http://confocal-microscopy-list.275.s1.nabble.com/Phase-Contrast-Microscopy-tp7590859p7590869.html
adherent cells or very thin unlabeled tissue sections). The camera on a
smartphone likely works too. Helps to use MetaMorph, ImageJ, Adobe
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.
See also Shinya's publications ... online or Collected Works,
... especially if you switch the light path from your (hopefully not
covid-19 contaminated) eyepiece to your camera port ... and then use
self-portrait may - or not - be inside the monitor figure ...
> *****
> 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>>
>