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What people seem to be missing in this thread is that glass and water
are both dispersive media so the critical angle will be different for
different wavelengths. You cannot expect to get optimal TIRF with two
colours coming in at the same angle.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
<http://www.guycox.com/optical.htm>
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
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From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Andreas Bruckbauer
Sent: Tuesday, 18 January 2011 9:06 AM
To: [log in to unmask]
Subject: Re: Objective lens chromatic aberration
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I guess the initial question about chromatic aberration and the new one
about TIRF got muddeled up. I guess chromatically different NA in TIRF
could arise when part of the correction is build in the tube lens? The
illumination light forTIRF "through the objective" does not pass the
tube lens and is thus is not well corrected. Furthermore slight
imperfections in the dichroic which relfects the laser light can lead to
distortions and these can be wavelength dependent.
best wishes
Andreas
-----Original Message-----
From: Craig Brideau <[log in to unmask]>
To: [log in to unmask]
Sent: Mon, 17 Jan 2011 19:01
Subject: Re: Objective lens chromatic aberration
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This would also lead to chromatically dependent NA, which would be a big
no-no for a corrected objective.
Craig
On Sun, Jan 16, 2011 at 2:16 PM, Mark Cannell
<[log in to unmask]>wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I suggest it is much more likely that the fiber/rear aperture coupling
lens
> was at fault. I must admit I don't see how with rear aperture
illumination
> the same and same focal length for the 3 lasers how the incident rays
could
> have different angles ...
>
> Cheers
>
>
>
> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Michael,
>>
>> There are a few factors that need to be compromised in TIRF. 1) The
TIRF
>> penetration depth is dependent upon the wavelength of the incident
>> illumination, the angle of incidence, and the refractive indices of
the
>> media at the interface. 2) An apochromatic lens brings lasers of
three
>> wavelengths on to the same focal plane, but may not to the same
incident
>> angle. Therefore, the incident angles for the three lasers should be
able
>> to be adjusted independently to get optimal TIRF images.
>>
>> You may want to take your sample off first, to see how different the
>> angles
>> of the three lasers projecting to the wall, to get an idea of the
incident
>> angles of your lasers coming out of the objective.
>>
>> Regards
>>
>> Gary G Li, PhD
>>
>>
>> On Sun, Jan 16, 2011 at 1:02 PM, Cammer, Michael <
>> [log in to unmask]
>>
>>> wrote:
>>>
>>
>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> I originally posted the message below to the microscopy listserv but
>>> reviewing the discussion here on chromatic aberration, I thought it
would
>>> be
>>> apropos here too even though it is not really regarding confocal.
>>>
>>> A few years ago we were having problems with the first commercial
Olympus
>>> TIRF system because we could not get consistent evanescent waves
with the
>>> one angle adjustment with the laser lines we had from 405 to 568 nm
that
>>> were delivered via a single fiber (it was worse when we later added
a 633
>>> nm
>>> laser). I suggested we pump each laser in through a separate path
that
>>> could be angled independently. We didn't build it, but I think
Olympus
>>> now
>>> sells a TIRF system that does this.
>>>
>>> Another issue is that when I first heard about TIRF maybe 15 years
ago,
>>> it
>>> was introduced as a ring illumination at the outer edge of the back
>>> aperture, not as a single point or crescent at the periphery on only
one
>>> side. A ring, or at least a series of points around the periphery,
seems
>>> like a better way to provide a uniform field due to aberrations from
>>> coherent light in the imperfect optics. Any thought on this?
>>>
>>> Sincerely,
>>>
>>> Michael
>>>
>>> -----------------------ORIGINAL
MESSAGE-------------------------------
>>> We have the Nikon TIRF system and have three laser lines
>>> going into the TIRF arm via a single fiber. When we project through
>>> the 100X objective through the sample onto the wall we see that the
>>> lines go through the sample at different angles. (You can see a
>>> picture of the projection at approx 45 degrees at
>>> http://www.flickr.com/photos/mcammer/5359189090/ .) It is also
>>> noticeable in the TIRF images that the field depth is different for
>>> each wavelength. Is this unavoidable due to the different
>>> wavelengths or is it possible to align the optics better so these
>>> spots would be more coincident?
>>>
>>>
>>>
>>> _________________________________________
>>> Michael Cammer, Assistant Research Scientist
>>> Skirball Institute of Biomolecular Medicine
>>> Lab: (212) 263-3208 Cell: (914) 309-3270
>>>
>>>
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