Has anyone tried this?

Microscopy Today (L), Nov. 2006, has an article on DLC -- Diffracted Light Contrast.
It seems it's a simple 'oblique' technique, using a knife-edge at the filter position of the condenser to cast a diffraction pattern on the specimen. It refers to this abstract:

Diffracted-light contrast enhancement: a re-examination of oblique illumination.   Piekos, WB.
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.

"A re-examination and modification of the very old oblique illumination technique has resulted in a method for contrast enhancement in microscopes, diffracted-light contrast (DLC), which provides high-contrast, high-resolution images of unstained biological material. The technique, which utilizes the diffracted light from the edge of a small, opaque plate, provides shadowcast images similar to those obtained by Nomarski DIC, anaxial illumination, modulation contrast, or single-sideband microscopy; however, it requires only a single additional component, which can be added to any bright field microscope. The contrast and three-dimensionality of the final image can be controlled by inserting differently shaped edges. Any bright field condenser will work with the technique and, consequently, it is a technique that may be especially useful with relatively basic, inexpensive laboratory and teaching microscopes although the image..."

See "poor man's dic" in this Micscape (L) article:
  Link : http://www.microscopy-uk.org.uk/mag/artdec03/wdonion2.html

Yes, I wondered if Pseudo-Phase was similar, but this DLC uses a diffraction edge with a de-focused condenser (similar to Pseudo-phase but there I think you are using phase shifts in the outer edge of the objective, not diffraction effects). It's -not- COL, or Oblique (necessarily) or Pseudo-Phase . . . I think . . . :-)

I put a picture in the Don Williams Photo Gallery of some of the plastic inserts I've used. They are arranged so that the points are on the same plane as the condenser diaphragm. Using the Lomo Aplanatic condenser they can be moved in and out to give varying degrees of diffraction of the cone of light. The two 'almost half circles' can be used as diffraction edges or taped together to make a slit. They are made of Mylar from an old 3.5" diskette. I have some five inch diskettes somewhere and they would make better circles. These are a few of the shapes I've tried. I can't find the others for the moment.

Quite good results for high magnification can be obtained by off-setting a phase condenser ring using either phase objectives or bright field. Many bizarre and sometimes quite spectacular results can be obtained messing about with these things. An extra lens above the field diaphragm can be used to 'tilt' the illumination either in Phase of BF. This saves shifting the condenser and is easier to adjust for high mag objectives. It also serves to adjust the diameter of the cone to fit the field. Closing the field diaphragm does the same, but most of the light is lost.

D

The patent, the paper in Microscopy Today, and the abstract, all refer to placing the edge at the field stop -- which I interpret to mean the field diaphragm. In other words, it is just working right at the light port underneath the microscope, very much below the condenser or its iris. So I guess you 'slap' this diffraction edge (the article says convex is best) right on top of the understage light source port, and defocus the condenser to that you are looking at the diffraction off the 'edge' of the insert.

FWIW, Steve

I would bet this was well understood 100 years ago

By putting the edge at the field stop it would be in focus in the same plane as the subject. It's not hard to understand with the illustration at:
  Link : http://micro.magnet.fsu.edu/primer/anatomy/kohler.html

the ray tracings cross at the field diaphragm and at the subjects. Unlike Köhler DLC has the edge in sharp focus at the back focal plane of the objective (you need a phase telescope, Optivar or pinhole to set up the lighting) and the condenser is wide open. I expect it will take a while to figure out what is going on. I don't believe we can directly extrapolate from what we think we know about this without repeating the experiment.

Köhler illumination is not hard or expensive to get with an external light source and mirror. The old cans with a focusing lens and diaphragm go for 30 or 40 bucks on eBay. The diaphragm is accessible on the front of the cans so it would be easy to mount the knife edge as a starting point for those that don't have Köhler.

Also Ted Clarke shows how to make with an iris, camera lens and fiber optic light source in Micscape:
  Link : http://www.microscopy-uk.org.uk/mag/artsep01/tcmacro.html

While his is made of machined metal it would work just as well using tape to hold a PVC part on aluminum channel stock from the hardware store to align the parts.

While I haven't really read the piece in depth, DLC is not the same as oblique lighting. It appears to be an interference method, not just a change in the structure of light that increases contrast.

Gordon

At least we can't complain about the lack of images, instructions or openness of the method used in US patent #6,600,598:
  Link : www.pat2pdf.org/patents/6600598.pdf

and the piece in Microscopy Today give clear instruction on how to set up scopes most of us have, or have the equivalent to, so we can see if we can duplicate his work.

I find it difficult to believe that what is little more than a bit of trash on the face of the field diaphragm window could have this much effect on the image and has not been noticed in the last 100 years. However, Abbe's limit is hammered home so hard most would dismiss the observation rather than step from behind Abbe's robes to risk facing the wrath heaped upon those that follow Rife's ways by large numbers of the community.

Unlike super resolution claims in the past this can be replicated by enough of us, and his claim of an order of magnitude improvement over the resolution of Abbe's limit should be something we can experimentally replicate and clearly prove or disprove his claims. I [believe] he disclosed all of the device. If he didn't and it's not too complex we should be able to figure it out.

I expect it to be one of the more interesting discussions we have had in a long time.

Gordon

Does this "DLC" method use only one knife-edge, or two? Mention of knife-edges makes me think of the Schlieren method to image variations in the refractive index of transparent media. This method usually uses two knife-edges in the conjugate focal planes of the illumination and imaging trains. If I understand this right, for a microscope system, the knife-edges would be at the condenser aperture stop and at the objective's back focal plane.

-kevin.

Isn't this somewhat the same as the technique where you mount a piece of copper wire diagonally in the objective?? Saw it years ago on the web, but no idea where.

René.

Hello,
This email message is a notification to let you know that a file has been uploaded to the Files area of the Microscope group. : /Piekos 1999 Diffracted-light contrast enhancement.pdf
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File : /Kachar 1985 Assymetric illumination contrast.pdf
Uploaded by : rvanwezel

The wire has to be at the back focal plane of the objective. This means you have to make a saddle shaped wire that goes down into the objective and is cemented in place at the top. Then you need to move it into the centre with a long tool of some kind. The wire thickness is important and varies with magnification. Its a fiddle, but the results seem to be worth the effort. I toyed with the idea for a while and then forgot.

D

"rvanwezel" wrote:
> Isn't this somewhat the same as the technique where you mount a piece
> of copper wire diagonally in the objective??

"Don Williams" wrote:
> The wire has to be at the back focal plane of the objective.

This is nonsense. The wire has to be anywhere but the back focal plane of the objective -- but where exactly I didn't know. I'll look for the original paper, if I can remember where I saw it in the first place.

D

Was it this one? I'm thinking of trying it...
  Link : http://www.schwaben.de/home/mathias/emikro/kontrast2.html

Regards, Daron

Correction: I say the back focal plane of the objective. This is nonsense. But I don't know where the wire should actually be -- I'll try to find the original reference.

D

This is the method, but not the article I read. It still doesn't tell us where the wire is supposed to be -- 5mm behind the uppermost element is rather vague -- but perhaps it doesn't matter? René might remember. Was it one of the UK websites perhaps? Maybe an objective that can be dissembled to get closer to the optics might be easier to work with. I have quite a few Phase Contrast Lomos I might [try] this some time.

D

"Daron Edie" wrote:
> Was it this one? I'm thinking of trying it...
> http://www.schwaben.de/home/mathias/emikro/kontrast2.html

> I find it difficult to believe that what is little more than a bit of trash
> on the face of the field diaphragm window could have this much effect on
> the image and not have been noticed in the last 100 years...
> Gordon

I have used some type of compound microscope since I was 8 - a LONG time ago. I can remember those inexpensive, at first plastics, then Edmond monoculars with a flip mirror. Having poor optics, no money for illuminators, etc, I experimented in every way imaginable seeking visual info. I remember a particularly good effect was reflecting the source light partially off the parabolic mirror and the stainless steel housing. LOL, ALL of my objectives were water dipping without any problems!

I still use techniques very similar to the ones discussed here for what I have called my poor mans, phase contast, DIC, etc. I think I even began a thread on such at my field forum.

So, hobbiest, take heart, you may get pro results for many of your applications with plastic filters, water coloring, Edmond prisms, and plastic polarizing sheets. Such methods are not only cheap, they are quick!

It's not the improved contrast but the increase in resolution claimed in the first and second image that I find hard to explain by the simple methods he used. He has published the pictures and methods he used for anyone to duplicate his work.

I have only read the patent once and the piece in Microscopy Today twice but the only thing I see that he did was put an odd shape stop described in the patent on the glass above a 90 degree prism and focus the stop in the back plane of the objective. His claim to be able to separate practicals 10 times closer together than the Abbe limit allows is a really big step if I read the text correctly.

The increased resolution is not what I would expect from that set up and the way the piece it written it wasn't what the author expected either. Since he had to borrow a resolution slide I don't expect he was working on increasing resolution.

If his work stands up it will require a rethinking of our conventional wisdom of transmitted light microscopy.

Gordon

"....Since he had to borrow a resolution slide I don't expect he was working on increasing resolution." ....

Heh! That's kinda funny. Eureka! I'll try to wade through the patent, but where I used to devour, I now peck because of a very small central vision blindness, just right of center, that hampers reading.

I saw a note somewhere, maybe yours or the forum's, where cutting slits in a black paper light field mask enhanced either contrast or, perhaps, resolution. If interference patterns can increase resolution, perhaps is what is happening.

If I can find the "slits" note again, I'll post the cite.

No sorry, never tried it, just remembered it. It seems like another 'grayfield' technique where most of the direct light (which is heavily aperture limited) is catched by the wire, only a bit goes around. Put a black dot on the middle of the objective and you have central darkfield illumination.

I have a natural aversion towards black box technique, I just don't know what exactly is happening.

Rene.

"Don Williams" wrote:
> This is the method, but not the article I read. It still doesn't tell us
> where the wire is supposed to be -- 5mm behind the uppermost element is
> rather vague -- but perhaps it doesn't matter? René might remember.

Haven't got access to Microscopy Today unfortunately, do they show images with super-resolution? What exactly do they say about it? I can only assume they can say that 25nm particles can be *detected* by this technique, not visualized in this scale.

Rene.