------------
>     *From:* Confocal Microscopy List
>     [mailto:[hidden email]] *On Behalf Of *Keith Morris
>     *Sent:* Wednesday, 1 April 2009 6:47 PM
>     *To:* [hidden email]
>     *Subject:* FW: Cleaning lens.
>
>     I think Zeiss’s comment that repeated use of ethanol will damage
>     Zeiss’s lens cement was just that – use it a few times a day and the
>     Zeiss objective will probably fail in 6 months or so [and this might
>     be the case for many solvents, ethanol is simply one more readily at
>     hand*]. Use ethanol every month or so and chances are the objective
>     will last a lot longer [and fail for another reason]. Use ethanol on
>     a very elderly microscope where the lenses are mounted in say gum
>     resin though and you will destroy the objective pretty much
>     instantly [or at least get a nasty smear of gum resin all over the
>     clear bit] - hence some people’s historical aversion is justified. I
>     did mention to a Zeiss rep why did he just use 70% ethanol on our
>     new 100x zillion NA TIRF objective when Zeiss say that repeated use
>     will damage the objective, he said, well repeated use will damage
>     the objective, but once or twice won’t matter [and then I thought
>     ‘well I suppose it’s not actually his £8,000 objective’]. Other
>     manufacturer’s actually recommend ethanol, e.g. Olympus. However
>     immersion oil doesn’t dissolve in ethanol that well, hence another
>     reason for the recommended use of other solvents, e.g. Petroleum
>     ether - see
>     http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379
>
>     *from the micro/primer site:  "In the past, solvents have been
>     routinely employed for nearly any cleaning task in microscopy, and
>     particularly for removal of immersion oil. Potential problems
>     associated with solvent cleaning are sufficiently serious that the
>     best current approach in cleaning the microscope is to use solvents
>     only when absolutely necessary, essentially as a last resort rather
>     than a first step. The issue of the use of solvents is further
>     complicated and confused by contradictory recommendations in the
>     scientific literature, as well as by differences in manufacturers'
>     technical publications. Although alcohol and xylene are widely
>     recommended as lens cleaning solvents, they are also named as being
>     harmful to both the mechanical and optical components of many
>     microscopes. Because of the variation in solvent recommendations,
>     and the likelihood that some of the materials used in the instrument
>     components are not known to the user, it is prudent to restrict use
>     of any solvent to an absolute minimum.”
>
>     i.e. have a look at:
>     http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html
>
>     Most of this discussion of solvents only applies to immersion oil
>     objectives, where the oil isn’t miscible in water. Our inverted
>     microscopes with all air objectives are rarely cleaned, otherwise
>     it’s just a blow with a puffer. On microscopes where oil and air
>     co-exist it’s often immersion oil contamination of the air objective
>     that’s the problem, so it’s solvents again. In the days when only my
>     group operated the microscopes, with all our oil immersion free
>     upright microscopes the objectives almost never needed cleaning.
>
>     For other spillages such as culture medium [inverted microscope
>     again] some solvents will probably fix biological muck onto the
>     lens, and there’s the salts content, so the use of water based
>     cleaners has been suggested [e.g. even breathing on the lens and
>     then lens tissue, using optical/glass cleaning solutions]. Water
>     drying onto the lens is a disaster though. Some even recommend
>     things like breaking polystyrene foam [to get a clean surface] and
>     gently rubbing the [oil free] lens with that. Or there’s Sparkle -
>     whatever that is, here in the UK it was a silicon based furniture
>     polish [yuk] not a commercial window glass cleaner. That’s the
>     problem with industrial cleaners, who knows what’s in them or
>     whether the constituents have been modified – you could try it on an
>     inconspicuous area of the objective lens first, I suppose.
>     Presumably optical lens cleaners are glass friendly though, and many
>     use glass cleaning products with no reported problems. All the links
>     in the previous posts [below] give loads of ideas for cleaning
>     objectives [when necessary].
>
>      
>
>     _http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf_
>
>
>     http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result
>     <http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result>
>
>     http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html//
>
>     /
>     <http://www.olympus.co.uk/microscopy/images/illum_cleaning.pdf>www.*olympus.co.uk/microscopy/images/illum_*cleaning.pdf**
>     <http://www.olympus.co.uk/microscopy/images/illum_cleaning.pdf>********/**/
>     /http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html**
>
>     ** **
>
>     **
>     Keith**
>
>     ** **
>     ** **
>
>     **---------------------------------------------------------------------------
>     Dr Keith J. Morris,
>     Molecular Cytogenetics and Microscopy Core,
>     Laboratory 00/069 and 00/070,
>     The Wellcome Trust Centre for Human Genetics,
>     Roosevelt Drive,
>     Oxford  OX3 7BN,
>     United Kingdom.
>
>     Telephone:  [phone]
>     Email:  [hidden email]
>     Web-pages: http://www.well.ox.ac.uk/cytogenetics/**
>
>     ** **
>     ** **
>     **
>     ------------------------------------------------------------------------
>     **
>     ** **
>
>     ***From:* Confocal Microscopy List
>     [mailto:[hidden email]] *On Behalf Of *Chris Tully
>     *Sent:* 30 March 2009 16:40
>     *To:* [hidden email]
>     *Subject:* Re: Cleaning lens.**
>
>     ** **
>
>     ** **
>
>     ** **
>
>     **Dear all,
>
>     While working for a Leica Microsystems dealer the local field
>     service engineer (factor trained) used a sequence of ethanol and
>     heptane to clean truly dirty lenses.  For standard cleaning a lens
>     wipe and Sparkle was his recommendation.  But for dried oil or the
>     like he would graduate to cotton swabs and either ethanol then
>     heptane or a 50:50 mix of the two.
>
>     Chris
>
>     Chris Tully
>     Microscopy and Image Analysis Expert
>     [hidden email] <mailto:[hidden email]>
>     240-888-1021
>     http://www.linkedin.com/in/christully**
>
>     ** **
>     ** **
>
>     **On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery
>     <[hidden email] <mailto:[hidden email]>>
>     wrote:**
>
>     ** **
>     ** **
>     ** **
>
>     **Keith,**
>
>     ** **
>
>     **            Methylated spirit that’s what he said, although I
>     still prefer and use ether when necessary.**
>
>     ** **
>     ** **
>
>     **Ian. **
>
>     ** **
>
>     ** **
>
>     ** **
>     ** **
>
>     **Dr. Ian Montgomery,**
>
>     ** **
>
>     **Histotechnology,**
>
>     ** **
>
>     **I.B.L.S. Support Unit,**
>
>     ** **
>
>     **Thomson Building,**
>
>     ** **
>
>     **University of Glasgow,**
>
>     ** **
>
>     **Glasgow,**
>
>     ** **
>
>     **G12 8QQ.**
>
>     ** **
>     ** **
>     **
>     ------------------------------------------------------------------------
>     **
>     ** **
>
>     ***From:* Confocal Microscopy List
>     [mailto:[hidden email]
>     <mailto:[hidden email]>] *On Behalf Of *Keith Morris
>     *Sent:* 30 March 2009 14:12**
>
>     ** **
>     ** **
>
>     **
>     *To:* [hidden email]
>     <mailto:[hidden email]>**
>
>     ** **
>
>     ***Subject:* Re: Cleaning lens.**
>
>     ** **
>     ** **
>     ** **
>
>     ** **
>
>     ** **
>
>     **Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’?
>     Methylated spirit is mainly ethanol, and so best avoided - the
>     Axiovert 100 manual says repeated use of 70% ethanol will damage the
>     objectives [but you can use it if you want]. Generally the faster
>     the solvent evaporation from the lens/cement area the better, hence
>     the suggestion of the solvent [pure] diethyl ether by many [and
>     that’s what I use].**
>
>     ** **
>
>     ** **
>
>     ** **
>
>     **‘Zeiss cleaning mixture L’, which the engineer’s now use since
>     diethyl ether has been withdrawn from their kit, is 90% by volume
>     ‘benzoline’ [petroleum ether sometimes called medical alcohol] and
>     10% ‘isopropanol’ [2-proponal, dimethyl carbinol,
>     2-hydroxyproparne]. The bottle says ‘Clean the optics by moving in
>     circles, slight pressure should be exerted on optics during
>     cleaning’. Petroleum ether or spirit isn’t the same as the diethyl
>     ether solvent/anaesthetic often used to clean objectives, but
>     apparently it does the job for Zeiss optics.  **
>
>     ** **
>
>     ** **
>
>     ** **
>
>     **Keith**
>
>     ** **
>     ** **
>
>     **---------------------------------------------------------------------------
>     Dr Keith J. Morris,
>     Molecular Cytogenetics and Microscopy Core,
>     Laboratory 00/069 and 00/070,
>     The Wellcome Trust Centre for Human Genetics,
>     Roosevelt Drive,
>     Oxford  OX3 7BN,
>     United Kingdom.
>
>     Telephone:  [phone]
>     Email:  [hidden email] <mailto:[hidden email]>
>     Web-pages: http://www.well.ox.ac.uk/cytogenetics/**
>
>     ** **
>     ** **
>     **
>     ------------------------------------------------------------------------
>     **
>     ** **
>
>     ***From:* Confocal Microscopy List
>     [mailto:[hidden email]
>     <mailto:[hidden email]>] *On Behalf Of *Ian Montgomery
>     *Sent:* 30 March 2009 12:28
>     *To:* [hidden email]
>     <mailto:[hidden email]>
>     *Subject:* Cleaning lens.**
>
>     ** **
>
>     ** **
>
>     ** **
>
>     **            In one of our teaching labs many years ago a student
>     complained they were having a problem with the x100 OI objective and
>     sure enough the image was lousy. I cleaned the objective and slide
>     then re-applied a spot of oil and still the image was lousy. I then
>     asked the student how exactly they had set up the microscope. Shock
>     horror, my world collapsed. They had unscrewed the objective, filled
>     it with oil, screwed it back on then put a drop on the slide. After
>     weeks of trying to clean the objective it went into the trash as
>     beyond economic repair.**
>
>     ** **
>
>     **            Cleaning objectives, I use the fluid recommended by
>     the local Zeiss engineer, 90% methylated spirit and 10% isopropanol.**
>
>     ** **
>
>     **Ian.   **
>
>     ** **
>
>     ** **
>
>     ** **
>
>     **Dr. Ian Montgomery,**
>
>     ** **
>
>     **Histotechnology,**
>
>     ** **
>
>     **I.B.L.S. Support Unit,**
>
>     ** **
>
>     **Thomson Building,**
>
>     ** **
>
>     **University of Glasgow,**
>
>     ** **
>
>     **Glasgow,**
>
>     ** **
>
>     **G12 8QQ.**
>
>     ** **
>
>     ** **
>
>     ** **
>
>     ** **
>
>     **
>     **
>
>     **No virus found in this incoming message.
>     Checked by AVG.
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>     31/03/2009 1:05 PM
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[Keith Morris]

Hi,

 

Not tried it with objectives, but I have always used Xylene to soak off DPX mounted coverslips. As DPX dries to essentially a plastic the razor blade or wooden stick idea sounds good too [perhaps with Xylene to loosen the DPX’s grip on the glass and save the lens coating]. DPX [polystyrene resin] comes dissolved in Xylene, so I guess that’s why it works [mostly] – you soak the slide for a fair time though. Xylene was used to used to clean objectives in the old days, so it might be OK for occasional use with them …oops drifting back to solvents again. Histomount using toluene as the base, plus Benzene has similar properties.

 

Keith

 

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Leigh Silvester
Sent: 01 April 2009 12:05
To: [hidden email]
Subject: Re: Cleaning lens.

 

Unrelated really but, our biggest problems is undergraduates on the teaching lab microscopes getting DPX on the lenses.

While they are always told how to avoid this, drop the stage before swinging a high magnification lens into place, in the excitement a fair number forget this after having tipped the contents of the DPX bottle onto the slide and floated a cover slip on the top.

 

I have been told that chloroform should get this off, however this has not been particularly successful when the DPX has dried on.

 

Of course I do get the DPX off but I hesitate to mention my technique.

 

Anyone come across a tried and tested method that doesn't involve careful application of a razor blade?

 

Leigh Silvester

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: 01 April 2009 11:00
To: [hidden email]
Subject: Re: Cleaning lens.

When I first learnt about microscopes (a very long time ago) I was taught that while dry objectives often had their front element mounted with some adhesive, oil immersion objectives didn't - the element was held by a screw ring.  Hence it was safe to clean them with solvent - we usually used xylene because that was handy - it was also used in mounting specimens (with canada balsam).  Of course this didn't offer any solution to the clumsy clot who gets oil on the x40.  But that wasn't actually quite so common then because oil immersion lenses were longer - ie not parfocal - so if someone swung the turret round the dry lenses would clear the oil.  On the other hand this didn't make using oil lenses too easy - and they didn't have spring noses then either. 

 

Nowadays I'd have thought that if any adhesive is used it would be epoxy which is pretty much immune to solvents. 

 

 

                                                                  Guy

 

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net

 

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, 1 April 2009 6:47 PM
To: [hidden email]
Subject: FW: Cleaning lens.

I think Zeiss’s comment that repeated use of ethanol will damage Zeiss’s lens cement was just that – use it a few times a day and the Zeiss objective will probably fail in 6 months or so [and this might be the case for many solvents, ethanol is simply one more readily at hand*]. Use ethanol every month or so and chances are the objective will last a lot longer [and fail for another reason]. Use ethanol on a very elderly microscope where the lenses are mounted in say gum resin though and you will destroy the objective pretty much instantly [or at least get a nasty smear of gum resin all over the clear bit] - hence some people’s historical aversion is justified. I did mention to a Zeiss rep why did he just use 70% ethanol on our new 100x zillion NA TIRF objective when Zeiss say that repeated use will damage the objective, he said, well repeated use will damage the objective, but once or twice won’t matter [and then I thought ‘well I suppose it’s not actually his £8,000 objective’]. Other manufacturer’s actually recommend ethanol, e.g. Olympus. However immersion oil doesn’t dissolve in ethanol that well, hence another reason for the recommended use of other solvents, e.g. Petroleum ether - see http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379

*from the micro/primer site:  "In the past, solvents have been routinely employed for nearly any cleaning task in microscopy, and particularly for removal of immersion oil. Potential problems associated with solvent cleaning are sufficiently serious that the best current approach in cleaning the microscope is to use solvents only when absolutely necessary, essentially as a last resort rather than a first step. The issue of the use of solvents is further complicated and confused by contradictory recommendations in the scientific literature, as well as by differences in manufacturers' technical publications. Although alcohol and xylene are widely recommended as lens cleaning solvents, they are also named as being harmful to both the mechanical and optical components of many microscopes. Because of the variation in solvent recommendations, and the likelihood that some of the materials used in the instrument components are not known to the user, it is prudent to restrict use of any solvent to an absolute minimum.”

i.e. have a look at: http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html

Most of this discussion of solvents only applies to immersion oil objectives, where the oil isn’t miscible in water. Our inverted microscopes with all air objectives are rarely cleaned, otherwise it’s just a blow with a puffer. On microscopes where oil and air co-exist it’s often immersion oil contamination of the air objective that’s the problem, so it’s solvents again. In the days when only my group operated the microscopes, with all our oil immersion free upright microscopes the objectives almost never needed cleaning.

For other spillages such as culture medium [inverted microscope again] some solvents will probably fix biological muck onto the lens, and there’s the salts content, so the use of water based cleaners has been suggested [e.g. even breathing on the lens and then lens tissue, using optical/glass cleaning solutions]. Water drying onto the lens is a disaster though. Some even recommend things like breaking polystyrene foam [to get a clean surface] and gently rubbing the [oil free] lens with that. Or there’s Sparkle - whatever that is, here in the UK it was a silicon based furniture polish [yuk] not a commercial window glass cleaner. That’s the problem with industrial cleaners, who knows what’s in them or whether the constituents have been modified – you could try it on an inconspicuous area of the objective lens first, I suppose. Presumably optical lens cleaners are glass friendly though, and many use glass cleaning products with no reported problems. All the links in the previous posts [below] give loads of ideas for cleaning objectives [when necessary].

 

http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf

http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html

www.olympus.co.uk/microscopy/images/illum_cleaning.pdf
http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Chris Tully
Sent: 30 March 2009 16:40
To: [hidden email]
Subject: Re: Cleaning lens.

 

Dear all,

While working for a Leica Microsystems dealer the local field service engineer (factor trained) used a sequence of ethanol and heptane to clean truly dirty lenses.  For standard cleaning a lens wipe and Sparkle was his recommendation.  But for dried oil or the like he would graduate to cotton swabs and either ethanol then heptane or a 50:50 mix of the two.

Chris

Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-888-1021
http://www.linkedin.com/in/christully

On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery <[hidden email]> wrote:

Keith,

            Methylated spirit that’s what he said, although I still prefer and use ether when necessary.

Ian.

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: 30 March 2009 14:12

To: [hidden email]

Subject: Re: Cleaning lens.

 

Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’? Methylated spirit is mainly ethanol, and so best avoided - the Axiovert 100 manual says repeated use of 70% ethanol will damage the objectives [but you can use it if you want]. Generally the faster the solvent evaporation from the lens/cement area the better, hence the suggestion of the solvent [pure] diethyl ether by many [and that’s what I use].

 

‘Zeiss cleaning mixture L’, which the engineer’s now use since diethyl ether has been withdrawn from their kit, is 90% by volume ‘benzoline’ [petroleum ether sometimes called medical alcohol] and 10% ‘isopropanol’ [2-proponal, dimethyl carbinol, 2-hydroxyproparne]. The bottle says ‘Clean the optics by moving in circles, slight pressure should be exerted on optics during cleaning’. Petroleum ether or spirit isn’t the same as the diethyl ether solvent/anaesthetic often used to clean objectives, but apparently it does the job for Zeiss optics.  

 

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ian Montgomery
Sent: 30 March 2009 12:28
To: [hidden email]
Subject: Cleaning lens.

 

            In one of our teaching labs many years ago a student complained they were having a problem with the x100 OI objective and sure enough the image was lousy. I cleaned the objective and slide then re-applied a spot of oil and still the image was lousy. I then asked the student how exactly they had set up the microscope. Shock horror, my world collapsed. They had unscrewed the objective, filled it with oil, screwed it back on then put a drop on the slide. After weeks of trying to clean the objective it went into the trash as beyond economic repair.

            Cleaning objectives, I use the fluid recommended by the local Zeiss engineer, 90% methylated spirit and 10% isopropanol.

Ian.   

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

 

 

 

No virus found in this incoming message.
Checked by AVG.
Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

 

No virus found in this outgoing message.
Checked by AVG.
Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation.

[Guy Cox]

Well, I'd say that is almost certainly through the spring mount.  Our protocol on inverted microscopes is to remove and clean all immersion objectives after each session and store them in their containers, tip down.  We made a stand in the drawer such that the lens boxes only fit that way up.  If that isn't feasible (or isn't successful)  in your lab then you'd better try a Steve Cody (adapt a condom to fit over the lens).

 

                                                    Guy

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, 1 April 2009 8:41 PM
To: [hidden email]
Subject: Re: Cleaning lens.

Well I guess modern epoxy glue is pretty immune to solvents, but it’s how well it sticks to the metal and glass that might be the problem [there is talk about micro-fissures]. All I know is that modern oil objectives have a tendency to let in liquids after a year or two in constant use [or is it abuse] on our inverted microscopes.

 

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: 01 April 2009 11:00
To: [hidden email]
Subject: Re: Cleaning lens.

 

When I first learnt about microscopes (a very long time ago) I was taught that while dry objectives often had their front element mounted with some adhesive, oil immersion objectives didn't - the element was held by a screw ring.  Hence it was safe to clean them with solvent - we usually used xylene because that was handy - it was also used in mounting specimens (with canada balsam).  Of course this didn't offer any solution to the clumsy clot who gets oil on the x40.  But that wasn't actually quite so common then because oil immersion lenses were longer - ie not parfocal - so if someone swung the turret round the dry lenses would clear the oil.  On the other hand this didn't make using oil lenses too easy - and they didn't have spring noses then either. 

 

Nowadays I'd have thought that if any adhesive is used it would be epoxy which is pretty much immune to solvents. 

 

 

                                                                  Guy

 

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net

 

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, 1 April 2009 6:47 PM
To: [hidden email]
Subject: FW: Cleaning lens.

I think Zeiss’s comment that repeated use of ethanol will damage Zeiss’s lens cement was just that – use it a few times a day and the Zeiss objective will probably fail in 6 months or so [and this might be the case for many solvents, ethanol is simply one more readily at hand*]. Use ethanol every month or so and chances are the objective will last a lot longer [and fail for another reason]. Use ethanol on a very elderly microscope where the lenses are mounted in say gum resin though and you will destroy the objective pretty much instantly [or at least get a nasty smear of gum resin all over the clear bit] - hence some people’s historical aversion is justified. I did mention to a Zeiss rep why did he just use 70% ethanol on our new 100x zillion NA TIRF objective when Zeiss say that repeated use will damage the objective, he said, well repeated use will damage the objective, but once or twice won’t matter [and then I thought ‘well I suppose it’s not actually his £8,000 objective’]. Other manufacturer’s actually recommend ethanol, e.g. Olympus. However immersion oil doesn’t dissolve in ethanol that well, hence another reason for the recommended use of other solvents, e.g. Petroleum ether - see http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379

*from the micro/primer site:  "In the past, solvents have been routinely employed for nearly any cleaning task in microscopy, and particularly for removal of immersion oil. Potential problems associated with solvent cleaning are sufficiently serious that the best current approach in cleaning the microscope is to use solvents only when absolutely necessary, essentially as a last resort rather than a first step. The issue of the use of solvents is further complicated and confused by contradictory recommendations in the scientific literature, as well as by differences in manufacturers' technical publications. Although alcohol and xylene are widely recommended as lens cleaning solvents, they are also named as being harmful to both the mechanical and optical components of many microscopes. Because of the variation in solvent recommendations, and the likelihood that some of the materials used in the instrument components are not known to the user, it is prudent to restrict use of any solvent to an absolute minimum.”

i.e. have a look at: http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html

Most of this discussion of solvents only applies to immersion oil objectives, where the oil isn’t miscible in water. Our inverted microscopes with all air objectives are rarely cleaned, otherwise it’s just a blow with a puffer. On microscopes where oil and air co-exist it’s often immersion oil contamination of the air objective that’s the problem, so it’s solvents again. In the days when only my group operated the microscopes, with all our oil immersion free upright microscopes the objectives almost never needed cleaning.

For other spillages such as culture medium [inverted microscope again] some solvents will probably fix biological muck onto the lens, and there’s the salts content, so the use of water based cleaners has been suggested [e.g. even breathing on the lens and then lens tissue, using optical/glass cleaning solutions]. Water drying onto the lens is a disaster though. Some even recommend things like breaking polystyrene foam [to get a clean surface] and gently rubbing the [oil free] lens with that. Or there’s Sparkle - whatever that is, here in the UK it was a silicon based furniture polish [yuk] not a commercial window glass cleaner. That’s the problem with industrial cleaners, who knows what’s in them or whether the constituents have been modified – you could try it on an inconspicuous area of the objective lens first, I suppose. Presumably optical lens cleaners are glass friendly though, and many use glass cleaning products with no reported problems. All the links in the previous posts [below] give loads of ideas for cleaning objectives [when necessary].

 

http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf

http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html

www.olympus.co.uk/microscopy/images/illum_cleaning.pdf
http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Chris Tully
Sent: 30 March 2009 16:40
To: [hidden email]
Subject: Re: Cleaning lens.

 

Dear all,

While working for a Leica Microsystems dealer the local field service engineer (factor trained) used a sequence of ethanol and heptane to clean truly dirty lenses.  For standard cleaning a lens wipe and Sparkle was his recommendation.  But for dried oil or the like he would graduate to cotton swabs and either ethanol then heptane or a 50:50 mix of the two.

Chris

Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-888-1021
http://www.linkedin.com/in/christully

On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery <[hidden email]> wrote:

Keith,

            Methylated spirit that’s what he said, although I still prefer and use ether when necessary.

Ian.

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: 30 March 2009 14:12

To: [hidden email]

Subject: Re: Cleaning lens.

 

Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’? Methylated spirit is mainly ethanol, and so best avoided - the Axiovert 100 manual says repeated use of 70% ethanol will damage the objectives [but you can use it if you want]. Generally the faster the solvent evaporation from the lens/cement area the better, hence the suggestion of the solvent [pure] diethyl ether by many [and that’s what I use].

 

‘Zeiss cleaning mixture L’, which the engineer’s now use since diethyl ether has been withdrawn from their kit, is 90% by volume ‘benzoline’ [petroleum ether sometimes called medical alcohol] and 10% ‘isopropanol’ [2-proponal, dimethyl carbinol, 2-hydroxyproparne]. The bottle says ‘Clean the optics by moving in circles, slight pressure should be exerted on optics during cleaning’. Petroleum ether or spirit isn’t the same as the diethyl ether solvent/anaesthetic often used to clean objectives, but apparently it does the job for Zeiss optics.  

 

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ian Montgomery
Sent: 30 March 2009 12:28
To: [hidden email]
Subject: Cleaning lens.

 

            In one of our teaching labs many years ago a student complained they were having a problem with the x100 OI objective and sure enough the image was lousy. I cleaned the objective and slide then re-applied a spot of oil and still the image was lousy. I then asked the student how exactly they had set up the microscope. Shock horror, my world collapsed. They had unscrewed the objective, filled it with oil, screwed it back on then put a drop on the slide. After weeks of trying to clean the objective it went into the trash as beyond economic repair.

            Cleaning objectives, I use the fluid recommended by the local Zeiss engineer, 90% methylated spirit and 10% isopropanol.

Ian.   

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

 

 

 

No virus found in this incoming message.
Checked by AVG.
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No virus found in this outgoing message.
Checked by AVG.
Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

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Checked by AVG.
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[Barry O'Brien]

A factory-trained service technician recommended to me that if a lens needed scraping in any way then the preferred implement was copper wire.  If you cut this with ordinary side-cutters there is a bevelled edge produced.

Barry O'Brien

At 12:04 a.m. 02/04/2009, you wrote:

Unrelated really but, our biggest problems is undergraduates on the teaching lab microscopes getting DPX on the lenses.
While they are always told how to avoid this, drop the stage before swinging a high magnification lens into place, in the excitement a fair number forget this after having tipped the contents of the DPX bottle onto the slide and floated a cover slip on the top.
 
I have been told that chloroform should get this off, however this has not been particularly successful when the DPX has dried on.
 
Of course I do get the DPX off but I hesitate to mention my technique.
 
Anyone come across a tried and tested method that doesn't involve careful application of a razor blade?
 
Leigh Silvester

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Guy Cox

Sent: 01 April 2009 11:00

To: [hidden email]

Subject: Re: Cleaning lens.

When I first learnt about microscopes (a very long time ago) I was taught that while dry objectives often had their front element mounted with some adhesive, oil immersion objectives didn't - the element was held by a screw ring.  Hence it was safe to clean them with solvent - we usually used xylene because that was handy - it was also used in mounting specimens (with canada balsam).  Of course this didn't offer any solution to the clumsy clot who gets oil on the x40.  But that wasn't actually quite so common then because oil immersion lenses were longer - ie not parfocal - so if someone swung the turret round the dry lenses would clear the oil.  On the other hand this didn't make using oil lenses too easy - and they didn't have spring noses then either. 

 

Nowadays I'd have thought that if any adhesive is used it would be epoxy which is pretty much immune to solvents. 

 

 

                                                                  Guy

 

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

    http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Electron Microscope Unit, Madsen Building F09,

University of Sydney, NSW 2006

______________________________________________

Phone [phone]     Fax [phone]

Mobile [phone]

______________________________________________

     http://www.guycox.net

 

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Keith Morris

Sent: Wednesday, 1 April 2009 6:47 PM

To: [hidden email]

Subject: FW: Cleaning lens.

I think Zeiss’s comment that repeated use of ethanol will damage Zeiss’s lens cement was just that – use it a few times a day and the Zeiss objective will probably fail in 6 months or so [and this might be the case for many solvents, ethanol is simply one more readily at hand*]. Use ethanol every month or so and chances are the objective will last a lot longer [and fail for another reason]. Use ethanol on a very elderly microscope where the lenses are mounted in say gum resin though and you will destroy the objective pretty much instantly [or at least get a nasty smear of gum resin all over the clear bit] - hence some people’s historical aversion is justified. I did mention to a Zeiss rep why did he just use 70% ethanol on our new 100x zillion NA TIRF objective when Zeiss say that repeated use will damage the objective, he said, well repeated use will damage the objective, but once or twice won’t matter [and then I thought ‘well I suppose it’s not actually his £8,000 objective’]. Other manufacturer’s actually recommend ethanol, e.g. Olympus. However immersion oil doesn’t dissolve in ethanol that well, hence another reason for the recommended use of other solvents, e.g. Petroleum ether - see http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379

*from the micro/primer site:  "In the past, solvents have been routinely employed for nearly any cleaning task in microscopy, and particularly for removal of immersion oil. Potential problems associated with solvent cleaning are sufficiently serious that the best current approach in cleaning the microscope is to use solvents only when absolutely necessary, essentially as a last resort rather than a first step. The issue of the use of solvents is further complicated and confused by contradictory recommendations in the scientific literature, as well as by differences in manufacturers' technical publications. Although alcohol and xylene are widely recommended as lens cleaning solvents, they are also named as being harmful to both the mechanical and optical components of many microscopes. Because of the variation in solvent recommendations, and the likelihood that some of the materials used in the instrument components are not known to the user, it is prudent to restrict use of any solvent to an absolute minimum.”

i.e. have a look at: http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html

Most of this discussion of solvents only applies to immersion oil objectives, where the oil isn’t miscible in water. Our inverted microscopes with all air objectives are rarely cleaned, otherwise it’s just a blow with a puffer. On microscopes where oil and air co-exist it’s often immersion oil contamination of the air objective that’s the problem, so it’s solvents again. In the days when only my group operated the microscopes, with all our oil immersion free upright microscopes the objectives almost never needed cleaning.

For other spillages such as culture medium [inverted microscope again] some solvents will probably fix biological muck onto the lens, and there’s the salts content, so the use of water based cleaners has been suggested [e.g. even breathing on the lens and then lens tissue, using optical/glass cleaning solutions]. Water drying onto the lens is a disaster though. Some even recommend things like breaking polystyrene foam [to get a clean surface] and gently rubbing the [oil free] lens with that. Or there’s Sparkle - whatever that is, here in the UK it was a silicon based furniture polish [yuk] not a commercial window glass cleaner. That’s the problem with industrial cleaners, who knows what’s in them or whether the constituents have been modified – you could try it on an inconspicuous area of the objective lens first, I suppose. Presumably optical lens cleaners are glass friendly though, and many use glass cleaning products with no reported problems. All the links in the previous posts [below] give loads of ideas for cleaning objectives [when necessary].

 

http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf

http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html

www. olympus.co.uk/microscopy/images/illum_cleaning.pdf

http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html

Keith

---------------------------------------------------------------------------

Dr Keith J. Morris,

Molecular Cytogenetics and Microscopy Core,

Laboratory 00/069 and 00/070,

The Wellcome Trust Centre for Human Genetics,

Roosevelt Drive,

Oxford  OX3 7BN,

United Kingdom.

Telephone:  [phone]

Email:  [hidden email]

Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Chris Tully

Sent: 30 March 2009 16:40

To: [hidden email]

Subject: Re: Cleaning lens.

 

Dear all,

While working for a Leica Microsystems dealer the local field service engineer (factor trained) used a sequence of ethanol and heptane to clean truly dirty lenses.  For standard cleaning a lens wipe and Sparkle was his recommendation.  But for dried oil or the like he would graduate to cotton swabs and either ethanol then heptane or a 50:50 mix of the two.

Chris

Chris Tully

Microscopy and Image Analysis Expert

[hidden email]

240-888-1021

http://www.linkedin.com/in/christully

On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery <[hidden email]> wrote:

Keith,

            Methylated spirit that’s what he said, although I still prefer and use ether when necessary.

Ian.

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Keith Morris

Sent: 30 March 2009 14:12

To: [hidden email]

Subject: Re: Cleaning lens.

 

Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’? Methylated spirit is mainly ethanol, and so best avoided - the Axiovert 100 manual says repeated use of 70% ethanol will damage the objectives [but you can use it if you want]. Generally the faster the solvent evaporation from the lens/cement area the better, hence the suggestion of the solvent [pure] diethyl ether by many [and that’s what I use].

 

‘Zeiss cleaning mixture L’, which the engineer’s now use since diethyl ether has been withdrawn from their kit, is 90% by volume ‘benzoline’ [petroleum ether sometimes called medical alcohol] and 10% ‘isopropanol’ [2-proponal, dimethyl carbinol, 2-hydroxyproparne]. The bottle says ‘Clean the optics by moving in circles, slight pressure should be exerted on optics during cleaning’. Petroleum ether or spirit isn’t the same as the diethyl ether solvent/anaesthetic often used to clean objectives, but apparently it does the job for Zeiss optics. 

 

Keith

---------------------------------------------------------------------------

Dr Keith J. Morris,

Molecular Cytogenetics and Microscopy Core,

Laboratory 00/069 and 00/070,

The Wellcome Trust Centre for Human Genetics,

Roosevelt Drive,

Oxford  OX3 7BN,

United Kingdom.

Telephone:  [phone]

Email:  [hidden email]

Web-pages: http://www.well.ox.ac.uk/cytogenetics/

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Ian Montgomery

Sent: 30 March 2009 12:28

To: [hidden email]

Subject: Cleaning lens.

 

            In one of our teaching labs many years ago a student complained they were having a problem with the x100 OI objective and sure enough the image was lousy. I cleaned the objective and slide then re-applied a spot of oil and still the image was lousy. I then asked the student how exactly they had set up the microscope. Shock horror, my world collapsed. They had unscrewed the objective, filled it with oil, screwed it back on then put a drop on the slide. After weeks of trying to clean the objective it went into the trash as beyond economic repair.

            Cleaning objectives, I use the fluid recommended by the local Zeiss engineer, 90% methylated spirit and 10% isopropanol.

Ian.  

 

Dr. Ian Montgomery,

Histotechnology,

I.B.L.S. Support Unit,

Thomson Building,

University of Glasgow,

Glasgow,

G12 8QQ.

 

 

No virus found in this incoming message.

Checked by AVG.

Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

No virus found in this outgoing message.

Checked by AVG.

Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date: 31/03/2009 1:05 PM

This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation.

Dr Barry O'Brien

Dept of Biological Sciences,

University of Waikato

Private Bag 3105

HAMILTON

New Zealand

Fax [phone]

Phone [phone]

[Mark Cannell]

I would be very cautious about this idea. The cutting of the wire will
work harden it and copper will scratch fluorite. I suppose you could
soften it again by flaming though. If any scraping is in order I would
definitely use a dissecting microscope to avoid lens damage. Pushing a
wadded lens tissue with liquid lubricant and forceps should a first
option I think.

my 2c

Regrds Mark Cannell

Barry O'Brien wrote:

> A factory-trained service technician recommended to me that if a lens
> needed scraping in any way then the preferred implement was copper
> wire. If you cut this with ordinary side-cutters there is a bevelled
> edge produced.
>
> Barry O'Brien
>
> At 12:04 a.m. 02/04/2009, you wrote:
>> Unrelated really but, our biggest problems is undergraduates on the
>> teaching lab microscopes getting DPX on the lenses.
>> While they are always told how to avoid this, drop the stage before
>> swinging a high magnification lens into place, in the excitement a
>> fair number forget this after having tipped the contents of the DPX
>> bottle onto the slide and floated a cover slip on the top.
>>
>> I have been told that chloroform should get this off, however this
>> has not been particularly successful when the DPX has dried on.
>>
>> Of course I do get the DPX off but I hesitate to mention my technique.
>>
>> Anyone come across a tried and tested method that doesn't involve
>> careful application of a razor blade?
>>
>> Leigh Silvester
>>
>> ------------------------------------------------------------------------
>>     From: Confocal Microscopy List [
>>     mailto:[hidden email]] On Behalf Of Guy Cox
>>     Sent: 01 April 2009 11:00
>>     To: [hidden email]
>>     Subject: Re: Cleaning lens.
>>
>>     When I first learnt about microscopes (a very long time ago) I
>>     was taught that while dry objectives often had their front
>>     element mounted with some adhesive, oil immersion objectives
>>     didn't - the element was held by a screw ring. Hence it was safe
>>     to clean them with solvent - we usually used xylene because that
>>     was handy - it was also used in mounting specimens (with canada
>>     balsam). Of course this didn't offer any solution to the clumsy
>>     clot who gets oil on the x40. But that wasn't actually quite so
>>     common then because oil immersion lenses were longer - ie not
>>     parfocal - so if someone swung the turret round the dry lenses
>>     would clear the oil. On the other hand this didn't make using oil
>>     lenses too easy - and they didn't have spring noses then either.
>>
>>     Nowadays I'd have thought that if any adhesive is used it would
>>     be epoxy which is pretty much immune to solvents.
>>
>>
>>     Guy
>>
>>
>>     Optical Imaging Techniques in Cell Biology
>>     by Guy Cox CRC Press / Taylor & Francis
>>     http://www.guycox.com/optical.htm
>>     ______________________________________________
>>     Associate Professor Guy Cox, MA, DPhil(Oxon)
>>     Electron Microscope Unit, Madsen Building F09,
>>     University of Sydney, NSW 2006
>>     ______________________________________________
>>     Phone [phone] Fax [phone]
>>     Mobile [phone]
>>     ______________________________________________
>>     http://www.guycox.net <http://www.guycox.net/>
>>
>>
>>     ------------------------------------------------------------------------
>>     From: Confocal Microscopy List [
>>     mailto:[hidden email]] On Behalf Of Keith Morris
>>     Sent: Wednesday, 1 April 2009 6:47 PM
>>     To: [hidden email]
>>     Subject: FW: Cleaning lens.
>>
>>     I think Zeiss’s comment that repeated use of ethanol will damage
>>     Zeiss’s lens cement was just that – use it a few times a day and
>>     the Zeiss objective will probably fail in 6 months or so [and
>>     this might be the case for many solvents, ethanol is simply one
>>     more readily at hand*]. Use ethanol every month or so and chances
>>     are the objective will last a lot longer [and fail for another
>>     reason]. Use ethanol on a very elderly microscope where the
>>     lenses are mounted in say gum resin though and you will destroy
>>     the objective pretty much instantly [or at least get a nasty
>>     smear of gum resin all over the clear bit] - hence some people’s
>>     historical aversion is justified. I did mention to a Zeiss rep
>>     why did he just use 70% ethanol on our new 100x zillion NA TIRF
>>     objective when Zeiss say that repeated use will damage the
>>     objective, he said, well repeated use will damage the objective,
>>     but once or twice won’t matter [and then I thought ‘well I
>>     suppose it’s not actually his £8,000 objective’]. Other
>>     manufacturer’s actually recommend ethanol, e.g. Olympus. However
>>     immersion oil doesn’t dissolve in ethanol that well, hence
>>     another reason for the recommended use of other solvents, e.g.
>>     Petroleum ether - see
>>     http://instrument-support.nikonusa.com/app/answers/detail/a_id/10379
>>
>>     *from the micro/primer site: "In the past, solvents have been
>>     routinely employed for nearly any cleaning task in microscopy,
>>     and particularly for removal of immersion oil. Potential problems
>>     associated with solvent cleaning are sufficiently serious that
>>     the best current approach in cleaning the microscope is to use
>>     solvents only when absolutely necessary, essentially as a last
>>     resort rather than a first step. The issue of the use of solvents
>>     is further complicated and confused by contradictory
>>     recommendations in the scientific literature, as well as by
>>     differences in manufacturers' technical publications. Although
>>     alcohol and xylene are widely recommended as lens cleaning
>>     solvents, they are also named as being harmful to both the
>>     mechanical and optical components of many microscopes. Because of
>>     the variation in solvent recommendations, and the likelihood that
>>     some of the materials used in the instrument components are not
>>     known to the user, it is prudent to restrict use of any solvent
>>     to an absolute minimum.”
>>
>>     i.e. have a look at:
>>     http://micro.magnet.fsu.edu/primer/anatomy/cleaning.html
>>
>>     Most of this discussion of solvents only applies to immersion oil
>>     objectives, where the oil isn’t miscible in water. Our inverted
>>     microscopes with all air objectives are rarely cleaned, otherwise
>>     it’s just a blow with a puffer. On microscopes where oil and air
>>     co-exist it’s often immersion oil contamination of the air
>>     objective that’s the problem, so it’s solvents again. In the days
>>     when only my group operated the microscopes, with all our oil
>>     immersion free upright microscopes the objectives almost never
>>     needed cleaning.
>>
>>     For other spillages such as culture medium [inverted microscope
>>     again] some solvents will probably fix biological muck onto the
>>     lens, and there’s the salts content, so the use of water based
>>     cleaners has been suggested [e.g. even breathing on the lens and
>>     then lens tissue, using optical/glass cleaning solutions]. Water
>>     drying onto the lens is a disaster though. Some even recommend
>>     things like breaking polystyrene foam [to get a clean surface]
>>     and gently rubbing the [oil free] lens with that. Or there’s
>>     Sparkle - whatever that is, here in the UK it was a silicon based
>>     furniture polish [yuk] not a commercial window glass cleaner.
>>     That’s the problem with industrial cleaners, who knows what’s in
>>     them or whether the constituents have been modified – you could
>>     try it on an inconspicuous area of the objective lens first, I
>>     suppose. Presumably optical lens cleaners are glass friendly
>>     though, and many use glass cleaning products with no reported
>>     problems. All the links in the previous posts [below] give loads
>>     of ideas for cleaning objectives [when necessary].
>>
>>
>>
>>     http://www.zeiss.com/C1256F8500454979/0/F9B766E00E70F4C4C1256F8D0054FFF8/$file/thecleanmicroscope.pdf
>>
>>
>>
>>     http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result
>>     <http://books.google.com/books?id=Dhn2KispfdQC&pg=PA51&lpg=PA51&dq=petroleum+spirit+cleaning+objectives&source=bl&ots=JxlHfCVuF5&sig=NTJt3Ol66sgsB8gluF1eKpeXLCc&hl=en&ei=DfvRSdGpI5WrjAflkvjlBg&sa=X&oi=book_result&resnum=1&ct=result>
>>
>>
>>     http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html
>>
>>
>>     www.
>>     <http://www.olympus.co.uk/microscopy/images/illum_cleaning.pdf>
>>     olympus.co.uk/microscopy/images/illum_cleaning.pdf
>>     <http://www.olympus.co.uk/microscopy/images/illum_cleaning.pdf>
>>     http://www.olympusmicro.com/primer/techniques/fluorescence/troubleshoot.html
>>
>>
>>
>>     Keith
>>
>>     ---------------------------------------------------------------------------
>>     Dr Keith J. Morris,
>>     Molecular Cytogenetics and Microscopy Core,
>>     Laboratory 00/069 and 00/070,
>>     The Wellcome Trust Centre for Human Genetics,
>>     Roosevelt Drive,
>>     Oxford OX3 7BN,
>>     United Kingdom.
>>
>>     Telephone: [phone]
>>     Email: [hidden email]
>>     Web-pages: http://www.well.ox.ac.uk/cytogenetics/
>>     ------------------------------------------------------------------------
>>     From: Confocal Microscopy List [
>>     mailto:[hidden email]] On Behalf Of Chris Tully
>>     Sent: 30 March 2009 16:40
>>     To: [hidden email]
>>     Subject: Re: Cleaning lens.
>>
>>
>>
>>     Dear all,
>>
>>     While working for a Leica Microsystems dealer the local field
>>     service engineer (factor trained) used a sequence of ethanol and
>>     heptane to clean truly dirty lenses. For standard cleaning a lens
>>     wipe and Sparkle was his recommendation. But for dried oil or the
>>     like he would graduate to cotton swabs and either ethanol then
>>     heptane or a 50:50 mix of the two.
>>
>>     Chris
>>
>>     Chris Tully
>>     Microscopy and Image Analysis Expert
>>     [hidden email] <mailto:[hidden email]>
>>     240-888-1021
>>     http://www.linkedin.com/in/christully
>>
>>     On Mon, Mar 30, 2009 at 10:12 AM, Ian Montgomery <
>>     [hidden email]
>>     <mailto:[hidden email]>> wrote:
>>
>>     Keith,
>>
>>     Methylated spirit that’s what he said, although I still prefer
>>     and use ether when necessary.
>>
>>     Ian.
>>
>>
>>
>>     Dr. Ian Montgomery,
>>
>>     Histotechnology,
>>
>>     I.B.L.S. Support Unit,
>>
>>     Thomson Building,
>>
>>     University of Glasgow,
>>
>>     Glasgow,
>>
>>     G12 8QQ.
>>     ------------------------------------------------------------------------
>>     From: Confocal Microscopy List [
>>     mailto:[hidden email]] On Behalf Of Keith Morris
>>     Sent: 30 March 2009 14:12
>>
>>
>>     To: [hidden email]
>>     <mailto:[hidden email]>
>>
>>     Subject: Re: Cleaning lens.
>>
>>
>>
>>     Are you sure the Zeiss Engineer didn’t say ‘petroleum spirit’?
>>     Methylated spirit is mainly ethanol, and so best avoided - the
>>     Axiovert 100 manual says repeated use of 70% ethanol will damage
>>     the objectives [but you can use it if you want]. Generally the
>>     faster the solvent evaporation from the lens/cement area the
>>     better, hence the suggestion of the solvent [pure] diethyl ether
>>     by many [and that’s what I use].
>>
>>
>>
>>     ‘Zeiss cleaning mixture L’, which the engineer’s now use since
>>     diethyl ether has been withdrawn from their kit, is 90% by volume
>>     ‘benzoline’ [petroleum ether sometimes called medical alcohol]
>>     and 10% ‘isopropanol’ [2-proponal, dimethyl carbinol,
>>     2-hydroxyproparne]. The bottle says ‘Clean the optics by moving
>>     in circles, slight pressure should be exerted on optics during
>>     cleaning’. Petroleum ether or spirit isn’t the same as the
>>     diethyl ether solvent/anaesthetic often used to clean objectives,
>>     but apparently it does the job for Zeiss optics.
>>
>>
>>
>>     Keith
>>
>>     ---------------------------------------------------------------------------
>>     Dr Keith J. Morris,
>>     Molecular Cytogenetics and Microscopy Core,
>>     Laboratory 00/069 and 00/070,
>>     The Wellcome Trust Centre for Human Genetics,
>>     Roosevelt Drive,
>>     Oxford OX3 7BN,
>>     United Kingdom.
>>
>>     Telephone: [phone]
>>     Email: [hidden email] <mailto:[hidden email]>
>>     Web-pages: http://www.well.ox.ac.uk/cytogenetics/
>>     ------------------------------------------------------------------------
>>     From: Confocal Microscopy List [
>>     mailto:[hidden email]] On Behalf Of Ian Montgomery
>>     Sent: 30 March 2009 12:28
>>     To: [hidden email]
>>     <mailto:[hidden email]>
>>     Subject: Cleaning lens.
>>
>>
>>
>>     In one of our teaching labs many years ago a student complained
>>     they were having a problem with the x100 OI objective and sure
>>     enough the image was lousy. I cleaned the objective and slide
>>     then re-applied a spot of oil and still the image was lousy. I
>>     then asked the student how exactly they had set up the
>>     microscope. Shock horror, my world collapsed. They had unscrewed
>>     the objective, filled it with oil, screwed it back on then put a
>>     drop on the slide. After weeks of trying to clean the objective
>>     it went into the trash as beyond economic repair.
>>
>>     Cleaning objectives, I use the fluid recommended by the local
>>     Zeiss engineer, 90% methylated spirit and 10% isopropanol.
>>
>>     Ian.
>>
>>
>>
>>     Dr. Ian Montgomery,
>>
>>     Histotechnology,
>>
>>     I.B.L.S. Support Unit,
>>
>>     Thomson Building,
>>
>>     University of Glasgow,
>>
>>     Glasgow,
>>
>>     G12 8QQ.
>>
>>
>>
>>
>>
>>     No virus found in this incoming message.
>>     Checked by AVG.
>>     Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date:
>>     31/03/2009 1:05 PM
>>
>>     No virus found in this outgoing message.
>>     Checked by AVG.
>>     Version: 7.5.557 / Virus Database: 270.11.35/2033 - Release Date:
>>     31/03/2009 1:05 PM
>>
>> This message has been checked for viruses but the contents of an
>> attachment may still contain software viruses, which could damage
>> your computer system: you are advised to perform your own checks.
>> Email communications with the University of Nottingham may be
>> monitored as permitted by UK legislation.
>
> Dr Barry O'Brien
> Dept of Biological Sciences,
> University of Waikato
> Private Bag 3105
> HAMILTON
> New Zealand
>
> Fax [phone]
> Phone [phone]

[Louis Villeneuve]

Hi to all,

Is it possible to detect luciferase activity  on cell with a standard confocal micriscope (let's say LSM 510 and LSM 710 equipped with  regular and spectral detector)?

 Emission of coelenterazine is at 470 nm.  

Thanks for you help!

Louis
Louis Villeneuve
Research Associate- Confocal Microscopy
Heart Montreal Institute- Research Center
5000 East Belanger
Montreal (Qc), Canada
H1T 1C8

514-376-3330 ext 3511
514-376-1355 (Fax)

[hidden email]

[Keith Morris]

No it probably isn't possible to use a confocal for luciferase. Just tried
it with our standard wide field inverted microscope, all lights off, lots
of tin foil, Orca ER peltier cooled camera, maximum gain and not a hint of
anything other than background noise. Our Orca ER has a 10 second max
exposure time, and the literature talks of 120 seconds plus exposure times
[and the max camera gain didn't help us]. We concluded we would need a
£25k EMCCD camera or better to use our microscope with our samples, so our
user is simply switching to an immunofluorescence method instead and
giving Luciferase a miss [just not worth spending the time developing the
method without the right microscope kit].

A point scanning confocal will probably be less capable than our Orca ER
camera, and certainly no better. You loose too much light in the optics,
as there's so little to begin with, and so you need a very expensive
low-noise sensitive camera. Normally you use one of those large
light-tight cabinet type microscope imagers, bit like a plate scanner.

Olympus do make the 'boxed' LV200 microscope for this type of work:

http://www.microscopy.olympus.eu/microscopes/Life_Science_Microscopes_Bioluminescence_Imaging_System_LV200_Applications.htm

and they talk of 15 minute exposure integration times for image capture.
If you have an expensive cooled B&W fluorescence microscope camera that
integrate for many minutes it might be worth a trying that though -
provided you can get the microscope stage into 'absolute darkness' [you
even have to get down to covering LEDs with insulating tape].

Our user was using a luceriferase kit that reduced light levels a tad to
hopefully keep it going brighter for far longer than the few seconds
Luciferase normally lasts.

Regards

Keith

-----------------------------------------------------------------

Dr Keith J Morris
Molecular Cytogenetics and Microscopy Core
The Wellcome Trust Centre for Human Genetics
Roosevelt Drive
Oxford
OX3 7BN
United Kingdom

> Hi to all,
>
> Is it possible to detect luciferase activity  on cell with a standard
> confocal micriscope (let's say LSM 510 and LSM 710 equipped with  regular
> and spectral detector)?
>
>  Emission of coelenterazine is at 470 nm.
>
> Thanks for you help!
>
> Louis
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8
>
> 514-376-3330 ext 3511
> 514-376-1355 (Fax)
>
> [hidden email]

[Keith Morris]

Sorry I should have said around 60 second exposure integration for
luciferase imaging [not 15 minutes] on the specially modified Olympus
LV200 'luciferase' microscope system. We do have a Zeiss 510 confocal by
the way, but rejected it as suitable for luciferase.

Keith

-----------------------------------------------------------------

Dr Keith J Morris
Molecular Cytogenetics and Microscopy Core
The Wellcome Trust Centre for Human Genetics
Roosevelt Drive
Oxford
OX3 7BN
United Kingdom

Tel:  +44   ( 0 ) 1865  287568
Email:   [hidden email]
HomePage:  http://www.well.ox.ac.uk/cytogenetics


> No it probably isn't possible to use a confocal for luciferase. Just
tried
> it with our standard wide field inverted microscope, all lights off,
lots
> of tin foil, Orca ER peltier cooled camera, maximum gain and not a hint
of
> anything other than background noise. Our Orca ER has a 10 second max
exposure time, and the literature talks of 120 seconds plus exposure
times
> [and the max camera gain didn't help us]. We concluded we would need a
£25k EMCCD camera or better to use our microscope with our samples, so
our
> user is simply switching to an immunofluorescence method instead and
giving Luciferase a miss [just not worth spending the time developing
the
> method without the right microscope kit].
>
> A point scanning confocal will probably be less capable than our Orca ER
camera, and certainly no better. You loose too much light in the optics,
as there's so little to begin with, and so you need a very expensive
low-noise sensitive camera. Normally you use one of those large
> light-tight cabinet type microscope imagers, bit like a plate scanner.
>
> Olympus do make the 'boxed' LV200 microscope for this type of work:
>
> http://www.microscopy.olympus.eu/microscopes/Life_Science_Microscopes_Bioluminescence_Imaging_System_LV200_Applications.htm
>
> and they talk of 15 minute exposure integration times for image capture.
If you have an expensive cooled B&W fluorescence microscope camera that
integrate for many minutes it might be worth a trying that though -
provided you can get the microscope stage into 'absolute darkness' [you
even have to get down to covering LEDs with insulating tape].
>
> Our user was using a luceriferase kit that reduced light levels a tad to
hopefully keep it going brighter for far longer than the few seconds
Luciferase normally lasts.

>
> Regards
>
> Keith
>
> -----------------------------------------------------------------
>
> Dr Keith J Morris
> Molecular Cytogenetics and Microscopy Core
> The Wellcome Trust Centre for Human Genetics
> Roosevelt Drive
> Oxford
> OX3 7BN
> United Kingdom
>
>
>> Hi to all,
>> Is it possible to detect luciferase activity  on cell with a standard

confocal micriscope (let's say LSM 510 and LSM 710 equipped with
regular

>> and spectral detector)?
>>  Emission of coelenterazine is at 470 nm.
>> Thanks for you help!
>> Louis
>> Louis Villeneuve
>> Research Associate- Confocal Microscopy
>> Heart Montreal Institute- Research Center
>> 5000 East Belanger
>> Montreal (Qc), Canada
>> H1T 1C8
>> 514-376-3330 ext 3511
>> 514-376-1355 (Fax)
>> [hidden email]
>

[rjpalmer]

You can do this with a true intensified
photon-counting camera, e.g., those from
Hamamatsu.  Can't locate on their web site the
model we used about 15 yrs ago, but it was based
on a multi-channel plate that provided about 10E6
amplification.  We mounted it on a side port on
our confocal and put the scope in a light box.
The thing was sensitive enough to detect
bacterial luciferase in single cells.  As noted,
lots of aluminum foil is recommend.  I assume you
are NOT trying to do three-D work, but I've
always been intrigued by that possibility.

At 9:39 AM -0400 4/3/09, Keith J Morris wrote:

>No it probably isn't possible to use a confocal for luciferase. Just tried
>it with our standard wide field inverted microscope, all lights off, lots
>of tin foil, Orca ER peltier cooled camera, maximum gain and not a hint of
>anything other than background noise. Our Orca ER has a 10 second max
>exposure time, and the literature talks of 120 seconds plus exposure times
>[and the max camera gain didn't help us]. We concluded we would need a
>£25k EMCCD camera or better to use our microscope with our samples, so our
>user is simply switching to an immunofluorescence method instead and
>giving Luciferase a miss [just not worth spending the time developing the
>method without the right microscope kit].
>
>A point scanning confocal will probably be less capable than our Orca ER
>camera, and certainly no better. You loose too much light in the optics,
>as there's so little to begin with, and so you need a very expensive
>low-noise sensitive camera. Normally you use one of those large
>light-tight cabinet type microscope imagers, bit like a plate scanner.
>
>Olympus do make the 'boxed' LV200 microscope for this type of work:
>
>http://www.microscopy.olympus.eu/microscopes/Life_Science_Microscopes_Bioluminescence_Imaging_System_LV200_Applications.htm
>
>and they talk of 15 minute exposure integration times for image capture.
>If you have an expensive cooled B&W fluorescence microscope camera that
>integrate for many minutes it might be worth a trying that though -
>provided you can get the microscope stage into 'absolute darkness' [you
>even have to get down to covering LEDs with insulating tape].
>
>Our user was using a luceriferase kit that reduced light levels a tad to
>hopefully keep it going brighter for far longer than the few seconds
>Luciferase normally lasts.
>
>Regards
>
>Keith
>
>-----------------------------------------------------------------
>
>Dr Keith J Morris
>Molecular Cytogenetics and Microscopy Core
>The Wellcome Trust Centre for Human Genetics
>Roosevelt Drive
>Oxford
>OX3 7BN
>United Kingdom
>
>
>>  Hi to all,
>>
>>  Is it possible to detect luciferase activity  on cell with a standard
>>  confocal micriscope (let's say LSM 510 and LSM 710 equipped with  regular
>>  and spectral detector)?
>>
>>   Emission of coelenterazine is at 470 nm.
>>
>>  Thanks for you help!
>>
>>  Louis
>>  Louis Villeneuve
>>  Research Associate- Confocal Microscopy
>>  Heart Montreal Institute- Research Center
>>  5000 East Belanger
>>  Montreal (Qc), Canada
>>  H1T 1C8
>>
>>  514-376-3330 ext 3511
>>  514-376-1355 (Fax)
>>
>>  [hidden email]

--
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax [phone]

[G. Esteban Fernandez]

We've also tried to see luminescence with an really good non-EM
non-intensified camera (Orca AG) on a widefield 'scope and saw
nothing, so I agree that a confocal would be much worse.  An
intensified camera worked though.

-Esteban


On Fri, Apr 3, 2009 at 8:39 AM, Keith J Morris <[hidden email]> wrote:

> No it probably isn't possible to use a confocal for luciferase. Just tried
> it with our standard wide field inverted microscope, all lights off, lots
> of tin foil, Orca ER peltier cooled camera, maximum gain and not a hint of
> anything other than background noise. Our Orca ER has a 10 second max
> exposure time, and the literature talks of 120 seconds plus exposure times
> [and the max camera gain didn't help us]. We concluded we would need a
> £25k EMCCD camera or better to use our microscope with our samples, so our
> user is simply switching to an immunofluorescence method instead and
> giving Luciferase a miss [just not worth spending the time developing the
> method without the right microscope kit].
>
> A point scanning confocal will probably be less capable than our Orca ER
> camera, and certainly no better. You loose too much light in the optics,
> as there's so little to begin with, and so you need a very expensive
> low-noise sensitive camera. Normally you use one of those large
> light-tight cabinet type microscope imagers, bit like a plate scanner.
>
> Olympus do make the 'boxed' LV200 microscope for this type of work:
>
> http://www.microscopy.olympus.eu/microscopes/Life_Science_Microscopes_Bioluminescence_Imaging_System_LV200_Applications.htm
>
> and they talk of 15 minute exposure integration times for image capture.
> If you have an expensive cooled B&W fluorescence microscope camera that
> integrate for many minutes it might be worth a trying that though -
> provided you can get the microscope stage into 'absolute darkness' [you
> even have to get down to covering LEDs with insulating tape].
>
> Our user was using a luceriferase kit that reduced light levels a tad to
> hopefully keep it going brighter for far longer than the few seconds
> Luciferase normally lasts.
>
> Regards
>
> Keith
>
> -----------------------------------------------------------------
>
> Dr Keith J Morris
> Molecular Cytogenetics and Microscopy Core
> The Wellcome Trust Centre for Human Genetics
> Roosevelt Drive
> Oxford
> OX3 7BN
> United Kingdom
>
>
>> Hi to all,
>>
>> Is it possible to detect luciferase activity  on cell with a standard
>> confocal micriscope (let's say LSM 510 and LSM 710 equipped with  regular
>> and spectral detector)?
>>
>>  Emission of coelenterazine is at 470 nm.
>>
>> Thanks for you help!
>>
>> Louis
>> Louis Villeneuve
>> Research Associate- Confocal Microscopy
>> Heart Montreal Institute- Research Center
>> 5000 East Belanger
>> Montreal (Qc), Canada
>> H1T 1C8
>>
>> 514-376-3330 ext 3511
>> 514-376-1355 (Fax)
>>
>> [hidden email]
>

--
G. Esteban Fernandez, Ph.D.
Associate Director
Molecular Cytology Core Facility
University of Missouri
120 Bond Life Sciences Center
Columbia, MO  65211

http://www.biotech.missouri.edu/mcc/

573-882-4895
573-884-9676 fax

[Monique Vasseur]

On your widefield microscope with camera, did you try binning of the pixels to image your luciferase?

For BRET imaging tests using luciferase, where very high resolution was not important, we used a CoolSnapHQ binning 4 (then giving a 26 um pixel size) on the side port with 60 to 120 sec exposure time to see where was the signal. Best resolution have been done with a Cascade 512B camera (EMCCD backilluminated) with 30 sec exposure and binning 1.  See the Julie Perroy article:
.
Biophys J. 2008 February 1; 94(3): 1001–1009.

Subcellular Imaging of Dynamic Protein Interactions by Bioluminescence Resonance Energy Transfer
Vincent Coulon,*† Martin Audet,‡ Vincent Homburger,* Joël Bockaert,* Laurent Fagni,* Michel Bouvier,‡ and Julie Perroy*


Monique Vasseur
Microscopie et imagerie
Département de biochimie
Université de Montréal
C.P. 6128, succursale Centre-ville
Montréal QC    H3C 3J7   Canada
tél. [phone] poste 5148
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Robert J. Palmer Jr.
Envoyé : 3 avril 2009 09:59
À : [hidden email]
Objet : Re: Luciferase (Gaussia) activity detection

You can do this with a true intensified
photon-counting camera, e.g., those from
Hamamatsu.  Can't locate on their web site the
model we used about 15 yrs ago, but it was based
on a multi-channel plate that provided about 10E6
amplification.  We mounted it on a side port on
our confocal and put the scope in a light box.
The thing was sensitive enough to detect
bacterial luciferase in single cells.  As noted,
lots of aluminum foil is recommend.  I assume you
are NOT trying to do three-D work, but I've
always been intrigued by that possibility.

At 9:39 AM -0400 4/3/09, Keith J Morris wrote:

>No it probably isn't possible to use a confocal for luciferase. Just tried
>it with our standard wide field inverted microscope, all lights off, lots
>of tin foil, Orca ER peltier cooled camera, maximum gain and not a hint of
>anything other than background noise. Our Orca ER has a 10 second max
>exposure time, and the literature talks of 120 seconds plus exposure times
>[and the max camera gain didn't help us]. We concluded we would need a
>£25k EMCCD camera or better to use our microscope with our samples, so our
>user is simply switching to an immunofluorescence method instead and
>giving Luciferase a miss [just not worth spending the time developing the
>method without the right microscope kit].
>
>A point scanning confocal will probably be less capable than our Orca ER
>camera, and certainly no better. You loose too much light in the optics,
>as there's so little to begin with, and so you need a very expensive
>low-noise sensitive camera. Normally you use one of those large
>light-tight cabinet type microscope imagers, bit like a plate scanner.
>
>Olympus do make the 'boxed' LV200 microscope for this type of work:
>
>http://www.microscopy.olympus.eu/microscopes/Life_Science_Microscopes_Bioluminescence_Imaging_System_LV200_Applications.htm
>
>and they talk of 15 minute exposure integration times for image capture.
>If you have an expensive cooled B&W fluorescence microscope camera that
>integrate for many minutes it might be worth a trying that though -
>provided you can get the microscope stage into 'absolute darkness' [you
>even have to get down to covering LEDs with insulating tape].
>
>Our user was using a luceriferase kit that reduced light levels a tad to
>hopefully keep it going brighter for far longer than the few seconds
>Luciferase normally lasts.
>
>Regards
>
>Keith
>
>-----------------------------------------------------------------
>
>Dr Keith J Morris
>Molecular Cytogenetics and Microscopy Core
>The Wellcome Trust Centre for Human Genetics
>Roosevelt Drive
>Oxford
>OX3 7BN
>United Kingdom
>
>
>>  Hi to all,
>>
>>  Is it possible to detect luciferase activity  on cell with a standard
>>  confocal micriscope (let's say LSM 510 and LSM 710 equipped with  regular
>>  and spectral detector)?
>>
>>   Emission of coelenterazine is at 470 nm.
>>
>>  Thanks for you help!
>>
>>  Louis
>>  Louis Villeneuve
>>  Research Associate- Confocal Microscopy
>>  Heart Montreal Institute- Research Center
>>  5000 East Belanger
>>  Montreal (Qc), Canada
>>  H1T 1C8
>>
>>  514-376-3330 ext 3511
>>  514-376-1355 (Fax)
>>
>>  [hidden email]

--
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax [phone]

[Keith Morris]

Hi Monique,

Yes we tried binning to the max 8, Gain to maximum, and exposure to
maximum. Sadly the Hammamatsu Orca 100 [C4742-95]* is limited to a 10
seconds maximum exposure [and we really needed that 120 seconds]. All we
got was camera noise [once we fully light proofed the microscope stage].

From measurements using some sort of luminosity scanner the user did
confirm there was weak light coming from the luciferase, but obviously
nowhere near enough for our Hammatsu Orca 100 camera to image anything. I
see frequent reference to the CoolSnapHQ & CoolSnap2 HQ being used for
imaging Luciferase luminescence, and all saying about 120 seconds exposure
[many modern Hammatsu and Photometrics interline CCD cameras offer similar
long exposure times these days, but our Orca 100 is four+ years old and
wasn't designed for very low light levels anyway]. Plus our inverted
microscope hasn't got the best objectives for fluorescence either [Phase
contrast, long working distance ones mostly], which no doubt put the tin
hat on it, even though we moved as much as possible out of the light path.

Thanks for the advice though.

You can see our setup at
http://www.well.ox.ac.uk/cytogenetics/nikon05.shtml

Keith

* Actually we haven't got an Orca ER - that was at the last place I worked
at, the ER is more sensitive than the 100.
-----------------------------------------------------------------

Dr Keith J Morris
Molecular Cytogenetics and Microscopy Core
The Wellcome Trust Centre for Human Genetics
Roosevelt Drive
Oxford
OX3 7BN
United Kingdom

Tel:  +44   ( 0 ) 1865  287568
Email:   [hidden email]
HomePage:  http://www.well.ox.ac.uk/cytogenetics

> On your widefield microscope with camera, did you try binning of the
> pixels to image your luciferase?
>
> For BRET imaging tests using luciferase, where very high resolution was
> not important, we used a CoolSnapHQ binning 4 (then giving a 26 um pixel
> size) on the side port with 60 to 120 sec exposure time to see where was
> the signal. Best resolution have been done with a Cascade 512B camera
> (EMCCD backilluminated) with 30 sec exposure and binning 1.  See the Julie
> Perroy article:
> .
> Biophys J. 2008 February 1; 94(3): 1001–1009.
>
> Subcellular Imaging of Dynamic Protein Interactions by Bioluminescence
> Resonance Energy Transfer
> Vincent Coulon,*† Martin Audet,‡ Vincent Homburger,* Joël Bockaert,*
> Laurent Fagni,* Michel Bouvier,‡ and Julie Perroy*
>
>
> Monique Vasseur
> Microscopie et imagerie
> Département de biochimie
> Université de Montréal
> C.P. 6128, succursale Centre-ville
> Montréal QC    H3C 3J7   Canada
> tél. [phone] poste 5148
> -----Message d'origine-----
> De : Confocal Microscopy List [mailto:[hidden email]] De
> la part de Robert J. Palmer Jr.
> Envoyé : 3 avril 2009 09:59
> À : [hidden email]
> Objet : Re: Luciferase (Gaussia) activity detection
>
> You can do this with a true intensified
> photon-counting camera, e.g., those from
> Hamamatsu.  Can't locate on their web site the
> model we used about 15 yrs ago, but it was based
> on a multi-channel plate that provided about 10E6
> amplification.  We mounted it on a side port on
> our confocal and put the scope in a light box.
> The thing was sensitive enough to detect
> bacterial luciferase in single cells.  As noted,
> lots of aluminum foil is recommend.  I assume you
> are NOT trying to do three-D work, but I've
> always been intrigued by that possibility.
>
> At 9:39 AM -0400 4/3/09, Keith J Morris wrote:
>>No it probably isn't possible to use a confocal for luciferase. Just
>> tried
>>it with our standard wide field inverted microscope, all lights off, lots
>>of tin foil, Orca ER peltier cooled camera, maximum gain and not a hint
>> of
>>anything other than background noise. Our Orca ER has a 10 second max
>>exposure time, and the literature talks of 120 seconds plus exposure
>> times
>>[and the max camera gain didn't help us]. We concluded we would need a
>>£25k EMCCD camera or better to use our microscope with our samples, so
>> our
>>user is simply switching to an immunofluorescence method instead and
>>giving Luciferase a miss [just not worth spending the time developing the
>>method without the right microscope kit].
>>
>>A point scanning confocal will probably be less capable than our Orca ER
>>camera, and certainly no better. You loose too much light in the optics,
>>as there's so little to begin with, and so you need a very expensive
>>low-noise sensitive camera. Normally you use one of those large
>>light-tight cabinet type microscope imagers, bit like a plate scanner.
>>
>>Olympus do make the 'boxed' LV200 microscope for this type of work:
>>
>>http://www.microscopy.olympus.eu/microscopes/Life_Science_Microscopes_Bioluminescence_Imaging_System_LV200_Applications.htm
>>
>>and they talk of 15 minute exposure integration times for image capture.
>>If you have an expensive cooled B&W fluorescence microscope camera that
>>integrate for many minutes it might be worth a trying that though -
>>provided you can get the microscope stage into 'absolute darkness' [you
>>even have to get down to covering LEDs with insulating tape].
>>
>>Our user was using a luceriferase kit that reduced light levels a tad to
>>hopefully keep it going brighter for far longer than the few seconds
>>Luciferase normally lasts.
>>
>>Regards
>>
>>Keith
>>
>>-----------------------------------------------------------------
>>
>>Dr Keith J Morris
>>Molecular Cytogenetics and Microscopy Core
>>The Wellcome Trust Centre for Human Genetics
>>Roosevelt Drive
>>Oxford
>>OX3 7BN
>>United Kingdom
>>
>>
>>>  Hi to all,
>>>
>>>  Is it possible to detect luciferase activity  on cell with a standard
>>>  confocal micriscope (let's say LSM 510 and LSM 710 equipped with
>>> regular
>>>  and spectral detector)?
>>>
>>>   Emission of coelenterazine is at 470 nm.
>>>
>>>  Thanks for you help!
>>>
>>>  Louis
>>>  Louis Villeneuve
>>>  Research Associate- Confocal Microscopy
>>>  Heart Montreal Institute- Research Center
>>>  5000 East Belanger
>>>  Montreal (Qc), Canada
>>>  H1T 1C8
>>>
>>>  514-376-3330 ext 3511
>>>  514-376-1355 (Fax)
>>>
>>>  [hidden email]
>
>
> --
> Robert J. Palmer Jr., Ph.D.
> Natl Inst Dental Craniofacial Res - Natl Insts Health
> Oral Infection and Immunity Branch
> Bldg 30, Room 310
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax [phone]
>