http://confocal-microscopy-list.275.s1.nabble.com/Phase-Contrast-Microscopy-tp7590859p7590871.html
to the objective lens NA. That's perfectly doable, but not widely practiced
> *****
> 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.
> *****
>
> It is true that bright field is still the resolution champion (especially
> with a DAPI filter cube in the light path). Although, why settle for poor
> visibility when you can use Rheinberg illumination and get the best of both
> worlds with both a high resolution brightfield image and a high contrast
> edge image (aka dark field).
>
> On Fri, May 1, 2020 at 3:15 PM George McNamara <
[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.
> > *****
> >
> > Ben wrote: "As others have said, phase contrast is fixing a fundamental
> > issue with brightfield imaging of poorly diffractive objects (such as
> > adherent cells)."
> >
> > In defense of brightfield microscopy:
> >
> > A correctly working brightfield microscope with a video camera or
> > digital camera, or the transmitted light path of a confocal microscope,
> > is perfectly capable of imaging very thin objects (such as very flat
> > adherent cells or very thin unlabeled tissue sections). The camera on a
> > smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe
> > Photoshop (has had math capability for a long time), etc, to be able to
> > subtract background, and add back offset. They key is contrast control.
> > Optional: unsharp masking. Some of the (GPU) deconvolution software
> > vendors even offer deconvolution of brightfield image data. Bonus: no
> halo.
> >
> > Reference:
> >
> > Shinya Inoue and Ken Spring 1997 Video Microscopy
> >
> >
https://www.springer.com/gp/book/9780306455315> >
> > I don't see the 1997 book online, the 1st edition, 1986 is
> >
> >
https://link.springer.com/book/10.1007%2F978-1-4757-6925-8> >
> > See also Shinya's publications ... online or Collected Works,
> >
https://www.worldscientific.com/worldscibooks/10.1142/6315> >
> > Bob and Nina Allen's papers.
> >
> > Also nanovid papers (single molecule imaging before STORM, PALM, etc).
> >
> > To quote (and add to) Yogi Berra: "You can see a lot by just looking"
> > ... especially if you switch the light path from your (hopefully not
> > covid-19 contaminated) eyepiece to your camera port ... and then use
> > your instrumentation well.
> >
> > For MetaMorph, see
http://mdc.custhelp.com/app/answers/detail/a_id/18800> > disclosure: I wrote most of the article (took over from Ted Inoue, whose
> > self-portrait may - or not - be inside the monitor figure ...
> > "Acquisition rules for Quantitative Fluorescence" section has stood up
> > pretty well since circa 1993).
> >
> >
> > On 5/1/2020 12:35 PM, Benjamin Smith 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.
> > > *****
> > >
> > > 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
> Tel (510) 642-9712
> Fax (510) 643-6791
> e-mail:
[hidden email]
>
>
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/>