NetNotes


the thinned area of the phase plate in the objective. However, in the web site indicated, and others, they use refracted light and diffracted light almost interchangeably in explaining phase contrast. To my “biologist” level of understanding, diffraction and refraction are very different phenomena and I did not think that diffraction changed the optical path length like refractive index differences. Refraction makes total sense to me in the context of phase contrast, but I don’t see how diffraction is relevant. Can someone explain what I am missing? Tanks, Dave Knecht david.knecht@uconn.edu


I am also a biologist and sometimes have a hard time understanding such complex physical phenomena. My understanding is:


– Diffraction is due to the interaction of the focused incoming light on the sample. Diffraction is the reason why an object is visible in light and electron microscopy.


– Refraction is related to the change in light speed through a change in the medium (lens) thickness, and the phase ring (which you must choose according to the objective used).


Te principle here is to separate in physical space the normal


(not diffracted) light from the light diffracted by the sample. If you look at the picture shown in the link you gave, you see only a yellow beam before the sample but aſter the sample you see a light pink color between (and also outside) the yellow beams (called direct surround light). Tis is the diffracted light (the lines point to the rosy color between the yellow light beams). I think I identified your problem: the phase difference doesn’t occur by passing through the sample, it occurs due to the phase ring placed aſter (or within) the objective lens. Te interaction of light with the sample gives diffracted, not refracted light (or perhaps some negligible refraction). Te diffracted light (by the specimen) will be refracted by the objective lens just as the non-diffracted light is BUT it will not strike the lens at the same place. Only the non-diffracted light will pass through the phase ring (because the ring is calculated to be in the path of non-diffracted light only) and this will result in a phase difference between the light diffracted by the specimen and the light that passes through it unchanged. Tis difference is then reconstructed on the image plane to give contrast. Stephane Nizets nizets2@yahoo.com


With due disclaimer that I am doing ion and electron beam


processing and my recollection of light optics is from loooong time ago – diffraction encompasses a multitude of phenomena, including separating spectral components of white light and possible interference between waves diffracted at different locations of the object. Separation of


the spectrum with white light illumination


and interference could both produce a delayed wavefront resulting in phase contrast. My feeling however is that most of phase contrast from thin, uniform, transparent biological specimens would be produced by differences in the index of refraction. Te “Diffracted light” notation on the Microscopyu page refers to separation of diffraction orders within the light path of the microscope. During diffraction contrast, imaging direct light (DC background) is blocked by the illumination aperture (condenser annulus) and the diffraction plate, while first-order diffracted light is passed to the image plane to form phase-contrast images. I’m sure that many people with more current and fundamental knowledge of optical microscopy will correct and expand my qualitative and exceedingly hand-waving explanation. Valery Ray vray@partbeamsystech.com


Perhaps, the confusing part here is that we usually consider


that diffraction occurs at sharp boundaries between transparent and opaque. But diffraction can also be caused by boundaries between parts


66


with different refractive indices. So, phase contrast utilizes diffraction which is caused by refraction. Such is at least my understanding. Mike Model mmodel@kent.edu


I think that your 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. Te illumination light passes through the annular 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 shiſt 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. Te smaller the object, the larger the angle of diffraction.


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 results 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 is a structure in the sample. Here is also a very nice iBiology talk on the subject: https://www.ibiology.org/talks/phase-contrast- microscopy. Kai Schleicher kai.schleicher@unibas.ch


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 set up properly and you are not getting phase contrast. 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 brightfield, and put it under the phase contrast microscope. Once you are focused on the sample, look at the focal plane (side without the ocular) and slide the annulus out of the way just a bit (it should look like the sun just peeking around the moon aſter a solar eclipse). Now look at your sample, and it should look like just brightfield (i.e., poorly contrasting). Ten put the annulus back into place, while looking at the sample, and you will see a sudden jump in contrast. Tis 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). Te issue is that brightfield microscopy works by having the diffracted light undergo a phase shiſt relative to the undiffracted light (as it follows a different path length in the microscope). Ten, these rays are allowed to interfere with each other at the image plane, producing an image. You can see this effect in brightfield in a similar manner to


www.microscopy-today.com • 2020 November


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76