http://confocal-microscopy-list.275.s1.nabble.com/Phase-Contrast-Microscopy-tp7590859p7590872.html
p.s. photo of the instrument looks a bit fuzzy to me.
> *****
> 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.
> *****
>
> According to this paper:
>
https://www.researchgate.net/publication/280600830_High_resolution_optical_microscope>
> provocatively titled " Resolution of 90 nm (lambda/5) in an optical
> transmission microscope with an annular condenser " (spoiler alert:
> misleading title), a brightfield microscope with annular condenser has
> astonishing resolution.
> Well, in this case the most likely cause for these astonishing results is
> just misunderstanding of the calibration patterns, according to the
> official Richardson test slide pattern description (
>
https://www.grayfieldoptical.com/files/innerpattern.pdf ) the "100 nm"
> pattern has a period of 200 nm, the same factor of two applies to the "99
> nm" horizontal and vertical bars.
> Arguably, a title stating "lambda/2.5 resolution" would not have the same
> distressing effect onto a regular microscopist...
> So, while transmitted light images look impressive with a well tuned
> instrument, it is well known that the resolution can match the resolution
> of fluorescence imaging only if the condenser NA is greater than or equal
> to the objective lens NA. That's perfectly doable, but not widely practiced
> these days...
> Best, zdenek
>
> On Fri, May 1, 2020 at 8:48 PM Benjamin Smith <
[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.
>> *****
>>
>> 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/>>
>