http://confocal-microscopy-list.275.s1.nabble.com/Phase-Contrast-Microscopy-tp7590859p7590870.html
with a DAPI filter cube in the light path). Although, why settle for poor
> *****
> 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.