Objective for confocal
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi All, I am new in confocal microscopy. We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any advantage to add a 100X/1.46 Oil objective? Ann Fook Yang, Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960 Boulevard Carling, Ottawa,Ontario K1A 0C6 [hidden email] Telephone/Téléphone: 613-759-1638 Facsimile/Télécopieur: 613-759-1701 |
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Hello Ann,
there may be different opinions on this, but here is my take:
With a confocal microscope, you can add magnification by zooming in and matching the image size (pixel dimensions) for optimal resolution. This means you can get 100 x magnification with a 40x at zoom 2.5, or a 63x at zoom 1.6. What the 100x/1.46 gives you in theory is greater resolution (20-30% more), since resolution increases with the square of the numerical aperture. In practice, you may not always achieve this extra resolution, unless you have quite bright samples that are relatively thin and properly mounted, in order to minimize spherical aberrations. You also need to consider the type of objective and the application. PlanApos are corrected for more colors, and therefore are best for precise colocalization of up to four dyes. Fluars or similar objectives are corrected for fewer colors, but tend to be brighter than PlanApos, because they are simpler lenses with less glass... these are often better for live-cell imaging, or for samples with fewer colors... for a more precise description, you may want to check the tutorial on lenses and chromatic aberrations here: We typically use objectives in the 40x-60x range for most of our confocal applications (in addition to a 10x or 20x for low-mag work, and possibly a long-working distance 40x for thick/unusual samples). We rarely use a 100x, except maybe to look at yeast cells, or some very fine cellular detail. A 100x/1.46 lens will often be less bright than a 40x/1.3, which means that your gain in resolution may be offset by increased bleaching. In addition, a 40x is more versatile since you can image a wider field of view at zoom 1, and then get a close-up at zoom 2-3 if you need more mag, without having to switch lenses. A 100x/1.46 lens, on the other hand, would be quite nice on a widefield microscope... also, this type of lens with high NA is what you need for applications such as TIRF (total internal Reflection microscopy)... -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 Tel: Office: 206-667-1215/ Lab: 206-667-4205 FAX: 206-667-6845 -------------------------------------------------- This message is confidential, intended only for the named recipient(s) and may contain information that is privileged or exempt from disclosure under applicable law. If you are not the intended recipient(s), you are notified that the dissemination, distribution or copying of this information is strictly prohibited. If you received this message in error, please notify the sender then delete this message. On Oct 26, 2007, at 1:09 PM, Yang, Ann-Fook wrote:
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In reply to this post by Yang, Ann-Fook
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I quite agree with what Julio said. There is one
more
point, though. The NA 1.46 lens is presumably
a
TIRF lens, and so may be handy if one later
wants to
add TIRF capabilities. As such, it will probably
also
have a correction collar, and if this is used
correctly it
will significantly improve performance. At
high NA,
minor differences in coverslip thickness and
room
temperature will noticeably impact on
resolution
unless they are corrected
for.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez Sent: Saturday, 27 October 2007 9:10 AM To: [hidden email] Subject: Re: Objective for confocal there may be different opinions on this, but here is my take:
With a confocal microscope, you can add magnification by zooming in and
matching the image size (pixel dimensions) for optimal resolution. This means
you can get 100 x magnification with a 40x at zoom 2.5, or a 63x at zoom
1.6.
What the 100x/1.46 gives you in theory is greater resolution (20-30% more),
since resolution increases with the square of the numerical aperture. In
practice, you may not always achieve this extra resolution, unless you have
quite bright samples that are relatively thin and properly mounted, in order to
minimize spherical aberrations.
You also need to consider the type of objective and the application.
PlanApos are corrected for more colors, and therefore are best for precise
colocalization of up to four dyes. Fluars or similar objectives are corrected
for fewer colors, but tend to be brighter than PlanApos, because they are
simpler lenses with less glass... these are often better for live-cell imaging,
or for samples with fewer colors... for a more precise description,
you may want to check the tutorial on lenses and chromatic aberrations
here:
We typically use objectives in the 40x-60x range for most of our confocal
applications (in addition to a 10x or 20x for low-mag work, and possibly a
long-working distance 40x for thick/unusual samples). We rarely use a 100x,
except maybe to look at yeast cells, or some very fine cellular detail. A
100x/1.46 lens will often be less bright than a 40x/1.3, which means that your
gain in resolution may be offset by increased bleaching. In addition, a 40x is
more versatile since you can image a wider field of view at zoom 1, and then get
a close-up at zoom 2-3 if you need more mag, without having to switch
lenses.
A 100x/1.46 lens, on the other hand, would be quite nice on a widefield
microscope... also, this type of lens with high NA is what you need for
applications such as TIRF (total internal Reflection microscopy)...
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
Tel: Office: 206-667-1215/ Lab: 206-667-4205
FAX: 206-667-6845
-------------------------------------------------- This message is confidential, intended only for the named recipient(s) and may contain information that is privileged or exempt from disclosure under applicable law. If you are not the intended recipient(s), you are notified that the dissemination, distribution or copying of this information is strictly prohibited. If you received this message in error, please notify the sender then delete this message. On Oct 26, 2007, at 1:09 PM, Yang, Ann-Fook wrote:
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Ann, To follow up from Julio's comments. The higher NA will give you improved resolution. A 1.42 NA object theoreticatly can resolve down to 210nm at 488nm exitation. That holds true for what ever the magnification the objective has. We have compared a 63x 1.42 NA to a 100x 1.42 NA objective. Both were Olympus UIS2 PlanApo objectives. Both could resolve the same structures. But the big difference was the amount of light that was needed to capture an image with the 100x objective. On average 7x more light input was required to get image with the 100x lens that was equivalent to the 63x lens. So my advice woudl be to get a 63x 1.42 NA objective, or better yet a 40x 1.4NA if you can find one (they are being made by Nikon i think but not sure about others). Of course this is only important if you want to b e able to resolve very small structures. If you do not want to be resolving down to 210nm then your 40x oil and 63x water imersion objective will be just fine. I can send you (and anyone else on the list) a copy of the reoprt with the comparison of the 63x vs the 100x lens if you want. Cheers Cam Cameron Nowell B.Sc (Hons) Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre 7 St Andrews Place East Melbourne, Victoria 3002 Phone: +61396561243 Mobile: +61422882700 Fax: +61396561411 ________________________________ From: Confocal Microscopy List on behalf of Yang, Ann-Fook Sent: Sat 27/10/2007 6:09 AM To: [hidden email] Subject: Objective for confocal Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi All, I am new in confocal microscopy. We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any advantage to add a 100X/1.46 Oil objective? Ann Fook Yang, Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960 Boulevard Carling, Ottawa,Ontario K1A 0C6 [hidden email] Telephone/Téléphone: 613-759-1638 Facsimile/Télécopieur: 613-759-1701 This email (including any attachments) may contain confidential and/or legally privileged information and is intended only to be read or used by the addressee. If you are not the intended addressee, any use, distribution, disclosure or copying of this email is strictly prohibited. Confidentiality and legal privilege attached to this email (including any attachments) are not waived or lost by reason of its mistaken delivery to you. If you have received this email in error, please delete it and notify us immediately by telephone or email. Peter MacCallum Cancer Centre provides no guarantee that this transmission is free of virus or that it has not been intercepted or altered and will not be liable for any delay in its receipt. |
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For what conditions the high NA oil immersion lenses (w/o the
correction collar) are optimized, room temperature or Live Cell Imaging (at
37 deg)? I recently compared NA 1.45 versus NA 1.49 (with the correction
collar), and haven't noticed any difference on fixed samples of 125-150 nm
fluorescent virus particles (at RT).
vitaly
nci-frederick
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Peter Gabriel Pitrone |
Re: Objective for confocal
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In reply to this post by Yang, Ann-Fook
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Magnification?!?!
On Oct 26, 2007, at 10:09 PM, Yang, Ann-Fook wrote:
Peter Gabriel Pitrone Light Microscopy Facility Max Planck Institute for Cell Biology and Genetics Pfotenhauerstrasse 108 01307 Dresden, Germany E-Mail: [hidden email] |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Magnification is increased with a 100x lens when compared to a 63x sure, but if you are using confocal then this isn't that important since you can digitally zoom in to get the same magnification. There are really no advantages of going to a 100x bjective if you have a high NA 63x available. Cheers Cam Cameron Nowell B. Sc (Hons) Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre 7 St Andrews Place East Melbourne, Victoria 3002 AUSTRALIA Phone: +61396561242 Mobile: +61422882700 Fax: +61396561411 ________________________________ From: Confocal Microscopy List on behalf of Peter Pitrone Sent: Mon 29/10/2007 8:24 PM To: [hidden email] Subject: Re: Objective for confocal Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Magnification?!?! On Oct 26, 2007, at 10:09 PM, Yang, Ann-Fook wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi All, I am new in confocal microscopy. We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any advantage to add a 100X/1.46 Oil objective? Ann Fook Yang, Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960 Boulevard Carling, Ottawa,Ontario K1A 0C6 [hidden email] Telephone/Téléphone: 613-759-1638 Facsimile/Télécopieur: 613-759-1701 Peter Gabriel Pitrone Light Microscopy Facility Max Planck Institute for Cell Biology and Genetics Pfotenhauerstrasse 108 01307 Dresden, Germany E-Mail: [hidden email] This email (including any attachments) may contain confidential and/or legally privileged information and is intended only to be read or used by the addressee. If you are not the intended addressee, any use, distribution, disclosure or copying of this email is strictly prohibited. Confidentiality and legal privilege attached to this email (including any attachments) are not waived or lost by reason of its mistaken delivery to you. If you have received this email in error, please delete it and notify us immediately by telephone or email. Peter MacCallum Cancer Centre provides no guarantee that this transmission is free of virus or that it has not been intercepted or altered and will not be liable for any delay in its receipt. |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Well, this is a excellent opportunity to use once more the cover-all answer: It depends... I did a pretty extensive comparison of the the objectives Apo 63x/1.4 Oil and Apo 100x/1.46. All tests were done with Leica objectives, results may differ for other brands. Test sample were beads 170nm in Glycergel close to the coverslip or homogeneously stained cells for the "Sensitivity" comparison. Results: Resolution was visibly and measurably better with the 100x. But: it is absolutely mandatory to adjust the correction collar with great care, otherwise you will do more harm than good. Not difficult in itself, but you simply have to do it. You will also gain in "Sensitivity": you will need less laser energy (measured in focal plane, not at AOTF) to achieve the same apparent image brightness. Under these conditions also a lower bleach rate was observed. So in principle I do think this is an excellent objective improving the performance of the system. Before this objective I had the opinion that a 100x would be allways less "bright" than a 63x. For a 63x/1.2 Water all this would be valid to an even larger degree, just consider the BUT... BUT: keep in mind that if you are working with thicker samples (meaning for me more than approx. 10-15um) the match of the RI (refractive index) becomes much more important for your imaging than pure NA. So if you work with living samples usually water immersion is the best, with fixed and embedded samples I would usually recommend Glycerol objectives since most commercial embedding media are very close in RI to this immersion medium. So it is very important to decide what is most important for your application: max. brightness/resolution in flat samples or maximum penetration/resolution/brightness in thick samples. Hope this comment is a bit helpfull. For background information go to Pawley's Handbook of confocal microscopy, read about RI match and spherical aberration. These factors have a huge impact on your image quality and can hardly be overestimated. Good luck Boris P.S. I am working for Leica Microsystems, but I think this makes no difference for this case... __________________________________________________ Boris Zarda Dr. rer. nat. Sales Manager Research Switzerland Leica Microsystems (Schweiz) AG Verkaufsgesellschaft Max Schmidheiny-Strasse 201 CH-9435 Heerbrugg Tel +41 44 768 36 30 Fax +41 71 726 34 44 "Yang, Ann-Fook" <[hidden email]> Gesendet von: An Confocal [hidden email] Microscopy List Kopie <CONFOCAL@LISTSER V.BUFFALO.EDU> Thema Objective for confocal 26.10.2007 22:09 Bitte antworten an Confocal Microscopy List <CONFOCAL@LISTSER V.BUFFALO.EDU> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi All, I am new in confocal microscopy. We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any advantage to add a 100X/1.46 Oil objective? Ann Fook Yang, Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960 Boulevard Carling, Ottawa,Ontario K1A 0C6 [hidden email] Telephone/Téléphone: 613-759-1638 Facsimile/Télécopieur: 613-759-1701 ______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email ______________________________________________________________________ |
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In reply to this post by Yang, Ann-Fook
Search the CONFOCAL archive at
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Search the CONFOCAL archive at
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Hi Julio,
Thank you for spending time to write. The information is very usefully to me.
Ann Fook -----Original
Message-----
Hello Ann,
there may be different opinions on this, but here is my take:
With a confocal microscope, you can add magnification by zooming in and matching the image size (pixel dimensions) for optimal resolution. This means you can get 100 x magnification with a 40x at zoom 2.5, or a 63x at zoom 1.6.
What the 100x/1.46 gives you in theory is greater resolution (20-30% more), since resolution increases with the square of the numerical aperture. In practice, you may not always achieve this extra resolution, unless you have quite bright samples that are relatively thin and properly mounted, in order to minimize spherical aberrations.
You also need to consider the type of objective and the application. PlanApos are corrected for more colors, and therefore are best for precise colocalization of up to four dyes. Fluars or similar objectives are corrected for fewer colors, but tend to be brighter than PlanApos, because they are simpler lenses with less glass... these are often better for live-cell imaging, or for samples with fewer colors... for a more precise description, you may want to check the tutorial on lenses and chromatic aberrations here:
We typically use objectives in the 40x-60x range for most of our confocal applications (in addition to a 10x or 20x for low-mag work, and possibly a long-working distance 40x for thick/unusual samples). We rarely use a 100x, except maybe to look at yeast cells, or some very fine cellular detail. A 100x/1.46 lens will often be less bright than a 40x/1.3, which means that your gain in resolution may be offset by increased bleaching. In addition, a 40x is more versatile since you can image a wider field of view at zoom 1, and then get a close-up at zoom 2-3 if you need more mag, without having to switch lenses.
A 100x/1.46 lens, on the other hand, would be quite nice on a widefield microscope... also, this type of lens with high NA is what you need for applications such as TIRF (total internal Reflection microscopy)...
-- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024
Tel: Office: 206-667-1215/ Lab: 206-667-4205 FAX: 206-667-6845
-------------------------------------------------- This message is confidential, intended only for the named recipient(s) and may contain information that is privileged or exempt from disclosure under applicable law. If you are not the intended recipient(s), you are notified that the dissemination, distribution or copying of this information is strictly prohibited. If you received this message in error, please notify the sender then delete this message.
On Oct 26, 2007, at 1:09 PM, Yang, Ann-Fook wrote:
Search the CONFOCAL archive at
Hi All,
I am new in confocal microscopy. We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any advantage to add a 100X/1.46 Oil objective?
Ann Fook Yang, Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960 Boulevard Carling, Ottawa,Ontario K1A 0C6
Telephone/Téléphone: 613-759-1638 Facsimile/Télécopieur: 613-759-1701
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In reply to this post by Yang, Ann-Fook
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Cameron, Thank you very much for sharing your experience. I would appreciate receiving the report you mentioned. Regards, Ann Fook -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Nowell, Cameron Sent: Friday, October 26, 2007 9:08 PM To: [hidden email] Subject: Re: Objective for confocal Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Ann, To follow up from Julio's comments. The higher NA will give you improved resolution. A 1.42 NA object theoreticatly can resolve down to 210nm at 488nm exitation. That holds true for what ever the magnification the objective has. We have compared a 63x 1.42 NA to a 100x 1.42 NA objective. Both were Olympus UIS2 PlanApo objectives. Both could resolve the same structures. But the big difference was the amount of light that was needed to capture an image with the 100x objective. On average 7x more light input was required to get image with the 100x lens that was equivalent to the 63x lens. So my advice woudl be to get a 63x 1.42 NA objective, or better yet a 40x 1.4NA if you can find one (they are being made by Nikon i think but not sure about others). Of course this is only important if you want to b e able to resolve very small structures. If you do not want to be resolving down to 210nm then your 40x oil and 63x water imersion objective will be just fine. I can send you (and anyone else on the list) a copy of the reoprt with the comparison of the 63x vs the 100x lens if you want. Cheers Cam Cameron Nowell B.Sc (Hons) Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre 7 St Andrews Place East Melbourne, Victoria 3002 Phone: +61396561243 Mobile: +61422882700 Fax: +61396561411 ________________________________ From: Confocal Microscopy List on behalf of Yang, Ann-Fook Sent: Sat 27/10/2007 6:09 AM To: [hidden email] Subject: Objective for confocal Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi All, I am new in confocal microscopy. We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any advantage to add a 100X/1.46 Oil objective? Ann Fook Yang, Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960 Boulevard Carling, Ottawa,Ontario K1A 0C6 [hidden email] Telephone/Téléphone: 613-759-1638 Facsimile/Télécopieur: 613-759-1701 This email (including any attachments) may contain confidential and/or legally privileged information and is intended only to be read or used by the addressee. If you are not the intended addressee, any use, distribution, disclosure or copying of this email is strictly prohibited. Confidentiality and legal privilege attached to this email (including any attachments) are not waived or lost by reason of its mistaken delivery to you. If you have received this email in error, please delete it and notify us immediately by telephone or email. Peter MacCallum Cancer Centre provides no guarantee that this transmission is free of virus or that it has not been intercepted or altered and will not be liable for any delay in its receipt. |
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In reply to this post by Yang, Ann-Fook
Search the CONFOCAL archive at
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Search the CONFOCAL archive at
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Hi Guy,
I appreciate your input, thank you.
Cheers, Ann Fook -----Original
Message-----
I quite agree with what Julio said. There is one more point, though. The NA 1.46 lens is presumably a TIRF lens, and so may be handy if one later wants to add TIRF capabilities. As such, it will probably also have a correction collar, and if this is used correctly it will significantly improve performance. At high NA, minor differences in coverslip thickness and room temperature will noticeably impact on resolution unless they are corrected for.
Guy
Optical Imaging Techniques in Cell Biology
From:
Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Ann,
there may be different opinions on this, but here is my take:
With a confocal microscope, you can add magnification by zooming in and matching the image size (pixel dimensions) for optimal resolution. This means you can get 100 x magnification with a 40x at zoom 2.5, or a 63x at zoom 1.6.
What the 100x/1.46 gives you in theory is greater resolution (20-30% more), since resolution increases with the square of the numerical aperture. In practice, you may not always achieve this extra resolution, unless you have quite bright samples that are relatively thin and properly mounted, in order to minimize spherical aberrations.
You also need to consider the type of objective and the application. PlanApos are corrected for more colors, and therefore are best for precise colocalization of up to four dyes. Fluars or similar objectives are corrected for fewer colors, but tend to be brighter than PlanApos, because they are simpler lenses with less glass... these are often better for live-cell imaging, or for samples with fewer colors... for a more precise description, you may want to check the tutorial on lenses and chromatic aberrations here:
We typically use objectives in the 40x-60x range for most of our confocal applications (in addition to a 10x or 20x for low-mag work, and possibly a long-working distance 40x for thick/unusual samples). We rarely use a 100x, except maybe to look at yeast cells, or some very fine cellular detail. A 100x/1.46 lens will often be less bright than a 40x/1.3, which means that your gain in resolution may be offset by increased bleaching. In addition, a 40x is more versatile since you can image a wider field of view at zoom 1, and then get a close-up at zoom 2-3 if you need more mag, without having to switch lenses.
A 100x/1.46 lens, on the other hand, would be quite nice on a widefield microscope... also, this type of lens with high NA is what you need for applications such as TIRF (total internal Reflection microscopy)...
-- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024
Tel: Office: 206-667-1215/ Lab: 206-667-4205 FAX: 206-667-6845
-------------------------------------------------- This message is confidential, intended only for the named recipient(s) and may contain information that is privileged or exempt from disclosure under applicable law. If you are not the intended recipient(s), you are notified that the dissemination, distribution or copying of this information is strictly prohibited. If you received this message in error, please notify the sender then delete this message.
On Oct 26, 2007, at 1:09 PM, Yang, Ann-Fook wrote:
Search the CONFOCAL archive at
Hi All,
I am new in confocal microscopy. We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any advantage to add a 100X/1.46 Oil objective?
Ann Fook Yang, Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960 Boulevard Carling, Ottawa,Ontario K1A 0C6
Telephone/Téléphone: 613-759-1638 Facsimile/Télécopieur: 613-759-1701
No virus found in this incoming message. No virus found in this outgoing message. |
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In reply to this post by Yang, Ann-Fook
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Cameron, I will be inetrested in the test report on the two Olympus lenses. Were your tests done in the wide-field or confocal mode? Our Olympus confocal had a 60x/1.42 PLAPON objective that turned out to be substantially worse in resolution than we expected from theoretical calculations. After testing several more of the same type we switched to the 100x/1.4 Universal Plan Apo (Thanks to our local Olympus office I was able to pick the best one among several of those objectives). It showed much better resolution and less chromatic aberration across the visible spectrum. I can e-mail test results to those who are interested (both mirror slide and sub-resolution beads were used). Regarding the signal intensity - I thought that in the point-scanning confocal mode, there should not be a big difference in excitation intensity and signal detected between different magnification objectives of the same NA - the size of the illuminated spot is determined by NA, as well as the amount of fluorescence signal collected. Since all the collected fluorescence signal is focused to a single spot in the pinhole plane, there again should not be much difference between, say, 60x and 100x objective, providing the pinhole size is set to the same Airy size and all other things are the same. Am I missing something fundamental? Stan Dr. Stanislav Vitha [hidden email] Microscopy and Imaging Center Texas A&M University BSBW 119 College Station, TX 77843-2257 tel: 979-845-1129 (main desk) tel: 979-845-1607 (direct link) fax: 979-847-8933 On Sat, 27 Oct 2007 11:08:29 +1000, Nowell, Cameron <[hidden email]> wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi Ann, > To follow up from Julio's comments. The higher NA will give you improved resolution. A 1.42 NA object theoreticatly can resolve down to 210nm at 488nm exitation. That holds true for what ever the magnification the objective has. > >We have compared a 63x 1.42 NA to a 100x 1.42 NA objective. Both were Olympus UIS2 PlanApo objectives. Both could resolve the same structures. But the big difference was the amount of light that was needed to capture an image with the 100x objective. On average 7x more light input was required to get image with the 100x lens that was equivalent to the 63x lens. > >So my advice woudl be to get a 63x 1.42 NA objective, or better yet a 40x 1.4NA if you can find one (they are being made by Nikon i think but not sure about others). > >Of course this is only important if you want to b e able to resolve very small structures. If you do not want to be resolving down to 210nm then your 40x oil and 63x water imersion objective will be just fine. > >I can send you (and anyone else on the list) a copy of the reoprt with the comparison of the 63x vs the 100x lens if you want. > > >Cheers > >Cam > > >Cameron Nowell B.Sc (Hons) >Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre >7 St Andrews Place >East Melbourne, Victoria 3002 > >Phone: +61396561243 >Mobile: +61422882700 >Fax: +61396561411 > >________________________________ > >From: Confocal Microscopy List on behalf of Yang, Ann-Fook >Sent: Sat 27/10/2007 6:09 AM >To: [hidden email] >Subject: Objective for confocal > > > >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi All, > >I am new in confocal microscopy. >We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any > >Ann Fook Yang, >Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada >EM Unit/ Unite EM >Edifice K.W. Neatby Building, >960 Carling Av /960 Boulevard Carling, >Ottawa,Ontario >K1A 0C6 > >[hidden email] >Telephone/Téléphone: 613-759-1638 >Facsimile/Télécopieur: 613-759-1701 > > > > >This email (including any attachments) may contain >confidential and/or legally privileged information and is >intended only to be read or used by the addressee. If you >are not the intended addressee, any use, distribution, >disclosure or copying of this email is strictly >prohibited. >Confidentiality and legal privilege attached to this email >(including any attachments) are not waived or lost by >reason of its mistaken delivery to you. >If you have received this email in error, please delete it >and notify us immediately by telephone or email. Peter >MacCallum Cancer Centre provides no guarantee that this >transmission is free of virus or that it has not been >intercepted or altered and will not be liable for any delay >in its receipt. >======================================================================== |
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In reply to this post by Nowell, Cameron
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi all,
Let's start by saying that I am all in favor of people constantly
monitoring the performance of their confocal microscopes (particularly
z-resolution and photon collection efficiency)...
Yes, strictly speaking, the size of the focus spot is
affected only by the NA and the wavelength (not the
objective lens magnification). While in single-beam confocal, the
magnification is controlled by the size of the scanned raster, in
disk-scanning of normal widefield, it is controlled by the objective
magnification. I won't talk much about disk-scanning/wiidefield today
except to note that when judging "image brightness" using
data obtained from a CCD, one must be fair and always correct for the
effective area of each CCD pixel, referred to the image plane: a pixel
that covers more area on a bright part of the specimen can be expected
to collect more light... This will happen if one uses an objective of
lower magnification unless one compensates by changing the
magnification of the phototube.
In addition, it is also true that the maximum NA that one
can use effectively varies with the refractive index
(RI) of the medium in which the specimen is suspended (or, if the
specimen is a transparent solid, the average RI of the specimen
itself): NA<1.2 for water specimens, NA<1.25 for glycerol
specimens and NA<1.49 for oil-embedded specimens. Using a NA 1.4
oil lens on a watery specimen will only be advantageous if one
confines ones interest to that part of the specimen which is within a
micron or two of the coverslip surface and even then, rays at angles
greater than about NA 1.3 will not contribute to the image because
they totally reflect at the water/glass interface.
To use any objective designed to work with one immersion medium
on a specimen having a different RI will produce spherical aberration:
a circumstance that will not only make the image blurry but will also
make the peak brightness of the image of any point object appear less
intense. In actual fact, the match between specimen RI and lens-design
RI is almost never perfect and this is why having a TIRF objective
with a correction collar is a great advantage (but only for
oil-embedded specimens or those within a micron or two of the
coverglass surface.) This RI-mismatch problem is MUCH more severe at
high NA, particularly for high-NA "dry" objectives (which as
a result, often have correction collars.)
However there are other important factors related to NA and
resolution that have not yet been discussed.
When one considers a laser confocal (single-beam or
disk-scanner), the most important of these is the matter of whether or
not the laser beam "fills" the back-focal plane (BFP) of a
particular objective lens. If you look at a 100x NA 1.4 and a 63x NA
1.4 from the back side, you will see that the diameter of the glass in
the latter is larger. That of a 40x NA 1.3 is larger
still.
A simple way to understand why this should be so is to imagine
that these are "simple" lenses (thin, one-element lenses).
In a thin lens, all the "focusing action" is thought of as
taking place at the principle plane in the middle of this thin lens.
In particular, that any light emerging from a point in the focal plane
on one side of the lens will leave the other side of the lens as a
parallel ray-bundle. If the point is on the optical axis, the emerging
parallel ray bundle will be parallel with this axis.
In round figures, the focal length of the 100x objective will be
about 2 mm and that of the 63x about 3 mm. Because they have the same
NA, they will both accept the same cone of light from a point on the
specimen. However, because of the different focal lengths, the high-NA
rays from the 63x will be ~50% farther from the axis when they reach
the principle plane than those from the 100x. Consequently, the part
of the back-focal plane that is used will have a larger diameter. If
one is to use this lens at its rated NA, the ray bundle in the BFP
must be at least this big.
Now imagine the light from the laser going in the opposite
direction. Clearly the bundle of light from the laser has some
diameter as it reaches the BFP of the objective. This diameter will
depend on the specifics of the beam expander used to convert the light
from the fiber into a ~parallel ray bundle. It is obvious that
if the diameter is large enough to "fill" the BFP of the
63x, then it will overfill that of the 100x (i.e., more than half of
the light will strike the metal around the edge of the glass elements
and so not progress to the specimen. It may be worse than this because
the obstructed light may reflect off components of the optical system
and end up being mistakenly collected as signal.) Although it is
possible to change the parameters of the beam expander in the confocal
scanner to compensate for this change, this is seldom done as it
requires fabricating a diffraction-limited zoom lens.
Consequently, each confocal is characterized by providing a ray
bundle or a certain size at the estimated location of the objective
BFP (An axial location, by the way, that depends on the objective lens
focal length. In the example above, the 63x BFP would be 6 mm from the
focus plane, that of the 100x, 4mm). If the ray bundle is large enough
to fill only the 100x, only this lens will operate at its rated NA
"on the way down" (Light coming back from the specimen will
be accepted by any lens to the full angle of its rated NA.). As a
result, it will produce a smaller spot in the specimen (because the
63x is NOT being operated at its rated NA) that is also has a higher
peak brightness (because it is smaller).
On the other hand, if the ray bundle is big enough to fill the
63x, both the 100x and the 63X will produce a spot of the same size
but much of the power in the laser beam will be strike the metal of
the 100x and consequently the not hit the specimen, making the image
will appear dimmer with the 100x.
So far so good. The bad part is that "the ray bundle"
from the beam expander doesn't actually have a constant brightness
from the center to its edge, and then go to zero. In general, its
intensity varies with distance from the axis in a Gaussian manner. In
other words it doesn't have "a diameter" but its intensity
always drops off away from the axis. The designer can decide how large
to make this Gaussian beam. He/she could design it so that only the
central 10% (in area) goes through the usable part of the 100x BFP (or
the central ~20% goes through the usable part of the 63x BFP). By
using only the central part of the Gaussian distribution in this way,
the intensity will be almost constant across the BFP and this means
that the spot really will be an Airy disk, not the somewhat larger
Gaussian spot that one gets when the BFP is filled by a Gaussian.
However, this will also mean that, 90% of the laser light is being
wasted. As lasers usually put out much more light (5-15 mW) than
one wants to expose a specimen to (1-20 microwatts), this only a
serious loss if one wishes to use the beam for intentional bleaching
or uncaging etc. Still, this is a strategy that few confocal
manufacturers pursue.
It is more common for the designer to set up the optics so that
about 80% of the light will sort-of fill the BFP of a 63x 1.4
(and that therefore a 40x NA 1.3 will be be severely under-filled). If
this is so, then the 100x will be filled with almost constant
intensity and will show slightly better resolution (assuming no
spherical aberration etc.) than the 63x because the latter will
produce an almost-Gaussian spot with a slightly larger
"full-width at half maximum". However, you would have to
look hard to see this change in resolution because, it is a small
change to start with (~10%) and in addition, the NA of the
signal-collection path will be the same on both systems. With a small
pinhole, the measured x-y-z resolution depends on both the
illumination and collection light paths. (i.e., If you want to see
this effect, image a planar array of fluorescent beads and use a large
pinhole size. Don't forget that the pinhole diameter should still be
~50% larger with the 100x.)
All this makes it hard to evaluate the optical performance of any
objective if you don't know the ray bundle diameter at the objective
BFP. The best way to measure this directly is to set up Kohler
illumination using an NA 1.4 oil-immersion condenser (and don't
forget to oil the condenser and use an oiled specimen!). Stop the
scanning beam near to the axis and hold a white card so that you
can see the shape of the light bundle that emerges from the condenser
on the side away from the specimen. You should see a circle of light
that has a sharp edge corresponding to the edge of the aperture
diaphragm. Open this diaphragm until the sharp edge is no longer
illuminated and then note the NA setting of the condenser. That is the
NA to which your objective was illuminated.
If you don't have an oil condenser (most inverted scopes don't),
you can make an approximate measurement using a low-mag dry lens with
an a NA that is high for its mag (say a 10x NA 0.45 or NA. 0.5) but is
still lower than that of the condenser mounted on your scope. Don't
forget to set up Kohler illumination. The active diameter of the BFP
of a 10x NA 0.45 will be about 2x larger than that of a 60x NA 1.4.
(The focal length is about 6x longer but the NA is ~3x smaller.)
However, as the BFP will occur at a plane that is optically about 40
mm from the specimen (rather than 6 mm for the 63x), it may not give a
totally accurate estimate of the size of the ray bundle for the higher
mag lens.
And then there is the matter of whether or not high-NA objectives
are actually diffraction limited. In Chapter 11 of the Handbook,
Rimas Juskaitis actually measures this and reports that he has yet to
test an objective in which diffraction-limited performance extended
beyond NA 1.35. In addition, although imaging performance was
often close to diffraction-limited on the axis, it became much worse
towards the edges of the field of view, especially on lower mag lenses
of a given NA. Although this work was done about 5 years ago and newer
lenses may be better, I think that it is unwise to assume that an
objective will produce optical performance that is limited only by the
NA engraved on the lens barrel.
I hope that this helps explain some of the inconsistencies that
are bound to crop up in measurements of this type, particularly if one
doesn't know what is happening at the objective BFP.
Good luck,
Jim P.
--
**********************************************
Prof. James B. Pawley, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC,
Vancouver Canada
Info:
http://www.3dcourse.ubc.ca/
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In reply to this post by Stanislav Vitha
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Our Olympus x100's have smaller back apertures and achieve higher effective NA's. If yours are the same, that may explain some of your results. Mike -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stanislav Vitha Sent: Monday, October 29, 2007 11:26 AM To: [hidden email] Subject: Re: Objective for confocal Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Cameron, I will be inetrested in the test report on the two Olympus lenses. Were your tests done in the wide-field or confocal mode? Our Olympus confocal had a 60x/1.42 PLAPON objective that turned out to be substantially worse in resolution than we expected from theoretical calculations. After testing several more of the same type we switched to the 100x/1.4 Universal Plan Apo (Thanks to our local Olympus office I was able to pick the best one among several of those objectives). It showed much better resolution and less chromatic aberration across the visible spectrum. I can e-mail test results to those who are interested (both mirror slide and sub-resolution beads were used). Regarding the signal intensity - I thought that in the point-scanning confocal mode, there should not be a big difference in excitation intensity and signal detected between different magnification objectives of the same NA - the size of the illuminated spot is determined by NA, as well as the amount of fluorescence signal collected. Since all the collected fluorescence signal is focused to a single spot in the pinhole plane, there again should not be much difference between, say, 60x and 100x objective, providing the pinhole size is set to the same Airy size and all other things are the same. Am I missing something fundamental? Stan Dr. Stanislav Vitha [hidden email] Microscopy and Imaging Center Texas A&M University BSBW 119 College Station, TX 77843-2257 tel: 979-845-1129 (main desk) tel: 979-845-1607 (direct link) fax: 979-847-8933 On Sat, 27 Oct 2007 11:08:29 +1000, Nowell, Cameron <[hidden email]> wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi Ann, > To follow up from Julio's comments. The higher NA will give you improved resolution. A 1.42 NA object theoreticatly can resolve down to 210nm at 488nm exitation. That holds true for what ever the magnification the objective has. > >We have compared a 63x 1.42 NA to a 100x 1.42 NA objective. Both were Olympus UIS2 PlanApo objectives. Both could resolve the same structures. But the big difference was the amount of light that was needed to capture an image with the 100x objective. On average 7x more light input was required to get image with the 100x lens that was equivalent to the 63x lens. > >So my advice woudl be to get a 63x 1.42 NA objective, or better yet a 40x 1.4NA if you can find one (they are being made by Nikon i think but not sure about others). > >Of course this is only important if you want to b e able to resolve very small structures. If you do not want to be resolving down to 210nm then your 40x oil and 63x water imersion objective will be just fine. > >I can send you (and anyone else on the list) a copy of the reoprt with the comparison of the 63x vs the 100x lens if you want. > > >Cheers > >Cam > > >Cameron Nowell B.Sc (Hons) >Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre >7 St Andrews Place >East Melbourne, Victoria 3002 > >Phone: +61396561243 >Mobile: +61422882700 >Fax: +61396561411 > >________________________________ > >From: Confocal Microscopy List on behalf of Yang, Ann-Fook >Sent: Sat 27/10/2007 6:09 AM >To: [hidden email] >Subject: Objective for confocal > > > >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi All, > >I am new in confocal microscopy. >We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any > >Ann Fook Yang, >Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada >EM Unit/ Unite EM >Edifice K.W. Neatby Building, >960 Carling Av /960 Boulevard Carling, >Ottawa,Ontario >K1A 0C6 > >[hidden email] >Telephone/Téléphone: 613-759-1638 >Facsimile/Télécopieur: 613-759-1701 > > > > >This email (including any attachments) may contain >confidential and/or legally privileged information and is >intended only to be read or used by the addressee. If you >are not the intended addressee, any use, distribution, >disclosure or copying of this email is strictly >prohibited. >Confidentiality and legal privilege attached to this email >(including any attachments) are not waived or lost by >reason of its mistaken delivery to you. >If you have received this email in error, please delete it >and notify us immediately by telephone or email. Peter >MacCallum Cancer Centre provides no guarantee that this >transmission is free of virus or that it has not been >intercepted or altered and will not be liable for any delay >in its receipt. >======================================================================== |
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In reply to this post by Stanislav Vitha
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi List, This is always a fun topic:) I should quantify the test we carried out here. The tests were carried out on the following lenses on an Olympus FV1000 single photon, non-spectral confocal - 63x NA 1.42 UPlanApo UIS2 - 100x NA 1.4 UPlanApo UIS2 The reason for the test were that we had a lab pushing to get a 100x objective because of the increased magnification. The tests were carried out to demonstrate that the 100x lens would be more detrimental to their samples (ie bleaching) and would not provide any greater resolution. The tests were carried out on drosophila embryos that were mounted in glycerol The end result was that laser power had to be boosted, leading to faster bleaching, to get an equivalent image with the 100x when compared to the 60x. This is all based on our confocal and result may be different for other machines and lens combinations. Cheers Cam -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stanislav Vitha Sent: Tuesday, 30 October 2007 2:26 AM To: [hidden email] Subject: Re: Objective for confocal Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Cameron, I will be inetrested in the test report on the two Olympus lenses. Were your tests done in the wide-field or confocal mode? Our Olympus confocal had a 60x/1.42 PLAPON objective that turned out to be substantially worse in resolution than we expected from theoretical calculations. After testing several more of the same type we switched to the 100x/1.4 Universal Plan Apo (Thanks to our local Olympus office I was able to pick the best one among several of those objectives). It showed much better resolution and less chromatic aberration across the visible spectrum. I can e-mail test results to those who are interested (both mirror slide and sub-resolution beads were used). Regarding the signal intensity - I thought that in the point-scanning confocal mode, there should not be a big difference in excitation intensity and signal detected between different magnification objectives of the same NA - the size of the illuminated spot is determined by NA, as well as the amount of fluorescence signal collected. Since all the collected fluorescence signal is focused to a single spot in the pinhole plane, there again should not be much difference between, say, 60x and 100x objective, providing the pinhole size is set to the same Airy size and all other things are the same. Am I missing something fundamental? Stan Dr. Stanislav Vitha [hidden email] Microscopy and Imaging Center Texas A&M University BSBW 119 College Station, TX 77843-2257 tel: 979-845-1129 (main desk) tel: 979-845-1607 (direct link) fax: 979-847-8933 On Sat, 27 Oct 2007 11:08:29 +1000, Nowell, Cameron <[hidden email]> wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi Ann, > To follow up from Julio's comments. The higher NA will give you improved resolution. A 1.42 NA object theoreticatly can resolve down to 210nm at 488nm exitation. That holds true for what ever the magnification the objective has. > >We have compared a 63x 1.42 NA to a 100x 1.42 NA objective. Both were Olympus UIS2 PlanApo objectives. Both could resolve the same structures. But the big difference was the amount of light that was needed to capture an image with the 100x objective. On average 7x more light input was required to get image with the 100x lens that was equivalent to the 63x lens. > >So my advice woudl be to get a 63x 1.42 NA objective, or better yet a >40x 1.4NA if you can find one (they are being made by Nikon i think but not sure about others). > >Of course this is only important if you want to b e able to resolve >very small structures. If you do not want to be resolving down to 210nm then your 40x oil and 63x water imersion objective will be just fine. > >I can send you (and anyone else on the list) a copy of the reoprt with the comparison of the 63x vs the 100x lens if you want. > > >Cheers > >Cam > > >Cameron Nowell B.Sc (Hons) >Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre >7 St Andrews Place >East Melbourne, Victoria 3002 > >Phone: +61396561243 >Mobile: +61422882700 >Fax: +61396561411 > >________________________________ > >From: Confocal Microscopy List on behalf of Yang, Ann-Fook >Sent: Sat 27/10/2007 6:09 AM >To: [hidden email] >Subject: Objective for confocal > > > >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi All, > >I am new in confocal microscopy. >We have a 40x/1.3 oil and a 63X/1.2 water objectives. Is there any > >Ann Fook Yang, >Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada >EM Unit/ Unite EM Edifice K.W. Neatby Building, 960 Carling Av /960 >Boulevard Carling, Ottawa,Ontario K1A 0C6 > >[hidden email] >Telephone/Téléphone: 613-759-1638 >Facsimile/Télécopieur: 613-759-1701 > > > > >This email (including any attachments) may contain confidential and/or >legally privileged information and is intended only to be read or used >by the addressee. If you are not the intended addressee, any use, >distribution, disclosure or copying of this email is strictly >prohibited. >Confidentiality and legal privilege attached to this email (including >any attachments) are not waived or lost by reason of its mistaken >delivery to you. >If you have received this email in error, please delete it and notify >us immediately by telephone or email. Peter MacCallum Cancer Centre >provides no guarantee that this transmission is free of virus or that >it has not been intercepted or altered and will not be liable for any >delay in its receipt. >======================================================================= >= This email (including any attachments) may contain confidential and/or legally privileged information and is intended only to be read or used by the addressee. If you are not the intended addressee, any use, distribution, disclosure or copying of this email is strictly prohibited. Confidentiality and legal privilege attached to this email (including any attachments) are not waived or lost by reason of its mistaken delivery to you. If you have received this email in error, please delete it and notify us immediately by telephone or email. Peter MacCallum Cancer Centre provides no guarantee that this transmission is free of virus or that it has not been intercepted or altered and will not be liable for any delay in its receipt. |
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