Fwd: Re: Optical fiber selection for uniform TIRF illumination--COMMERCIAL RESPONSE

[Martin Wessendorf-2]

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Posted on behalf of Dr. Alexander Asanov.

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Re: Optical fiber selection for uniform TIRF illumination

Dear John,

Your consideration does not take into account the issue of stray light,
which is very important for objective-TIRF.  Stray light in the case of
objective-based geometry significantly affects the notation of the depth
of penetration.  In 2006, Mattheyses and Axelrod reported 10-15% of
stray light relative to the intensity of the evanescent wave at the TIRF
interface. In TIRF Labs we tested 11 high NA Nikon and Olympus
objectives and obtained from 15% to 45% of stray light. There are
several other authors who reported on the detrimental effect of stray
light; the group of Martin Oheim and their 2014 papers in Biophysical J
are among them.

Before I comment on the depth of penetration, I would like to use your
numbers to additionally answer to Dr. Joshua Vaughan on his issue of
“objective lens laser damage” and explain his picture of the damaged
lens. You logically commented that single mode fiber is necessary for
better focusing the excitation light into a 5-15 micron spot at the back
of the objective. Let us assume 10 microns and take a 100 mW laser.  
Focusing of a 100 mW beam into a 10-micron spot produces >100,000
Watt/cm2 intensity, which significantly exceeds the laser-burning level
for organic materials. In fact, such intensity might cause damage to
some inorganic compounds used for optical coatings. Thus, the estimate
based on your numbers sheds additional light to the picture of damaged
objective posted by Dr. Joshua Vaughan at
http://i63.tinypic.com/mmemvl.jpg.  Although Dr. Vaughan used multimode
lasers and fibers, even they caused the damage. Using of single mode
lasers and fibers with larger values of the radiance will impose even
more risks.

Return back to the issue of the penetration depth. Unlike in prism- and
lightguide-based TIRF geometries, in the case of objective-type TIRF,
the stray light travels along the same optical path and significantly
contaminates the exponential decay of the evanescent wave. The intensity
of stray light 10-40% is not negligible. Instead of true exponential
decay, in the case of objective-TIRF we obtain the exponent with maximum
of intensity at the TIRF interface, plus the intensity of stray light.
The latter is a complex function of numerous factors. Among them is the
X, Y, Z position of the focusing spot and the radiance parameters of the
beam. If there is a microscopic inclusion on the way of the ~100,000
Watt/cm2, it will make its bad job for additional deviations from the
non-ideal behavior.

Despite the significant literature on the unfavorable effect of stray
light in objective-TIRF, some researchers ignore the fact of stray light
and report the calculated theoretical depth of penetration. This is
incorrect and can lead to misconceptions. Unfortunately, at the moment,
there is no general recommendation how to report the error of
penetration depth caused by stray light. The error can be large, up to
40-80% in certain practical cases. I believe that the entire TIRF
community should carefully work on this issue. Rapid progress of
super-resolution methods adds a color of urgency to this issue.

Best regards,

Alex Asanov, Ph.D.

TIRF Labs

[hidden email] <mailto:[hidden email]>

*****

Kyle,

There's a good reason why TIRF systems of this type do not use
multi-mode optical fibres. Though it is true the multi-mode fibre would
offer a higher throughput of light from the laser source and it can be
made fringe-free and speckle-free if shaken sufficiently, there is
fundamental property of light that will prevent you from focusing down
the light from this type of fibre to a small enough size at the
back-focal plane of the objective lens (etendue - the optical equivalent
of conservation of energy). You need to consider not only the core size,
but also the NA of the fibre. The product of the fibre NA and the
cross-sectional area of the fibre core is a constant.

For a well defined TIRF penetration depth, you need to focus the light
down onto the back-focal plane to as small a size as possible. The
reasoning behind this is as follows: Having a well-defined penetration
depth means that all of the illumination light must approach the
water-glass interface at a well defined angle. That angle is in turn
defined by the radial position of the light focused at the back-focal
plane, and hence the size/area of the spot of light located there. A
larger spot focused at the back of the objective will cause the light to
approach the interface at many different angles of incidence, and thus
the sectioning ability of the evanescent wave will be compromised.

You can work through the mathematics associated with this scenario and
you'll find that having an error of less than 10 nm associated with the
penetration depth requires that the spot size at the back of the
objective be on the order of 5-15 um. You cannot achieve that spot size
with a multi-mode fibre because of etendue (believe me, I've tried). You
can, get to sizes of that order with a single-mode fibre (which has a
core size of about 5 um). Again, the importance of this is that if your
beam is well defined in terms of angle of incidence (ie: a very
collimated beam at the front focal plane of objective), you will be able
to achieve very thin sectioning at the lowest penetration depths offered
by the objective lens (50-90 nm depending on the NA of the objective).

Now I say all that, and then present to you the following paper which
appeared fairly recently (open-access):

http://onlinelibrary.wiley.com/doi/10.1002/jbio.201500324/abstract

This paper presents an TIRF illumination method that does utilize
multi-mode fibres, but if you read the fine print, you'll see that they
do sacrifice some of the sectioning ability that could be achieved with
a single-mode fibre. The main focus of this article is to use the
multi-mode illumination method for localization microscopy where thin
sectioning is not always or not strictly needed, and so in that sense
there is some innovation here. But it's difficult from the images
presented to say how this really would compare to TIRF implemented in
the traditional sense using a single-mode fibre. The raw images are not
available, and I'm not familiar with the structures being imaged in the
cell samples they use. My instinct says that this just won't be as good
as what can be achieved the "normal" way, but it may be worth revisiting
in the lab. It's also questionable whether or not they really get the
benefit of the higher throughput again because of etendue arguments.
They have to dump most of the light outside of the objective aperture to
get a TIRF image without any non-evanscent (widefield / sub-critical)
light. And who knows where all that unused scattered light goes. If any
of the authors of that paper are present here on the listserver, perhaps
they can offer their perspective on this, I'd be interested to know.

Well, hope that sets on the right path (whatever that is). Do let us
know how you get on if you end up trying this.

Cheers,

John Oreopoulos

Staff Scientist

Spectral Applied Research

A Division of Andor Technologies

www.spectral.ca <http://www.spectral.ca>


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