Re: Phase Contrast Microscopy

Posted by Benjamin Smith on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Phase-Contrast-Microscopy-tp7590859p7590864.html

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I've found the best way to understand phase contrast is on a phase contrast
microscope.  On a phase contrast microscope, take out one of the oculars so
you can see the focal planes.  In the path without the ocular, you should
see a ring of light perfectly aligned to an annular neutral density filter
(switch between brightfield and phase contrast to see how the illumination
perfectly aligns with the ND filter).  If they are not aligned, then the
annulus is not setup properly and you are not getting phase contrast (check
your manual to fix this).

Next. make a slide with a Kim-Wipe or lens paper on it, or grab an
unstained test slide (although tissue paper makes this effect very clear) .
As you move the sample into the light path, you will see all the diffracted
light miss the annular ring of light and fill the rest of the focal plane.
Move the sample out of the light path, and you will see this diffracted
light disappear.

Next, get a poor phase object like adherent cells or anything else that is
hard to see with bright field, and put it under the phase contrast
microscope.  Once you are focused on the sample, look at the focal plane
(side without the occular) and slide the annulus out of the way just a bit
(it should look like the sun just peaking around the moon after a solar
eclipse).  Now look at your sample, and it should look like just
brightfield (i.e. poorly contrasting).  Then put the annulus back into
place, while looking at the sample, and you see a sudden jump in contrast.
This is one of the striking features of phase contrast where it really is
all or none, and if the annulus is at all out of alignment you just get
brightfield.

As others have said, phase contrast is fixing a fundamental issue with
brightfield imaging of poorly diffractive objects (such as adherent
cells).  The issue is that bright field microscopy works by having the
diffracted light undergo a phase shift relative to the undiffracted light
(as it follows a different path length in the microscope).  Then, these
rays are allowed to interfere with each other at the image plane, producing
an image.  You can see this effect in bright field in a similar manner to
the phase contrast trick described above, but instead close the aperture
stop and you will again be able to see the diffracted light separated from
the non-diffracted light in the focal plane.  The problem with poorly
diffracting objects is two fold, 1) very little light gets diffracted
leaving not much light for interference, and 2) the phase shift of the
diffracted light is very small.  The end result being that samples like
adherent cells only cause a very small decreases in intensity, and since we
perceive light on a logarithmic scale, this is very hard for us to see.

Therefore, phase contrast fixes both of these issues.  One way (which you
can see by looking at the focal planes) is that it uses a cone of light as
the illumination pattern (due to the annulus at the front focal plane), and
this cone perfectly lines up with an annular neutral density filter (the
phase plate) at the back focal plane.  This ring illumination is a clever
way to spatially separate the diffracted light from the undiffracted light
at the focal plane, allowing us to attenuate the undiffracted light without
impacting the diffracted light, balancing the amount of diffracted and
undiffracted light.  Why use a ring to do this instead of say an aperture
stop?  The ring illumination simply preserves more of the illumination NA
and therefore preserves more of your resolution.

However, the phase plate doesn't stop there, as its name suggests, it also
enhances the phase difference between the diffracted and undiffracted light
(ideally such that the highest diffractive orders are phase shifted to
180°) such that the sharpest edges get perfect destructive interference.

Now at this point, you may be asking yourself, how do they know how much to
attenuate the undiffracted light (i.e. how dark to make the annular ND
filter) and how much of a phase shift to impose, as some samples may be
more diffractive than others.  And this is why there are actually many
different phase contrast objectives with different phase plates.  In life
sciences, most of the time we're only using phase contrast to check cell
culture, so we wind up only ever dealing with just one phase plate
optimized for that task.

Hope this helps, and I really do recommend checking it out on your own
microscope.  I've found with students, looking at the correlation between
the focal plane and image plane with and without sample can really inspire
that "aha" moment.

Cheers,
   Ben Smith

On Fri, May 1, 2020 at 12:35 AM Kai Schleicher <[hidden email]>
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.
> *****
>
> Hi David,
>
> I think that you understanding of phase contrast is correct and that you
> have explained it yourself properly and in a concise way.
> Diffraction and refraction are different indeed. It is in fact the
> diffraction of light that plays an important role in phase contrast
> microscopy.
>
> Have a look at the Leica tutorial:
> https://www.leica-microsystems.com/science-lab/the-principles-of-phase-contrast
> and figure 4 in the link you shared:
>
> 1. The illumination light passes through the annual ring in the condenser,
> resulting in a hollow cone of illumination.
> 2.1 A higher refractive index in the sample causes retardation, on average
> generating a phase shift of -1/4 λ in the light that interacted with the
> specimen compared to freely passing light.
> 2.2 Light interacting with the specimen (cell, granule, nucleus...) is
> diffracted to the outside of the illuminating light cone.The smaller the
> object, the larger the angle of diffraction [1].
> 2.3 Light that does not interact with the specimen is not diffracted and
> hence stays on the inside of the illumination cone.
> 3. In the phase plate, only the light on the inside of the light-cone is
> then advanced +1/4  λ .
> 4. This result in a phase difference between illuminating light and sample
> light of 1/2 λ, generating destructive interference and hence maximum
> contrast in the imaging plane wherever there was a structure in the sample
>
> Here is also a very nice iBology talk on the subject:
> https://www.ibiology.org/talks/phase-contrast-microscopy
> Hope this helps!
>
> [1] Abbe Diffraction:  https://www.youtube.com/watch?v=d8Tqoo0S6gc
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
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e-mail: [hidden email]
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