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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
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