http://confocal-microscopy-list.275.s1.nabble.com/Phase-Contrast-Microscopy-tp7590859p7590873.html
Our microbiologists love the 100x 1.4 NA phase contrast objectives for combined phase and widefield fluorescence imaging. I always wondered how the phase ring on the objective is actually made and what influence it has on the fluorescence signal? When it just shifts the phase, it should be relatively transparent and let the fluorescence pass without losses, but when I look at the ring from the back, it is quite reflective, indicating that some of the fluorescence signal will be lost. HAs anyone compared objectives with and without phase ring? What will be the influence of the phase shift on the PSF?
Subject: Re: Phase Contrast Microscopy ... defense of brightfield microscopy
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 ...
> *****
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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>>
>