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