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Hi, all
Great discussion!
Re: the 1/4 condition
This is an engineering condition that is also tied to the fact that
most phase kits are used for looking a biological entities. Here's the deal:
There is a 1/8th wave optical path difference between most biological
entities and the fluid in which they are mounted. I've only met 2
people in my life who are able to explain the mathematics, but
somehow, that 1/8th optical path difference is converted into a 1/4
wave phase shift between light going through the specimen and light
going through the background.
The microscope is designed to take advantage of this 1/4 wave
phase. The ultimate goal here is two fold: (a) to cut the amplitude
of the background illumination so that it more closely matches the
amplitude of the light coming from the sample (ie: waves of
approximately the same height) and (b) to put the waves 1/2 wave
length out of step (or phase) so that they can destructively
interfere, creating a dark area against the bright background (ie: contrast).
Here is how it works:
The lighted annulus in the phase plate in the condenser is used to
carefully place the background illumination to that dark smokey ring
on the phase plate which is placed in the back focal plane (BFP) of
the objective.
That dark smokey ring has two functions:
a. It acts as a neutral density filter (about 18%) to cut the
intensity of the background down to more closely match the intensity
of the light going through the sample
b. It is typically CUT so that the light going through it has less
light to go through and therefore has a "jump" on light going through
bright area of the phase plate. Remember: the light going through the
sample is retarded by 1/4 of a wave versus the light going through
the background. When that illumination then goes through the thicker
part of the phase plate, it is retarded another 1/4 wave.
The result of step a is that wave amplitude of the background
illumination closely matches the amplitude of the light going through
the sample, to optimize interference.
The result of step b is that the two waves are out of phase by 1/2
wave: the ideal situation specifically for destructive interference.
Also, Phase Kits come with
a. telescopes to align the image of the lighted annulus in the
condenser with the "smokey ring" in the phase plate. You can see the
same image using the "bertrand lens" in an Optivar or mag changer, or
just taking the eyepiece out and peering down into the BFP of the objective.
b. 546nm green filters because that is the wavelength for which most
phase kits are optimized ... and, happily, also is a sensitive range
for both our eyes and many cameras. You can test this yourself by
setting up a phase kit and observing the image of the sample with and
without the green filter. NOTE: Some phase kits are optimized for
589, so make sure to check.
Re: variable phase - yes, this was a delightful addition back when
more of us were "fiddlers" and "Tweekers". I also have a Reichert
Zetopan (now more than 50 years old) which has "anopteral phase"
which was adjustable.
For further details see:
Ross, K. F. A., "Phase Contrast and Interference Microscopy for Biologists" and
Pluta, M., "Advanced Light Microscopy: Vol 2 - Specialized Methods"
Hope this was helpful.. and that you can now at least moderately tune
your phase systems for better results.
Best regards,
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education*
www.MicroscopyEducation.com
*A subsidiary of The Microscopy & Imaging Place, Inc.
7101 Royal Glen Trail, Suite A
McKinney, TX 75070
P: 972-924-5310
F: 214-592-0277
MME is currently scheduling courses for the Fall 2013. Call us today
for a free training evaluation.
At 01:33 PM 1/21/2013, JOEL B. SHEFFIELD wrote:
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>I, too, am teaching a course in microscopy, and have run across the same
>issue. It has occurred to me that we actually can't see what is happening
>to light when it is within an area of high refractive index until it
>re-emerges into our normal world. At that point, it resumes its original
>speed/frequency, and so it's a bit like Schrodinger's cat. At the same
>time, I have come across much more detailed versions of the cause of the
>phase effect. Take a look at Murphy and Davidson's new book, "Fundamentals
>of Light Microscopy and Electronic Imaging" for a discussion of a dual wave
>model (the S and P waves) that derives from a diffraction-based analysis
>rather than a velocity of light analysis. I have to admit that I am still
>struggling with that one, and would welcome any enlightenment.
>
>As to your second question, the answer is "no". Different structures will
>cause different amounts of phase shift. This is why the phase contrast
>image is not binary, but shows gradations. The 1/4 wavelength appears to
>be just a convenient average, and a way to set the phase plate somewhere in
>the middle. In an early Reichert microscope that I had a chance to see
>many years ago, the phase system was continuous, so that you could vary the
>added shift from + to - 1/4, and reverse the contrast at will.
>
>Joel
>
>On Mon, Jan 21, 2013 at 1:53 PM, MODEL, 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
> > *****
> >
> > Hi Claire - the speed of light does change but the eye responds only to
> > frequency, it doesn't know anything about wavelength. And the frequency
> > remains the same throughout all transformations of the wave.
> >
> > Mike
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[log in to unmask]]
> > On Behalf Of Claire Brown, Dr.
> > Sent: Monday, January 21, 2013 1:30 PM
> > To: [log in to unmask]
> > Subject: Refraction and Dispersion-phase contrast
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > I am teaching a class on light microscopy and have two questions:
> >
> > 1) If higher refractive indices materials slow down the speed of light
> > does the wavelength also change so that frequency and energy are conserved?
> > If this is true does is the wavelength shift so small that the colour does
> > not change a great deal? The other explanation I had is that the speed of
> > light never changes but short wavelengths take longer to travel through
> > high NA materials because they interact with the material and travel along
> > a longer path to reach the other side of the material. So the speed does
> > not change, the wavelength does not change but the light takes longer to
> > get through the material.
> >
> > 2) Does diffracted light shift by exactly 1/4 a wavelength in phase from
> > incident light? If so why is it exactly 1/4 of a wavelength?
> >
> > Sorry for my basic questions but these sometimes seem harder to explain
> > and understand than more complex concepts.
> >
> > Sincerely,
> >
> > Claire
> >
>
>
>
>--
>
>
>Joel B. Sheffield, Ph.D
>Department of Biology
>Temple University
>Philadelphia, PA 19122
>Voice: 215 204 8839
>e-mail: [log in to unmask]
>URL: http://astro.temple.edu/~jbs
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