Posted by
Shalin Mehta on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Refraction-and-Dispersion-phase-contrast-tp7579532p7579549.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Hi Claire and Joel,
As Joel points out, 1/4 wavelength is the convenient average phase-shift.
It is worth emphasizing that the phase-shift in question is between direct
light and the diffraction orders produced by transparent object. Zernike in
his nobel lecture shares many insights on how he first came up with and
then refined the phase contrast method - (
http://www.nobelprize.org/nobel_prizes/physics/laureates/1953/zernike-lecture.pdf).
He points out that when studying phase-gratings (not under microscope, but
macroscopically), he found that when he used a telecope to precisely focus
on the grating it disappeared. The phase-grating reappeared when the
telescope was slightly defocused. He explains this behavior thus,
a. To see the structure of phase grating, the diffracted light must be
"thrown onto coherent background".
b. The phase of diffracted light adds or cancels the amplitude of coherent
background (i.e., uniform undiffracted light). When the phase-grating is in
focus, the relative amplitudes and phase are such that the coherent
background is not changed. Out of focus, the phases align better so that
coherent background is visibly changed.
The phasor diagram in above lecture points out that the average phase
difference between direct and diffracted light is 1/4*Wavelength.
Zernike first used a 'phase-slit' to achieve both a and b. Then to create a
circularly symmetric contrast, he developed annulus. The lecture points out
that microscopists following Abbe's theory remained wedded to thinking in
terms of amplitude gratings and missed the point about phase-gratings.
In short, Zernike's lecture is an absolutely fascinating read. I think all
of the above is logically (rather than chronologically) laid out in Murphy
and Davidson chapter - I tried looking up on Google Books, but page 120 is
not part of preview!
Cheers
Shalin
website:
http://mshalin.com(office) Lillie 110, (ph) 508-289-7374.
HFSP Postdoctoral Fellow,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA
On Mon, Jan 21, 2013 at 2:57 PM, JOEL B. SHEFFIELD <
[hidden email]> 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 <
[hidden email]> 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:
[hidden email]]
> > On Behalf Of Claire Brown, Dr.
> > Sent: Monday, January 21, 2013 1:30 PM
> > To:
[hidden email]
> > 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:
[hidden email]
> URL:
http://astro.temple.edu/~jbs>