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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:

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>
> 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
>
>
>
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>> To join, leave or search the confocal microscopy listserv, go to:
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>>
>> 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:
>>>
>>
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>>>
>>> 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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