Given the recent posts suggesting that perhaps the Group should "have another go at evaluating DLC", and while acknowledging Ross' comment that it's quite likely a rehash of a technique that was known many years ago, I found the time today to study the Barry Piekos patent (US 6,600,598). I chose the patent as a starting point because quite often detailed information is included that is not available within publications in scientific journals of some form, where the emphasis is usually on experimental results (images).

A synopsis is available at:
  http://www.kscitech.com/MicGroup/Msg_07.2.htm
Although not yet complete, it brings up some points that may be worth further discussion.

In particular, reference to an adjustment away from a standard Köhler setup to give an enhanced illumination region close to the active (shadow) edge having 'monochromatic blue light' seems to beg comparison with:
1) Gregor's discussion of the significance of using blue light to allow resolution of A.pellucida structure at the limit of LM resolution, based upon sound physics;
2) Peter's superb A.pellucida image that required blue LED illumination; and
3) One of Don's most recent A.pellucida images with a blue filter in the illumination path.

But, of course, this connection may well be a gross over-simplification?

Also, at least in the patent, it seems that there is no claim re "super-resolution" - only an enhancement of contrast. But I may have missed something here.

Lastly, it would be very interesting to know exactly what dimensions and form factor were used by Barry for his edge plate?

Regards, Keith

This patent pertains to a special illumination system for brightfield microscopes. Light control within the optical path of the microscope is achieved by placement of an opaque, convex shaped element across the light path to produce a "highly beneficial contrast enhancement" - referred to as Diffracted Light Contrast (DLC).

A section of Fig.1 is shown here to include only the microscope components most important to a discussion of the patent's claims.

Typically, the light source for a microscope is a tungsten filament lamp (122). This lamp is combined with a condenser lens (124) and a mirror (126) to efficiently direct a beam of light on to the underside of a refractive specimen (150), via the aperture diaphragm (140) and condenser (142).

The normal field diaphragm (128) is used to regulate the amount of light delivered to the specimen. In the preferred patent embodiment this diaphragm is kept in a wide-open position, i.e. maximum aperture and maximum specimen illumination. It is claimed that reducing this aperture will diminish the 3D shadowcast effect which predominates DLC.

An edge plate (130), as shown in Fig.2, is now added to the optical path adjacent to the field diaphragm and orthogonal to the microscope's optical axis (designated by the vertical dotted line in Fig.1). This plate, in general, is positioned to block only a small percentage of the light beam. After interacting with the edge plate the light passes through the aperture diaphragm, which is also kept close to wide open. If this aperture is reduced, it has the effect of 'stopping down' the condenser. At low magnifications it is claimed that this diminishes the 3D shadowcast effect, while at higher magnifications it has the undesirable effect of increasing the depth of field. Condenser (142) serves to focus light through a small area of the specimen and into the objective lens (160) to form an intermediate image (virtual) of the specimen.

The general form of the edge plate is shown in Fig.2 as a plan view (orthogonal to the microscope's optical axis). In a simple setup, the edge plate is manipulated by hand using a small diameter rod handle (132). This handle reduces the asymmetric disruption of the illuminating light beam, with can lead to induced astigmatism.

The plate body(134) has inactive edges designated as (135), (136) and (138). These edges "must be sufficiently spaced" from the convex active edge (137) to prevent any optical interaction within the region of light cast by this active edge. The "thickness of the active edge is not critical", but the plate is typically fabricated from thin aluminum sheet with black anodized surfaces.

Assuming that the microscope has condenser(142) positioned for Köhler illumination, the field diaphragm will be in focus. As the edge plate is immediately adjacent to this diaphragm, it is also in focus and will cast a sharp shadow on to the specimen. As viewed through the eyepiece, conventional brightfield and darkfield regions are seperated by a chromatic region, which can be adjusted in form and color by moving the condenser away, by a small amount, from the exact Köhler position. It is observed that the chromatic region takes on a monochromatic blue color when the condenser-edge plate combination is below the Köhler position, and changes to a red color when above the Köhler position. Thus it may be theorized that "the monochromatic light results from diffraction along the active edge(137) of the edge plate."

"scitech200" wrote:
> ...away from a standard Köhler setup to give an enhanced illumination region
> close to the active (shadow) edge having 'monochromatic blue light' seems to beg comparison...

Hi!
I would class Köhler illumination as a quality and form of light, rather than a color of light. You can certainly have monochromatic Köhler.

And yes, blue (actually violet) light at about 410nm has much more resolving power than red light at about 680nm.

Good luck, Don

Hi Don,
You wrote:
> You can certainly have monochromatic Köhler.

Yes. But the intriquing aspect of Barry's setup (if I'm understanding the patent info correctly) is that a monochromatic blue illumination area is generated, and this seems to be the area that leads to "enhanced contrast". The illumination area is very close to the active edge of his edge plate and it requires moving the condenser-plate combination away from an exact Köhler setup. This could be considered (maybe incorrectly) analogous to tilting a diffraction grating in the white illumination beam to produce monochromatic light?

Does he actually have a built-in monochromator? If so, a reasonable question may be: how does the DLC image compare with that obtained, for the same specimen, by simply illuminating with monochromatic blue light?

Most microscopists accept the Abbe criterion for determining the minimum observable seperation (resolution) of features for a refractive specimen. That is, high NA (numerical aperture) and short wavelength illumination can be expected to produce the highest resolution. I suppose that, strictly, it's an "effective NA" value that determines the resolution; with contributing values for the condenser optics, slide and coverslip glass, specimen mountant, objective optics and the medium seperating both the condenser and objective from the slide/specimen/cover glass assembly.

But what about contrast for a refractive specimen? It seems that microscopists recognize that even though the NA and illumination wavelength may allow a resolution defined by the Abbe criterion, an actual observation may be limited by lack of contrast. Come to think about it - I'm not at all clear on what determines contrast for differing illumination techniques.

From what I have seen so far, Barry was studying a technique for contrast enhancement during the 1999-2003 period for the patent filing, but only very recently published the so-called super-resolution images using basically the same DLC setup. It may be an interesting exercise to track these contrast versus super-resolution experiments?

Thank you for your interest in this topic. Maybe other group members will jump back in and help us better understand Barry's DLC technique.

Regards, Keith

Keith
In the lower image of the one that shows an image of the gratings. The one that show enhancement of the 0.4 lines in the bottom panel of the top picture shows much worse resolution of the 1.0 lines than picture above it.

[These images are referenced in message [33851] on page [1] of a previous Section titled: Diffracted Light Contrast (DLC)].

Some have speculated that may be due to a periodic feature in the light.

may have doubling in the lines around the edge as they come in pairs a lot.

Here are the papers I have in my file on the subject.
1. Diffracted-Light Contrast Enhancement: A Re-Examination of Oblique Illumination, Barry Piekos, Microscopy Research and Technique 46:334–337 (1999)
2. Diffracted Light Contrast: Improving the Resolution of a Basic Light Microscope by an Order of Magnitude, Barry Piekos, Microscopy Today
3. Variable Coherence Scattering Microscopy, Erwan Baleine and Aristide Dogariu PRL 95, 193904 (2005) Physical Review Letters week ending 4 November 2005
4. US Patent 6,600,598 Barry Piekos
5. Zero-Cost-Mod-BF-Microscope for Phase-Grad-Schlieren, Daniel Axelrod
6. Cell-Substrate Contacts Illuminated by Total Internal Reflection Fluorescence, Daniel Axelrod, Biophysics Research Division and Department of Physics, University of Michigan, Ann Arbor, Michigan 48109
7. What Everyone Needs to Know About Evanescent Fields Tom Hunt 1-01- 03
8. Graded-field microscopy with white light Ran Yi, Kengyeh K. Chu, Jerome Mertz Boston University, Department of Biomedical Engineering, Boston, MA, 02215
9. Super-resolution imaging through a planar silver layer David O.S. Melville and Richard J. Blaikie
10. A piece on Single Sideband Microscopy

Gordon

Hi Keith,
Is Axelrod's paper available in electronic format? I've searched extensively for such but haven't come across it.

Barry's latest picture is interesting. I have to take his word for the interpretation but it seems plausible. This brings me back to the need for comparisons to be made using higher and lower NA objectives on specimens the light microscopy properties of which are known. For the time being we should leave attempting to push the boundaries of the highest NA objectives to people like Barry who are familiar with fine structure from an EM perspective.

Regards, Selwyn

Hi Selwyn,
> Is Axelrod's paper available in electronic format? I've searched
> extensively for such but haven't come across it.

You are right - only misplaced links and an abstract. It should be in the public domain because the work was done under a grant from the NIH.

Gordon lists it as one of his reference files, so we'll see what is actually available without copyright issues.

In the meantime, here is an optical path diagram:
  Link : http://www.kscitech.com/Temp/Pic_07.htm
for the Axelrod Schlieren microscope using phase gradients.

> This brings me back to the need for comparisons to be made using
> higher and lower NA objectives on specimens the light microscopy
> properties of which are known.

I'm getting specific recommendations on how best to replicate Barry's DLC results, using a readily available specimen. It seems that we may need something a little more stable than a hand held edge plate. I'll keep you informed as this progresses.

There are some very experienced group members that have good reasons to discount this as an insignificant technique, but as a follow on to Steve Neeley's comments while starting this thread:
"The fairest thing we can experience is the mysterious. It is the fundamental emotion which stands at the cradle of true art and science. He who knows it not and can no longer wonder, no longer feel amazement, is but a snuffed out candle".
This quote by a rather well known fellow that spent his latter years at Princeton's Institute for Advanced Studies.

Regards, Keith

Thanks Keith

Selwyn