Objectives for live cell imaging
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Edelmann, Richard E. Dr. |
Objectives for live cell imaging
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O.k., obviously live cell imaging would be best done with either (1)
an upright with a water immersion lens (dipping) or (2) cells adherent to a cover slip and an oil immersion lens on an inverted scope. So what about live tissue imaging on an inverted scope? (I.e. whole or dissected insect neuro-muscular development). How do you best deal with spherical aberration due to samples in a watery medium a distance from the coverslip? With say a 40 to 60X magnification. Scope is a Zeiss 710. Building, some sort of a flexible damn system for using a water immersion lens on an inverted really does not seem practical. Thanks for any help. Richard E. Edelmann, Ph.D. Electron Microscopy Facility Director 364 Pearson Hall Miami University, Oxford, OH 45056 Ph: 513.529.5712 Fax: 513.529.4243 E-mail: [hidden email] http://www.emf.muohio.edu |
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John Oreopoulos |
Re: Objectives for live cell imaging
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Actually, I think most people on this listserver would say use a
water lens for any live-cell application, even deep tissue imaging on an inverted microscope since this will reduce the aberrations due to refractive index mismatch. Steven Cody and Guy Cox I believe have documented on their websites some nice and simple ways to create water dams. John Oreopoulos On 2-Dec-09, at 10:50 AM, Richard E. Edelmann wrote: > O.k., obviously live cell imaging would be best done with either (1) > an upright with a water immersion lens (dipping) or (2) cells > adherent to a cover slip and an oil immersion lens on an inverted > scope. > > So what about live tissue imaging on an inverted scope? (I.e. whole > or dissected insect neuro-muscular development). How do you best > deal with spherical aberration due to samples in a watery medium a > distance from the coverslip? With say a 40 to 60X magnification. > Scope is a Zeiss 710. > > Building, some sort of a flexible damn system for using a water > immersion lens on an inverted really does not seem practical. > > Thanks for any help. > > > Richard E. Edelmann, Ph.D. > Electron Microscopy Facility Director > 364 Pearson Hall > Miami University, Oxford, OH 45056 > Ph: 513.529.5712 Fax: 513.529.4243 > E-mail: [hidden email] > http://www.emf.muohio.edu |
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In reply to this post by Edelmann, Richard E. Dr.
Hi!
Look up the Greiner Lumox dishes. NOT cheap but quite a while back I read that their RI is very close to that of water. It works fine with beads and a 63x water immersion (no coverslip) but you might want to test them yourself (my users have yet to buy them). No direct financial interests. Good luck! Sophie ____________________________________________________ Sophie M. K. Brunet, Ph. D. Research Officer Optical Spectroscopy, Laser Systems and Applications [hidden email] 306-966-1719 (office) 306-966-1702 (fax) ____________________________________________________ Saskatchewan Structural Sciences Centre University of Saskatchewan Thorvaldson Bldg. 110 Science Place Saskatoon, Sk S7N 5C9 ____________________________________________________ Quoting "Richard E. Edelmann" <[hidden email]>: > O.k., obviously live cell imaging would be best done with either (1) > an upright with a water immersion lens (dipping) or (2) cells > adherent to a cover slip and an oil immersion lens on an inverted > scope. > > So what about live tissue imaging on an inverted scope? (I.e. whole > or dissected insect neuro-muscular development). How do you best > deal with spherical aberration due to samples in a watery medium a > distance from the coverslip? With say a 40 to 60X magnification. > Scope is a Zeiss 710. > > Building, some sort of a flexible damn system for using a water > immersion lens on an inverted really does not seem practical. > > Thanks for any help. > > > Richard E. Edelmann, Ph.D. > Electron Microscopy Facility Director > 364 Pearson Hall > Miami University, Oxford, OH 45056 > Ph: 513.529.5712 Fax: 513.529.4243 > E-mail: [hidden email] > http://www.emf.muohio.edu > |
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In reply to this post by Edelmann, Richard E. Dr.
In this case, for inverted microscope, water immersion (non-dipping) lens is better, preferable 40X 1.2 NA for fluorescence, you don't want a too high magnification, since fluorescence light throughput will be lower with higher magnification. Essentially, you need high NA, low magnification, turns out 40X 1.2NA objective is the sweet spot.
Using water immersion (non-dipping) lens for long time-lapse, use 1.33 RI immersion oil (the same RI of water) is better than real water, since it won't evaporate. I usually use Bioptechs Delta-T dish (Fisher Scientific cat-# 12-071-34), or Thermo Scientific Nunc Lab-Tek Chamber Cover Glass (cat# 12-565-401) with appropriate stage adapters on inverted scopes. Good luck. Tao On Wed, Dec 2, 2009 at 10:50 AM, Richard E. Edelmann <[hidden email]> wrote: O.k., obviously live cell imaging would be best done with either (1) -- http://tongtao.com |
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Armstrong, Brian |
Re: Objectives for live cell imaging
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In reply to this post by Edelmann, Richard E. Dr.
We often image live cells on inverted microscopes using water immersion
lenses. It is not a problem, and in fact you should image live cells in aqueous with a water immersion lens. You can also use a thicker water from Zeiss Immersol W (and others) that has an RI similar to W. We use this for long term live cell imaging at 37degrees. Live cell imaging is best done through a glass coverslip bottom dish using water immersion lenses on an inverted scope (in my humble opinion). Cheers, Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Hope Dept of Neuroscience 1450 E Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag ing/Pages/default.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Richard E. Edelmann Sent: Wednesday, December 02, 2009 7:51 AM To: [hidden email] Subject: Objectives for live cell imaging O.k., obviously live cell imaging would be best done with either (1) an upright with a water immersion lens (dipping) or (2) cells adherent to a cover slip and an oil immersion lens on an inverted scope. So what about live tissue imaging on an inverted scope? (I.e. whole or dissected insect neuro-muscular development). How do you best deal with spherical aberration due to samples in a watery medium a distance from the coverslip? With say a 40 to 60X magnification. Scope is a Zeiss 710. Building, some sort of a flexible damn system for using a water immersion lens on an inverted really does not seem practical. Thanks for any help. Richard E. Edelmann, Ph.D. Electron Microscopy Facility Director 364 Pearson Hall Miami University, Oxford, OH 45056 Ph: 513.529.5712 Fax: 513.529.4243 E-mail: [hidden email] http://www.emf.muohio.edu --------------------------------------------------------------------- SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. --------------------------------------------------------------------- |
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Goodhouse, Joseph G. |
Re: Objectives for live cell imaging
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The perfect optical system will have no refractive mismatch in
the system. This can be accomplished when imaging a fixed biological specimen if it has been prepared properly and mounted in a medium that has an RI of 1.515 and is imaged with an oil immersion objective. If this can't be accomplished such as with a live sample then the rule is to have the fewest refractive changes and to have them to be minimum. When imaging cultured cells this is done by using an oil immersion optic. One then has only 1 refractive mismatch, from 1.515 to 1.33. Empirical observation has demonstrate that for cultured cells one will obtain higher resolved images with an oil optic. However when imaging a live sample where one has to get as deep into the sample, such as fish and fly embryos it has been our experience that a water optic will do better for depth. Joe Goodhouse Confocal Core Lab Manager Dept. of Molecular Biology Princeton University Washington Road Princeton, NJ. 08544-1014 609-258-5432 Visit us at http://www.molbio1.princeton.edu/facility/confocal/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian Sent: Wednesday, December 02, 2009 12:06 PM To: [hidden email] Subject: Re: Objectives for live cell imaging We often image live cells on inverted microscopes using water immersion lenses. It is not a problem, and in fact you should image live cells in aqueous with a water immersion lens. You can also use a thicker water from Zeiss Immersol W (and others) that has an RI similar to W. We use this for long term live cell imaging at 37degrees. Live cell imaging is best done through a glass coverslip bottom dish using water immersion lenses on an inverted scope (in my humble opinion). Cheers, Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Hope Dept of Neuroscience 1450 E Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag ing/Pages/default.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Richard E. Edelmann Sent: Wednesday, December 02, 2009 7:51 AM To: [hidden email] Subject: Objectives for live cell imaging O.k., obviously live cell imaging would be best done with either (1) an upright with a water immersion lens (dipping) or (2) cells adherent to a cover slip and an oil immersion lens on an inverted scope. So what about live tissue imaging on an inverted scope? (I.e. whole or dissected insect neuro-muscular development). How do you best deal with spherical aberration due to samples in a watery medium a distance from the coverslip? With say a 40 to 60X magnification. Scope is a Zeiss 710. Building, some sort of a flexible damn system for using a water immersion lens on an inverted really does not seem practical. Thanks for any help. Richard E. Edelmann, Ph.D. Electron Microscopy Facility Director 364 Pearson Hall Miami University, Oxford, OH 45056 Ph: 513.529.5712 Fax: 513.529.4243 E-mail: [hidden email] http://www.emf.muohio.edu --------------------------------------------------------------------- SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. --------------------------------------------------------------------- |
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Keith Morris |
Re: Objectives for live cell imaging
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In reply to this post by Edelmann, Richard E. Dr.
We occasionally image one 'large' fly related organ: whole Drosphila brains
using a long working distance 40x water objective on our Zeiss 510 confocal with inverted Axiovert 200. I believe Zeiss can provide a 'drip tray' to fit around the top of the objective or you can make your own dam [for one-offs I have simply used clean PTFE washers rested into place on top of the objective, surface tension helping out, and you can put a bit of tissue round the side of the objective just in case, or perhaps use a 'raised up' Zeiss 'live cell' foam tube that are made to fit tight round the objective]. Zeiss can also provide a special 'water' immersion fluid [Immersol W] to help: it's apparently mostly a derivative of 'perfluorinepolyether' rather than water as such - the safety sheet talks of 40oC, but ask Zeiss about its evaporation rates at 37oC. When using Immersol W our user doesn't normally bother with sophisticated dams and such [he's just careful with it and probably relies a lot on surface tension and lens tissues]. However as our drosphila tissue is fixed so there's no problem doing a large [150+] z-stack [no movement], and as there's no time-lapse we don't hang around with the sample/immersion fluid. We use a 40x water immersion objective borrowed from another facility at the moment as they cost around £7k*. Generally such water objectives are kept locked in a cupboard and only used by experienced users [the owners], so we get less far problems with water immersion that with oil immersion and culture fluid spillages on our inverted microscopes. Adventurous types have even built a water feed tube and dam system to keep the water immersion liquid in place [but I've never seen that, I've only heard...]. So it may be easier with an upright in some situations, but, as others here have said, it is quite practical to use an inverted configuration microscope with water objectives [sometimes helped by little ingenuity]. Just try it with a water objective for a few hours on loan via a Zeiss rep. We invert the slide for the fixed brains, but I imagine a Mattek type Petri dish would be fine [i.e. better] for live work with an inverted. I assume your insects don't need 37oC incubators - we always use the water objective at room temperature [i.e. on a mammalian live cell free day]. A confocal 3D reconstruction of a drosophila brain imaged as described above can be seen at: http://www.well.ox.ac.uk/volocity Regards, Keith * <a href="https://www.micro-shop.zeiss.com/us/us_en/objektive.php?cp_sid=&s=t&f=od&p%5">https://www.micro-shop.zeiss.com/us/us_en/objektive.php?cp_sid=&s=t&f=od&p%5 b%5d=421767-9970-000 --------------------------------------------------------------------------- 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: +44 (0)1865 287568 Email: [hidden email] Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Richard E. Edelmann Sent: 02 December 2009 15:51 To: [hidden email] Subject: Objectives for live cell imaging O.k., obviously live cell imaging would be best done with either (1) an upright with a water immersion lens (dipping) or (2) cells adherent to a cover slip and an oil immersion lens on an inverted scope. So what about live tissue imaging on an inverted scope? (I.e. whole or dissected insect neuro-muscular development). How do you best deal with spherical aberration due to samples in a watery medium a distance from the coverslip? With say a 40 to 60X magnification. Scope is a Zeiss 710. Building, some sort of a flexible damn system for using a water immersion lens on an inverted really does not seem practical. Thanks for any help. Richard E. Edelmann, Ph.D. Electron Microscopy Facility Director 364 Pearson Hall Miami University, Oxford, OH 45056 Ph: 513.529.5712 Fax: 513.529.4243 E-mail: [hidden email] http://www.emf.muohio.edu |
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George McNamara |
Re: Objectives for live cell imaging
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In reply to this post by Edelmann, Richard E. Dr.
Hi Richard,
I agree with many of the items mentioned be previous responders. Here's my take: Zeiss makes a very nice 25x/0.8 NA multi-immersion lens ("IMM"). It can be dialed for any refractive index from 1.33 to 1.515. I usually leave it at RI 1.33 on our LSM510/UV, but change as needed. This, combined with a 35 mm imaging dish (e.g. mattek.com = glassbottomdishes.com or WPI or Greiner bio-One, or do-it-yourself), RI matched immersion and mounting media, and especially the ability to zoom up (or down to 0.6x) on the LSM710 makes for a very robust imaging platform. Zeiss also has a wide range of 20x, 32x and 40x NA 1.0 and NA 1.2 lenses, and some interesting glycerol lenses (e.g. 63x/1.3NA), if you really need to push the resolution (and have the money). I am now recommending the coverglass bottom dishes to all my users for both live specimens and standard cell culture immunofluorescence - I've seen too many coverglass-slide preparation leaking all kinds of mounting media and think $2.10 for an imaging dish is an excellent investment (on the researcher's part) to avoid ruining any expensive objective lenses on our core's microscopes. Best wishes and happy holidays to all, George p.s. speaking of Zeiss, while checking out latest objective lenses on their website I noticed this in their news: Carl Zeiss Expects Recovery Fiscal Year 2008/09 Ended with a Loss This is not to single out Zeiss in these economic times - I doubt the other companies in our field had a spectacular 2009. At 10:50 AM 12/2/2009, you wrote:
George McNamara, Ph.D. Image Core Manager Analytical Imaging Core Facility University of Miami, Miller School of Medicine Miami, FL 33136 [hidden email] [hidden email] 305-243-8436 office http://www.sylvester.org/AICF (Analytical Imaging Core Facility) http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file) http://home.earthlink.net/~geomcnamara |
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