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Refraction and Dispersion-phase contrast

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Claire Brown

Refraction and Dispersion-phase contrast

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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

mmodel

Re: Refraction and Dispersion-phase contrast

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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:
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*****

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

Craig Brideau

Re: Refraction and Dispersion-phase contrast

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To join, leave or search the confocal microscopy listserv, go to:
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Wavelength doesn't really exist; it's a metric we came up with to describe
specific frequencies in a numerically convenient manner, more or less. If
you could 'freeze time' and hold a ruler up to monochromatic light then in
theory you could measure the length of one oscillation of the light. In
air, this follows f = c/lambda where f = frequency, c = speed of light in
vacuum, and lambda is wavelength. In glass you have to modify the speed of
light by no/nmat where no is 1 (index of refraction of vacuum) and nmat is
index of refraction of the material the light is propagating through. If
you do the 'freeze time' trick again and try to measure the wave as it
passes through glass, you will find the wave is 'bunched up' a bit as it
propagates slower through matter than vacuum. However, if you observe at
an infinitely small point as the wave goes by, and unfreeze time, you will
record exactly the same frequency regardless of the material you are in.
It's Monday where I am so please take what I say with a grain of salt.
@:-)

Craig

On Mon, Jan 21, 2013 at 11:53 AM, 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

Re: Refraction and Dispersion-phase contrast

In reply to this post by mmodel

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

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:

--

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

Zac Arrac Atelaz

Re: Refraction and Dispersion-phase contrast

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Joel: Here you have 2 more links so you can keep asking "basic" things and pushing science (and scientist) to new questions, and great new answers thank you for sharing with us, and letting us rethink all this. http://www.sciencedaily.com/releases/1999/10/991005114024.htm http://lcogt.net/spacebook/speed-light Best regards Gabriel OH
> Date: Mon, 21 Jan 2013 14:57:05 -0500

> From: [hidden email]
> Subject: Re: Refraction and Dispersion-phase contrast
> To: [hidden email]
>
> *****
> 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

Barbara Foster

Re: Refraction and Dispersion-phase contrast

In reply to this post by JOEL B. SHEFFIELD

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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 <[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

Andreas Bruckbauer

Re: Refraction and Dispersion-phase contrast

In reply to this post by Craig Brideau

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The "freezing in time" can be done experimentally by creating a standing wave. It was a standard experiment for biology/medicine students to measure the wavelength of acoustic standing waves using a little microphone. This shows that wavelength is very real! Frrequency stays the same but wavelengh changes in material of higher refractive index.

best wishes

Andreas

-----Original Message-----
From: Craig Brideau <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Mon, 21 Jan 2013 19:58
Subject: Re: 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
*****

Wavelength doesn't really exist; it's a metric we came up with to describe
specific frequencies in a numerically convenient manner, more or less. If
you could 'freeze time' and hold a ruler up to monochromatic light then in
theory you could measure the length of one oscillation of the light. In
air, this follows f = c/lambda where f = frequency, c = speed of light in
vacuum, and lambda is wavelength. In glass you have to modify the speed of
light by no/nmat where no is 1 (index of refraction of vacuum) and nmat is
index of refraction of the material the light is propagating through. If
you do the 'freeze time' trick again and try to measure the wave as it
passes through glass, you will find the wave is 'bunched up' a bit as it
propagates slower through matter than vacuum. However, if you observe at
an infinitely small point as the wave goes by, and unfreeze time, you will
record exactly the same frequency regardless of the material you are in.
It's Monday where I am so please take what I say with a grain of salt.
@:-)

Craig

On Mon, Jan 21, 2013 at 11:53 AM, MODEL, MICHAEL <[hidden email]> wrote:

George McNamara

Re: Refraction and Dispersion-phase contrast

In reply to this post by mmodel

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi mike and Claire,

with respect to 'eye response', instead of frequency or wavelength, I
think it is easier to deal with this in terms of the photoreceptor
molecule (opin, rhodopsin, other opsin, depending on cell) and the
photon energy packet is just absorbed. After absorption, the photon no
longer exists, so at this time point it does not have either frequency
or wavelength. On the other hand, the absorbed energy results in the
chromophore-protein to do work.

Enjoy,

George

On 1/21/2013 1:53 PM, MODEL, MICHAEL 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
>
>

Shalin Mehta

Re: Refraction and Dispersion-phase contrast

In reply to this post by JOEL B. SHEFFIELD

*****
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*****

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
>

JOEL B. SHEFFIELD

Re: Refraction and Dispersion-phase contrast

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Shalin,

Thank you so much for clearing this up so well. I think that the paradox
that pervades this subject is the idea of "background" vs "sample", since
the "background" light does not reintegrate with the "sample" if one is
under the impression that the background is extracellular and the sample
is within the cell. Eventually, I realized that this idea is incorrect,
and the destructive interference had to occur with light from the one
source point, and that there had, therefore to be essentially two waveforms
that emanate from the single spot. In Murphy's original book, and now in
the new one, that point is discussed in terms of S and P waves. --and
finally makes sense.

Joel

On Tue, Jan 22, 2013 at 12:15 PM, Shalin Mehta <[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 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
> >
>

--

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

Casal Moreno, Carmen

FISH and Immunofluorescence

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Hi everyone,

I am Carmen Casal, and I am working in the Microscopy Unit of Cancer Epigenetics and Biology Program in Barcelona, Spain.
I am wondering if some of you have done an Immunofluorescence in combination with FISH. I know that there are some papers describing the combined method but my question is that if I have to detect an hybrid structure formed with DNA and RNA (RLoop) and for this motive I can't denaturalize the structure because I am sure that it will broke with the treatment, and also I have to do an Immunofluorescence to detect the gene where this kind of structure is located, how can I do it?

Thank you very much.
Best regards,
Carmen.

Carmen Casal Moreno, PhD
Microscopy Unit
Programa d'Epigenètica i Biologia del Càncer (PEBC)
Fundació Institut d'Investigació Biomèdica de Bellvitge (IDIBELL)
Av.Gran Via de L'Hospitalet, 199 - 203
08908 L'Hospitalet de Llobregat
Barcelona
932607500 ext 3246
[hidden email]

Steffen Dietzel

Re: FISH and Immunofluorescence

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Hi Carmen,

I am not sure I see where the problem is. I guess you want to detect RNA
in the sample with a DNA probe, right? Then you can denature the DNA and
cool it down before you add it to the sample for hybridization, e.g.
overnight. Then, antibody staining would be the next day.

What do you mean by doing Immunofluorescence to detect the gene? Is
DNA-DNA FISH also involved?

Steffen

On 23.01.2013 13:38, Casal Moreno, Carmen wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
>
> I am Carmen Casal, and I am working in the Microscopy Unit of Cancer Epigenetics and Biology Program in Barcelona, Spain.
> I am wondering if some of you have done an Immunofluorescence in combination with FISH. I know that there are some papers describing the combined method but my question is that if I have to detect an hybrid structure formed with DNA and RNA (RLoop) and for this motive I can't denaturalize the structure because I am sure that it will broke with the treatment, and also I have to do an Immunofluorescence to detect the gene where this kind of structure is located, how can I do it?
>
> Thank you very much.
> Best regards,
> Carmen.
>
>
> Carmen Casal Moreno, PhD
> Microscopy Unit
> Programa d'Epigenètica i Biologia del Càncer (PEBC)
> Fundació Institut d'Investigació Biomèdica de Bellvitge (IDIBELL)
> Av.Gran Via de L'Hospitalet, 199 - 203
> 08908 L'Hospitalet de Llobregat
> Barcelona
> 932607500 ext 3246
> [hidden email]
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

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