Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
***** COMMERCIAL RESPONSE ******
Although a software fix may not be the ideal solution for Chromatic
aberration, in the current version of the Advanced Fluorescence Acquisition
plug-in module for Image-Pro Plus, we have implemented a per channel focus
offset, that allows you to automatically shift the focus level as you switch
from one channel to the next in software.
If anyone is interested in evaluating this feature or getting more
information on our products, please feel free to contact me directly.
Chris Tully
Applications Engineer
Media Cybernetics, Inc. "From Images to Answers"
8484 Georgia Avenue, Suite 200
Silver Spring, MD 20910
Tel: +1 (301) 495-3305 ext. 202 Fax: +1 (301) 495-5964
E-mail: [log in to unmask]
Websites: http://www.MediaCy.com
http://www.Solutions-Zone.com
http://Shop.MediaCy.com
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Kate Luby-Phelps
Sent: Friday, June 03, 2005 12:54 PM
To: [log in to unmask]
Subject: Re: [CONFOCAL] CONFOCAL Digest - 1 Jun 2005 to 2 Jun 2005
(#2005-127)
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Funny you should post this today as I was just discussing the same
issue. A user has been noticing a big focal difference between FITC
and Cy5 using the 10x lens with the Perkin Elmer Ultraview ERS on a Zeiss
Axiovert 200M. The Cy5 channel is completely out of focus when FITC is in
focus. There is no noticeable focus difference between the two channels with
the 100x lens. I assume the chromatic aberration arises from the microlenses
in the Yokogawa head, but it may also be compounded by the fact that the
pinhole diameter is fixed and is not correct for 10x resulting in a big
difference in total light throughput between the two channels. I suppose
that the magnitude of the aberration will depend on how much correction is
built into the objective you are using.
Kate
On Jun 2, 2005, at 11:03 PM, Automatic digest processor wrote:
> There are 5 messages totalling 837 lines in this issue.
>
> Topics of the day:
>
> 1. How much chromatic aberration is expected... (5)
>
> ----------------------------------------------------------------------
>
> Date: Thu, 2 Jun 2005 12:15:45 -0400
> From: Stephen Bunnell <[log in to unmask]>
> Subject: Re: How much chromatic aberration is expected...
>
>> This message is in MIME format. Since your mail reader does not
>> understand
> this format, some or all of this message may not be legible.
>
> --B_3200559345_221672
> Content-type: text/plain;
> charset="ISO-8859-1"
> Content-transfer-encoding: quoted-printable
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
>
>> Hi everyone-
>> =20
>> As a practical matter, I understand that axial chromatic
>> aberration i=
> s
>> pretty much built in to imaging systems. Nevertheless, it was my
>> belief =
> that
>> the current generations on lenses were supposed to be quite good at
>> corre=
> cting
>> for it. However, I can't find much information on exactly how good
>> they =
> are
>> in real world applications.
>> =20
>> I am routinely imaging live, rapidly moving, samples in aqueous
>> media using a Perkin-Elmer Ultraview/Yokogawa spinning-disc confocal
>> system.
>> Typically, I image small (width & depth of ~100-200nm) structures
>> constra=
> ined
>> to the cell-coverslip interface. I am having significant problems
>> with a=
> xial
>> chromatic aberration. The problem seems to be lens-specific. The
>> axial =
> CA I
>> see between CFP & YFP is often larger than the features in question
>> -up t=
> o
>> 500nm! I also see axial CA between Alexa 488 and 647 in some of the
>> lense=
> s- of
>> up to 800nm. The total axial CA between CFP and Alexa 647 with the
>> worst =
> of my
>> lenses is ~1.3 =B5m, and with the best it is 200nm. Of four lenses,
>> one sh=
> ows a
>> positive linear correlation between displacement and wavelength,
>> another =
> a
>> negative linear correlation between displacement and wavelength, a
>> third =
> shows
>> a curve, and one, blissfully, is flat. I am seriously concerned
>> about ho=
> w
>> these axial aberrations would affect FRET calculations, etc.
>> However, on=
> a
>> more mundane level, axial CA this bad simply makes it difficult to
>> rapidl=
> y
>> obtain pictures of multiple colors at consistent brightness ratios,
>> and a=
> t a
>> consistent plane within the cell, as very small movements in Z
>> dramatical=
> ly
>> influence the quality of my images.
>> =20
>> I would like to know what kind of axial aberration people see in
>> prac=
> tice.
>> What can you really expect to achieve in the real world? Before I go
>> demanding new lenses from the supplier, I=B9d like to know the best I
>> can e=
> xpect
>> to achieve. Ideally, I=B9d like to know how the different makers=B9
>> (Olympus, Zeiss, Nikon, etc.) lenses compare in this regard.
>> =20
>> Best regards,
>> =20
>> Steve Bunnell
>> =20
>> *********************************************************************
>> *
>> ***=
> ***
>> Stephen C. Bunnell, Ph.D.
>> Assistant Professor
>> Tufts University Medical School
>> Department of Pathology
>> Jaharis Bldg., Room 512
>> 150 Harrison Ave.
>> Boston, MA 02111
>> =20
>> Phone: (617) 636-2174
>> Fax: (617) 636-2990
>> Email: [log in to unmask]
>
>
>
> --B_3200559345_221672
> Content-type: text/html;
> charset="ISO-8859-1"
> Content-transfer-encoding: quoted-printable
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
> <HTML>
> <HEAD>
> <TITLE>Re: How much chromatic aberration is expected...</TITLE>
> </HEAD> <BODY> <FONT FACE=3D"Lucida Grande, Verdana, Helvetica,
> Arial"><SPAN STYLE=3D'font-siz= e:12.0px'><BR>
> </SPAN></FONT><BLOCKQUOTE><FONT FACE=3D"Lucida Grande, Verdana,
> Helvetica, Ar= ial"><SPAN STYLE=3D'font-size:12.0px'>Hi everyone-<BR>
> <BR> As a practical matter, I understand that
> axial chro= matic aberration is pretty much built in to imaging
> systems.
> Neverthel=
> ess, it was my belief that the current generations on lenses were
> supposed t= o be quite good at correcting for it. However, I
> can't find much infor= mation on exactly how good they are in real
> world applications.<BR> <BR>
> I am routinely imaging live, rapidly moving,
> sample= s in aqueous media using a Perkin-Elmer Ultraview/Yokogawa
> spinning-disc con= focal system. Typically, I image small (width &
> depth of
> ~100-200nm) str=
> uctures constrained to the cell-coverslip interface. I am having
> signi= ficant problems with axial chromatic aberration. The
> problem seems to = be <B>lens-specific</B>. The axial CA I see
> between CFP & YFP is o= ften larger than the features in question
> -up to 500nm! I also see axial CA = between Alexa 488 and 647 in some
> of the lenses- of up to 800nm. The total a= xial CA between CFP and
> Alexa 647 with the worst of my lenses is ~1.3 µ= ;m, and with the
> best it is 200nm. Of four lenses, one shows a positiv= e linear
> correlation between displacement and wavelength, another a negative=
> linear correlation between displacement and wavelength, a third shows
> a cur= ve, and one, blissfully, is flat. I am seriously
> concerned about how t= hese axial aberrations would affect FRET
> calculations, etc.
> However, o=
> n a more mundane level, axial CA this bad simply makes it difficult to
> rapid= ly obtain pictures of multiple colors at consistent brightness
> ratios, and a= t a consistent plane within the cell, as very small
> movements in Z dramatica= lly influence the quality of my images.<BR>
> <BR> I would like to know what kind of axial
> aberration = people see in practice. What can you really expect to
> achieve in the real wo= rld? Before I go demanding new lenses
> from the supplier, I’d lik= e to know the best I can expect to
> achieve. Ideally, I’d like to know = how the different
> makers’ (Olympus, Zeiss, Nikon, etc.) lenses compare= in this
> regard.<BR> <BR> Best regards,<BR> <BR>
> Steve Bunnell<BR> <BR>
> **********************************************************************
> *
> ****=
> *<BR>
> Stephen C. Bunnell, Ph.D.<BR>
> Assistant Professor<BR>
> Tufts University Medical School<BR>
> Department of Pathology<BR>
> Jaharis Bldg., Room 512<BR>
> 150 Harrison Ave.<BR>
> Boston, MA 02111<BR>
> <BR>
> Phone: (617) 636-2174<BR>
> Fax: (617) 636-2990<BR>
> Email: [log in to unmask]<BR>
> </SPAN></FONT></BLOCKQUOTE><FONT FACE=3D"Lucida Grande, Verdana,
> Helvetica, A= rial"><SPAN STYLE=3D'font-size:12.0px'><BR>
> </SPAN></FONT> </BODY> </HTML>
>
>
> --B_3200559345_221672--
>
> ------------------------------
>
> Date: Thu, 2 Jun 2005 13:27:19 -0400
> From: Andrew Resnick <[log in to unmask]>
> Subject: Re: How much chromatic aberration is expected...
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> Steve,
>
> Longitudinal chromatic aberration (LCA- the type you mention below,
> the=20 other is transverse chromatic aberration) in high-end objective
> lenses=20
> (apochromats) is much smaller than what you describe. The
> manufacturer=20 should be able to provide you with specific
> information about how much=20 chromatic aberration your lenses have,
> given the serial number- it's part=20 of the quality assurance
> testing. I would definitely contact the sales rep= =20
> or factory rep and try and get the test results of your objectives.
> As a=20
> quick check, try doing some normal brightfield on a resolution
> target,=20 using various color filters in the illumination path to
> check the LCA of=20 the microscope- see if the plane of focus changes.
> Or even widefield=20 fluorescence, if you can.
>
> The Nipkow system, on the other hand, may not be so well-corrected.
> It=20
> could be a number of things, from the way light is coupled to the
> scan=20 head, to an incorrect specification of the coupling between
> the scan head=20 and microscope. I would recommend starting by making
> sure everything is=20 connected properly, and that the Yokogawa field
> lens and spacer are=20 correctly specified to work with your
> microscope.
>
> Hope this helps,
>
> Andy
>
>
> At 12:15 PM 6/2/2005, you wrote:
>> Search the CONFOCAL archive at=20
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>> Hi everyone-
>>
>> As a practical matter, I understand that axial chromatic
>> aberration=20 is pretty much built in to imaging systems.
>> Nevertheless, it was my=20 belief that the current generations on
>> lenses were supposed to be quite=20 good at correcting for it.
>> However, I can't find much information on=20 exactly how good they
>> are in real world applications.
>>
>> I am routinely imaging live, rapidly moving, samples in aqueous
>> media=
> =20
>> using a Perkin-Elmer Ultraview/Yokogawa spinning-disc confocal
>> system.=20 Typically, I image small (width & depth of ~100-200nm)
>> structures=20 constrained to the cell-coverslip interface. I am
>> having significant=20 problems with axial chromatic aberration. The
>> problem seems to be=20 lens-specific. The axial CA I see between CFP
>> & YFP is often larger than=
> =20
>> the features in question -up to 500nm! I also see axial CA between
>> Alexa=
> =20
>> 488 and 647 in some of the lenses- of up to 800nm. The total axial
>> CA=20 between CFP and Alexa 647 with the worst of my lenses is ~1.3
>> =B5m, and=20 with the best it is 200nm. Of four lenses, one shows a
>> positive linear=20 correlation between displacement and wavelength,
>> another a negative=20 linear correlation between displacement and
>> wavelength, a third shows a=20 curve, and one, blissfully, is flat.
>> I am seriously concerned about how=
> =20
>> these axial aberrations would affect FRET calculations, etc.
>> However, on=
> =20
>> a more mundane level, axial CA this bad simply makes it difficult
>> to=20 rapidly obtain pictures of multiple colors at consistent
>> brightness=20 ratios, and at a consistent plane within the cell, as
>> very small=20 movements in Z dramatically influence the quality of my
>> images.
>>
>> I would like to know what kind of axial aberration people see
>> in=20 practice. What can you really expect to achieve in the real=20
>> world? Before I go demanding new lenses from the supplier, I=92d
>> like to=
> =20
>> know the best I can expect to achieve. Ideally, I=92d like to know
>> how the=
> =20
>> different makers=92 (Olympus, Zeiss, Nikon, etc.) lenses compare in
>> this=
> regard.
>>
>> Best regards,
>>
>> Steve Bunnell
>>
>
> Andrew Resnick, Ph. D.
> Instructor
> Department of Physiology and Biophysics Case Western Reserve
> University
> 216-368-6899 (V)
> 216-368-4223 (F)=20
>
> ------------------------------
>
> Date: Fri, 3 Jun 2005 03:33:08 +1000
> From: Guy Cox <[log in to unmask]>
> Subject: Re: How much chromatic aberration is expected...
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> This is all a very interesting question. First of all, you do really
> mean axial CA not lateral CA? I would have thought that the axial
> resolution of any Nipkow-disk system would not reach 500nm so that it
> would be hard to measure such an amount of CA.
>
> With an achromat you are normally bringing blue & red to the same
> focus so green emission will be quite noticeably out. A fluorite lens
> narrows the gap a lot but still has the same basic correction. An
> apochromat should be pretty good so long as you stay within its
> correction range - violet and far-red will definitely screw things up.
> In a confocal system it all gets more complex since the question of
> your excitation focus and your emission focus being identical comes
> into the equation.
>
> As for lateral CA, there you can be in big trouble with a spinning
> disk. The trouble is that most manufacturers use the tube lens and/or
> the eyepiece to correct it - but your disk comes before either of
> these. So you will see strange colour effects at the edge of your
> image since the imaged spot will miss the pinhole. Nikon reportedly
> does all its correction in the objective so if this is true a Nikon
> microscope should be the best one to use for any spinning disk
> application. Any comment from Nikon or other manufacturers here?
>
> Guy
>
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>
>>> Hi everyone-
>>>
>>> As a practical matter, I understand that axial chromatic
>>> aberration is pretty much built in to imaging systems.
>>> Nevertheless, it was my belief that the current generations on
>>> lenses were supposed to be quite good at correcting for it.
>>> However, I can't find much information on exactly how good they are
>>> in real world applications.
>>>
>>> I am routinely imaging live, rapidly moving, samples in aqueous
>>> media using a Perkin-Elmer Ultraview/Yokogawa spinning-disc confocal
>>> system.
>>> Typically, I image small (width & depth of ~100-200nm) structures
>>> constrained to the cell-coverslip interface. I am having
>>> significant problems with axial chromatic aberration. The problem
>>> seems to be lens-specific. The axial CA I see between CFP & YFP is
>>> often larger than the features in question -up to 500nm! I also see
>>> axial CA between Alexa 488 and 647 in some of the
>>> lenses- of
>>> up to 800nm. The total axial CA between CFP and Alexa 647 with the
>>> worst of my lenses is ~1.3 µm, and with the best it is 200nm. Of
>>> four lenses, one shows a positive linear correlation between
>>> displacement and wavelength, another a negative linear correlation
>>> between displacement and wavelength, a third shows a curve, and one,
>>> blissfully, is flat. I am seriously concerned about how
>>> these axial aberrations would affect FRET calculations, etc.
>>> However,
>>> on a
>>> more mundane level, axial CA this bad simply makes it difficult to
>>> rapidly obtain pictures of multiple colors at consistent brightness
>>> ratios, and at a consistent plane within the cell, as very small
>>> movements in Z dramatically influence the quality of my images.
>>>
>>> I would like to know what kind of axial aberration people see in
>>> practice.
>>> What can you really expect to achieve in the real world? Before I
>>> go demanding new lenses from the supplier, Iąd like to know the best
>>> I can expect to achieve. Ideally, Iąd like to know how the different
>>> makersą (Olympus, Zeiss, Nikon, etc.) lenses compare in this regard.
>>>
>>> Best regards,
>>>
>>> Steve Bunnell
>>>
>>> ********************************************************************
>>> *
>>> *******
>>> Stephen C. Bunnell, Ph.D.
>>> Assistant Professor
>>> Tufts University Medical School
>>> Department of Pathology
>>> Jaharis Bldg., Room 512
>>> 150 Harrison Ave.
>>> Boston, MA 02111
>>>
>>> Phone: (617) 636-2174
>>> Fax: (617) 636-2990
>>> Email: [log in to unmask]
>>
>>
>>
>
>
> --
> Associate Professor Guy Cox
> Electron Microscope Unit,
> University of Sydney,
> NSW 2006, Australia
>
> Phone:+61 2 9351 3176 Fax:+61 2 9351 7682
> http://www.guycox.net
>
> ------------------------------
>
> Date: Thu, 2 Jun 2005 11:03:46 -0700
> From: Mike Adams <[log in to unmask]>
> Subject: Re: How much chromatic aberration is expected...
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> ***COMMERCIAL RESPONSE***
>
> Hi all! Regarding Nikon CFI60 lenses, all corrections are made within
> = the objective. As pointed out by Guy, some manufacturers do make
> some = corrections in the tube lens. Also, the Nikon line of VC
> (violet =
> corrected) Plan Apo optics should excel in such applications.
>
> Cheers!
> Mike
>
> Michael C Adams
> Microscopy & Imaging Specialist - San Diego A.G. Heinze, Inc 6370 Lusk
> Blvd, Ste F107 San Diego, CA 92121
> General: 800.325.2246
> Fax: 858.452.7730
> Mobile: 858.361.9110
> Email: [log in to unmask]
>
> www.agheinze.com=20
> www.nikonusa.com
> www.photomet.com
> www.universal-imaging.com
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On =
> Behalf Of Guy Cox
> Sent: Thursday, June 02, 2005 10:33 AM
> To: [log in to unmask]
> Subject: Re: How much chromatic aberration is expected...
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> This is all a very interesting question. First of all, you do really
> mean axial CA not lateral CA? I would have thought that the axial
> resolution of any Nipkow-disk system would not reach 500nm so that it
> would be hard to measure such an amount of CA.
>
> With an achromat you are normally bringing blue & red to the same
> focus so green emission will be quite noticeably out. A fluorite lens
> narrows the gap a lot but still has the same basic correction. An
> apochromat should be pretty good so long as you stay within its
> correction range - violet and far-red will definitely screw things up.
> In a confocal system it all gets more complex since the question of
> your excitation focus and your emission focus being identical comes
> into the equation.
>
> As for lateral CA, there you can be in big trouble with a spinning
> disk. The trouble is that most manufacturers use the tube lens and/or
> the eyepiece to correct it - but your disk comes before either of
> these. So you will see strange colour effects at the edge of your
> image since the imaged spot will miss the pinhole. Nikon reportedly
> does all its correction in the objective so if this is true a Nikon
> microscope should be the best one to use for any spinning disk
> application. Any comment from Nikon or other manufacturers here?
>
> Guy
>
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>>
>>
>>> Hi everyone-
>>>
>>> As a practical matter, I understand that axial chromatic =
> aberration
>>> is
>>> pretty much built in to imaging systems. Nevertheless, it was my =
> belief
>>> that
>>> the current generations on lenses were supposed to be quite good at
>>> correcting for it. However, I can't find much information on
>>> exactly how good =
> they
>>> are
>>> in real world applications.
>>>
>>> I am routinely imaging live, rapidly moving, samples in aqueous
>>> media using a Perkin-Elmer Ultraview/Yokogawa spinning-disc confocal
>>> =
> system.
>>> Typically, I image small (width & depth of ~100-200nm) structures
>>> constrained to the cell-coverslip interface. I am having
>>> significant problems =
> with
>>> axial
>>> chromatic aberration. The problem seems to be lens-specific. The =
> axial
>>> CA I
>>> see between CFP & YFP is often larger than the features in question
>>> =
> -up
>>> to
>>> 500nm! I also see axial CA between Alexa 488 and 647 in some of the
>>> lenses- of
>>> up to 800nm. The total axial CA between CFP and Alexa 647 with the =
> worst
>>> of my
>>> lenses is ~1.3 =B5m, and with the best it is 200nm. Of four lenses,
>>> =
> one
>>> shows a
>>> positive linear correlation between displacement and wavelength, =
> another
>>> a
>>> negative linear correlation between displacement and wavelength, a =
> third
>>> shows
>>> a curve, and one, blissfully, is flat. I am seriously concerned =
> about
>>> how
>>> these axial aberrations would affect FRET calculations, etc. =
> However,
>>> on a
>>> more mundane level, axial CA this bad simply makes it difficult to
>>> rapidly obtain pictures of multiple colors at consistent brightness
>>> ratios, =
> and
>>> at a
>>> consistent plane within the cell, as very small movements in Z
>>> dramatically influence the quality of my images.
>>>
>>> I would like to know what kind of axial aberration people see in
>>> practice.
>>> What can you really expect to achieve in the real world? Before I
>>> go demanding new lenses from the supplier, I=B9d like to know the
>>> best I =
> can
>>> expect
>>> to achieve. Ideally, I=B9d like to know how the different makers=B9
>>> (Olympus, Zeiss, Nikon, etc.) lenses compare in this regard.
>>>
>>> Best regards,
>>>
>>> Steve Bunnell
>>>
>>> =
> **********************************************************************
> *
> **=
> ***
>>> Stephen C. Bunnell, Ph.D.
>>> Assistant Professor
>>> Tufts University Medical School
>>> Department of Pathology
>>> Jaharis Bldg., Room 512
>>> 150 Harrison Ave.
>>> Boston, MA 02111
>>>
>>> Phone: (617) 636-2174
>>> Fax: (617) 636-2990
>>> Email: [log in to unmask]
>>
>>
>>
>
>
> --
> Associate Professor Guy Cox
> Electron Microscope Unit,
> University of Sydney,
> NSW 2006, Australia
>
> Phone:+61 2 9351 3176 Fax:+61 2 9351 7682
> http://www.guycox.net
>
> ------------------------------
>
> Date: Fri, 3 Jun 2005 09:13:48 +0800
> From: Paul Rigby <[log in to unmask]>
> Subject: Re: How much chromatic aberration is expected...
>
> This is a multi-part message in MIME format.
>
> ------_=_NextPart_001_01C567D9.7EB4882E
> Content-Type: text/plain;
> charset="iso-8859-1"
> Content-Transfer-Encoding: quoted-printable
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> Hi Stephen,
> You don't mention exactly which objectives you are using. Correct me
> if = I am wrong, but isn't the PE Ultraview/Yokogawa designed to work
> = optimally with a 100x oil immersion objective (NA 1.3 or 1.4)? I
> note = that you are using live cells in aqueous media, so I would
> expect that = you would get significant aberration (chromatic and
> spherical) if you = were using an oil immersion objective in this
> situation. Perhaps one = mitigating fact is that you are working very
> close to the coverslip = interface, so these effects might not be as
> pronounced. Alternatively, = if you are using a high NA water
> immersion objective, make sure the = coverslip correction collar is
> correctly set. I would expect significant = aberration problems if
> this were not set optimally.
> Hope this helps.
> =20
> Cheers
> Paul
> ---------------
>
> Dr Paul J Rigby
> Director and Senior Lecturer
> Biomedical Imaging & Analysis Facility (M510) The University of
> Western Australia
> 35 Stirling Highway, Crawley, WA, 6009 Australia Ph (61-8) 9346 2819
> Fx (61-8) 9346 3469
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]]On =
> Behalf Of Stephen Bunnell
> Sent: Friday, 3 June 2005 12:16 AM
> To: [log in to unmask]
> Subject: Re: How much chromatic aberration is expected...
>
>
> Search the CONFOCAL archive at =
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal=20
>
>
> Hi everyone-
>
> As a practical matter, I understand that axial chromatic
> aberration =
> is pretty much built in to imaging systems. Nevertheless, it was my =
> belief that the current generations on lenses were supposed to be
> quite =
> good at correcting for it. However, I can't find much information on =
> exactly how good they are in real world applications.
> =20
> I am routinely imaging live, rapidly moving, samples in aqueous =
> media using a Perkin-Elmer Ultraview/Yokogawa spinning-disc confocal =
> system. Typically, I image small (width & depth of ~100-200nm) =
> structures constrained to the cell-coverslip interface. I am having =
> significant problems with axial chromatic aberration. The problem
> seems =
> to be lens-specific. The axial CA I see between CFP & YFP is often =
> larger than the features in question -up to 500nm! I also see axial CA
> =
> between Alexa 488 and 647 in some of the lenses- of up to 800nm. The =
> total axial CA between CFP and Alexa 647 with the worst of my lenses
> is =
> ~1.3 =B5m, and with the best it is 200nm. Of four lenses, one shows a
> =
> positive linear correlation between displacement and wavelength,
> another =
> a negative linear correlation between displacement and wavelength, a =
> third shows a curve, and one, blissfully, is flat. I am seriously =
> concerned about how these axial aberrations would affect FRET =
> calculations, etc. However, on a more mundane level, axial CA this
> bad =
> simply makes it difficult to rapidly obtain pictures of multiple
> colors =
> at consistent brightness ratios, and at a consistent plane within the =
> cell, as very small movements in Z dramatically influence the quality
> of =
> my images.
>
> I would like to know what kind of axial aberration people see in =
> practice. What can you really expect to achieve in the real world? =
> Before I go demanding new lenses from the supplier, I'd like to know
> the =
> best I can expect to achieve. Ideally, I'd like to know how the =
> different makers' (Olympus, Zeiss, Nikon, etc.) lenses compare in this
> =
> regard.
>
> Best regards,
>
> Steve Bunnell
>
> ***********************************************************************
> **=
> ***
> Stephen C. Bunnell, Ph.D.
> Assistant Professor
> Tufts University Medical School
> Department of Pathology
> Jaharis Bldg., Room 512
> 150 Harrison Ave.
> Boston, MA 02111
>
> Phone: (617) 636-2174
> Fax: (617) 636-2990
> Email: [log in to unmask]
>
>
>
>
>
>
> ------_=_NextPart_001_01C567D9.7EB4882E
> Content-Type: text/html;
> charset="iso-8859-1"
> Content-Transfer-Encoding: quoted-printable
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0 Transitional//EN">
> <HTML><HEAD>
> <META HTTP-EQUIV=3D"Content-Type" CONTENT=3D"text/html; =
> charset=3Diso-8859-1">
> <TITLE>Re: How much chromatic aberration is expected...</TITLE>
>
> <META content=3D"MSHTML 6.00.2800.1498" name=3DGENERATOR></HEAD>
> <BODY>
> <DIV><SPAN class=3D788405500-03062005><FONT face=3DArial
> color=3D#0000ff =
> size=3D2>Hi=20
> Stephen,</FONT></SPAN></DIV>
> <DIV><SPAN class=3D788405500-03062005><FONT face=3DArial
> color=3D#0000ff =
> size=3D2>You=20
> don't mention exactly which objectives you are using. Correct me if I
> am =
> wrong,=20
> but isn't the PE Ultraview/Yokogawa designed to work optimally with a =
> 100x oil=20
> immersion objective (NA 1.3 or 1.4)? I note that you are using live =
> cells in=20
> aqueous media, so I would expect that you would get significant =
> aberration=20
> (chromatic and spherical) if you were using an oil immersion objective
> =
> in this=20
> situation. Perhaps one mitigating fact is that you are working very =
> close to the=20
> coverslip interface, so these effects might not be as
> pronounced.=20
> Alternatively, if you are using a high NA water immersion objective, =
> make sure=20
> the coverslip correction collar is correctly set. I would expect =
> significant=20
> aberration problems if this were not set optimally.</FONT></SPAN></DIV>
> <DIV><SPAN class=3D788405500-03062005><FONT face=3DArial
> color=3D#0000ff =
> size=3D2>Hope=20
> this helps.</FONT></SPAN></DIV>
> <DIV><SPAN class=3D788405500-03062005><FONT face=3DArial
> color=3D#0000ff =
>
> size=3D2></FONT></SPAN> </DIV>
> <DIV><SPAN class=3D788405500-03062005><FONT face=3DArial
> color=3D#0000ff =
>
> size=3D2>Cheers</FONT></SPAN></DIV>
> <DIV><SPAN class=3D788405500-03062005><FONT face=3DArial
> color=3D#0000ff =
>
> size=3D2>Paul</FONT></SPAN></DIV>
> <DIV><SPAN class=3D788405500-03062005></SPAN><FONT=20
> size=3D2>---------------<BR><BR>Dr Paul J Rigby<BR>Director and
> Senior=20
> Lecturer<BR>Biomedical Imaging & Analysis Facility (M510)<BR>The =
> University=20
> of Western Australia<BR>35 Stirling Highway, Crawley, WA,=20
> 6009<BR>Australia<BR>Ph (61-8) 9346 2819<BR>Fx (61-8) 9346 =
> 3469<BR></DIV></FONT>
> <BLOCKQUOTE>
> <DIV class=3DOutlookMessageHeader dir=3Dltr align=3Dleft><FONT =
> face=3DTahoma=20
> size=3D2>-----Original Message-----<BR><B>From:</B> Confocal =
> Microscopy List=20
> [mailto:[log in to unmask]]<B>On Behalf Of </B>Stephen=20
> Bunnell<BR><B>Sent:</B> Friday, 3 June 2005 12:16 AM<BR><B>To:</B>=20
> [log in to unmask]<BR><B>Subject:</B> Re: How much =
> chromatic=20
> aberration is expected...<BR><BR></FONT></DIV>Search the CONFOCAL =
> archive at=20
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal <FONT=20
> face=3D"Lucida Grande, Verdana, Helvetica, Arial"><SPAN=20
> style=3D"FONT-SIZE: 12px"><BR></SPAN></FONT>
> <BLOCKQUOTE><FONT face=3D"Lucida Grande, Verdana, Helvetica, =
> Arial"><SPAN=20
> style=3D"FONT-SIZE: 12px">Hi =
> everyone-<BR><BR> As a=20
> practical matter, I understand that axial chromatic aberration is =
> pretty=20
> much built in to imaging systems. Nevertheless, it was my =
> belief that=20
> the current generations on lenses were supposed to be quite good
> at=20
> correcting for it. However, I can't find much information on
> =
> exactly=20
> how good they are in real world=20
> =
> applications.<BR> <BR> I
> =
> am=20
> routinely imaging live, rapidly moving, samples in aqueous media =
> using a=20
> Perkin-Elmer Ultraview/Yokogawa spinning-disc confocal system. =
> Typically, I=20
> image small (width & depth of ~100-200nm) structures
> constrained =
> to the=20
> cell-coverslip interface. I am having significant problems =
> with axial=20
> chromatic aberration. The problem seems to be =
> <B>lens-specific</B>.=20
> The axial CA I see between CFP & YFP is often larger
> than =
> the=20
> features in question -up to 500nm! I also see axial CA between
> Alexa =
> 488 and=20
> 647 in some of the lenses- of up to 800nm. The total axial CA =
> between CFP=20
> and Alexa 647 with the worst of my lenses is ~1.3 =B5m, and with
> the =
> best it=20
> is 200nm. Of four lenses, one shows a positive linear =
> correlation=20
> between displacement and wavelength, another a negative linear =
> correlation=20
> between displacement and wavelength, a third shows a curve, and
> one, =
>
> blissfully, is flat. I am seriously concerned about how
> these =
> axial=20
> aberrations would affect FRET calculations, etc. However, on
> a =
> more=20
> mundane level, axial CA this bad simply makes it difficult to =
> rapidly obtain=20
> pictures of multiple colors at consistent brightness ratios, and
> at =
> a=20
> consistent plane within the cell, as very small movements in Z =
> dramatically=20
> influence the quality of my
> images.<BR><BR> I =
> would=20
> like to know what kind of axial aberration people see in practice.
> =
> What can=20
> you really expect to achieve in the real world? Before I go =
> demanding=20
> new lenses from the supplier, I’d like to know the best I
> can =
> expect to=20
> achieve. Ideally, I’d like to know how the different =
> makers’ (Olympus,=20
> Zeiss, Nikon, etc.) lenses compare in this=20
> regard.<BR><BR> Best=20
> regards,<BR><BR> Steve=20
> =
> Bunnell<BR><BR>********************************************************
> **=
> ******************<BR>Stephen=20
> C. Bunnell, Ph.D.<BR>Assistant Professor<BR>Tufts University
> Medical =
>
> School<BR>Department of Pathology<BR>Jaharis Bldg., Room
> 512<BR>150 =
> Harrison=20
> Ave.<BR>Boston, MA 02111<BR><BR>Phone: (617) 636-2174<BR>Fax:=20
> (617) 636-2990<BR>Email:=20
> [log in to unmask]<BR></SPAN></FONT></BLOCKQUOTE><FONT=20
> face=3D"Lucida Grande, Verdana, Helvetica, Arial"><SPAN=20
> style=3D"FONT-SIZE: =
> 12px"><BR></BLOCKQUOTE></SPAN></FONT></BODY></HTML>
>
> ------_=_NextPart_001_01C567D9.7EB4882E--
>
> ------------------------------
>
> End of CONFOCAL Digest - 1 Jun 2005 to 2 Jun 2005 (#2005-127)
> *************************************************************
>
>
Kate Luby-Phelps, Ph.D.
Director, Live Cell Imaging Core Facility
Associate Professor, Dept. of Cell Biology
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Dallas, TX 75390-9039
T: 214-648-0429
F: 214-648-8694
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