Re: Condenser lens choice for a given objective

Posted by Kyle Michael Douglass on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Condenser-lens-choice-for-a-given-objective-tp7584901p7584908.html

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Thanks for the feedback, Barbara. It is very helpful.

I have heard the advice before about the condenser NA needing to be
greater than or equal to the objective NA. Can you offer some physical
explanation or intuition for why this is?

One admittedly incomplete explanation I can think of for the
recommendation goes like this: the light collected by the objective
consists of two parts. One part is the transmitted light that is not
scattered by the sample. The other part is the light scattered by the
sample. If the condenser's working NA is smaller than the objective's,
then the unscattered, transmitted light fills only a portion of the
objective's back focal plane. However, the light scattered by the sample
will probably be dispersed across the entire back focal plane because it
will encode all the spatial frequencies of the sample.

I wonder if it's the inhomogeneous distribution of light from the two
components in the objective's back focal plane that leads to the matched
NA requirements of the condenser and objective. Does this make sense?

Thanks!
Kyle

On 03/17/2016 08:22 PM, Barbara Foster wrote:

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>
> Hi, Kyle
>
> The general rule of thumb is that the NA on the condenser should meet
> or exceed that of the objective.
>
> If you are using oil immersion objectives, ideally, to achieve that
> goal, you should use an oil immersion condenser, otherwise you are
> limited to an NA of 0.9.
>
> Also, remember that the aperture iris in the condenser adjusts the
> condenser's WORKING  numerical aperture.  Just because the condenser
> is marked 1.4 NA doesn't mean that, in a practical experiment, it will
> be operating at 1.4.  I follow the guidelines set down by Frits
> Zernicke (inventor of Phase contrast):  gently close the aperture iris
> to the "Oomph" position: that delicate balance between sufficient edge
> definition and optimum resolution (Yes, the condenser does contribute
> to resolution).
>
> As for planning for growth:
> You might want to invest in a turret condenser early on.  That will
> give you the option to add those other contrast techniques as you grow
> into them.
>
> And just one more reminder, specifically regarding DIC:
> If you are going to use plastic vessels (petri dishes, multi-well
> plates, growth flasks), use Hoffman Modulation Contrast instead of
> DIC.  DIC uses polarized light.  The plastic will affectt the shear
> and cause effects that will be hard to interpret.  Some HMC set-ups do
> use pol to control the width of the slit in the condenser, but all of
> that is on the incoming side of the sample and will not be affected by
> plastic containers.
>
> Good hunting!
> Barbara Foster, President & Chief Consultant
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>
> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your
>> posting.
>> *****
>>
>> Hello listers,
>>
>>
>> I have a couple of questions about condensers for you. I'd like to do
>> some transmitted light imaging in an inverted microscope using high
>> magnification, oil-immersion objectives. For the moment, I don't need
>> to do anything other than brightfield with a high power LED light
>> source. It might be nice to do phase contrast or DIC in the future,
>> but I don't need it now.
>>
>>
>> My questions are:
>>
>>
>> 1) What are the rules of thumb for matching a brightfield condenser
>> to an objective? I won't be using anything but oil immersion
>> objectives with NA's greater than 1.4.
>>
>>
>> 2) If I do want to do phase contrast or DIC in the future, should I
>> put special consideration into the condenser lens selection now? I
>> imagine the condenser NA will determine what phase contrast rings I
>> can use, but does it impact DIC?
>>
>>
>> Thanks!
>>
>> Kyle
>>
>>
>> Dr. Kyle M. Douglass
>> Post-doctoral Researcher
>> EPFL - The Laboratory of Experimental Biophysics
>> http://leb.epfl.ch/
>> http://kmdouglass.github.io

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch