Apparently, the "long-standing theory" has been shaken up, but via another method. I ran across this article regarding a new method of fluorescence 3-D light microscopy
  Link : www.uchsc.edu/lightmicroscopy/sted4pi.htm

One can google on STED microscopy for other articles about this new type of light microscopy that can apparently image details as small as 30-35nm.

I have been experimenting with DLC with no great success so far. I use the "Mathias arrow" to move between dark field and oblique illumination on my Lomo Biolam with great success. If I am successful with a DLC edge plate, I will post the results.

Regards, Duane Frybarger

Duane,
You wrote:
> ...I use the "Mathias arrow" to move between dark field and oblique
> illumination on my Lomo Biolam with great success. If I am
> successful with a DLC edge plate, I will post the results.

Per the Micscape (L) articles (2003) by Walter Dioni:

  Link : www.microscopy-uk.org.uk/mag/artoct03/wdoblique.html
  Link : www.microscopy-uk.org.uk/mag/artnov03/wdonion.html

it seems that the "Mathias arrow" device allows relatively simple, but versatile experiments with microscope illumination.

I don't recall this device being discussed previously - but it may have been referred to by some other name. Do you have any additional references to this technique?

Also, do you have any images for a specimen different from Dioni's?If you could share these with the group we could see what to expect with the "move between dark field and oblique illumination".

Btw: Micscape has published an update (Feb 3) from Barry Piekos, [re his DLC technique,] including a new image of 'microvilli on a kidney cell'.

Thank you for any assistance.
Regards, Keith

Hi Duane,
Contrast in DLC is quite impressive and I think better than the method of oblique illumination. I have experimented with (shifting the filter holder on a Zeiss Standard so that it partially obstructs the light, condenser best oiled to slide if using 40x and above objective - Wessenberg and Reed, 'The use of oblique illumination in microscopic observations of living protozoa', Transactions of the American Microscopal Society, Vol 90, No. 44, 449-457 (1971)).

However it is fiddly and neither method is as pleasing as DIC if you've got it. However, that's the strength of Piekos raising the DLC issue: it's a very acceptable alternative for those who haven't invested in DIC and theoretical reasons have been published by other authors suggesting that an edge plate near the field diaphragm is one of two optimal positions in the light path (these can be combined).

See: Graded-field microscopy with white light - Yi, Chu and Mertz:
  Link : http://www.opticsexpress.org/DirectPDFAccess/...

As for enhanced resolution from DLC, and its explanation if it is in fact achieved, the issue is moot with the onus on those suggesting it to prove their case; note that a convincing explanation is not required as a prerequisite for establishing it as an empirical finding.

I was able to photograph dots on F. rhomboides (posted to this group) with a 40x 0.75 NA objective. I don't know the distribution of dot sizes among specimens but suspect that this challenge is not exceedingly great for such an objective lens when special (already understood) diffraction effects are allowed for as against a standard image.

My image of Amphipleura is very dodgy. If it is something more than ghosting then the apparent size of the structure is, maybe, consistent with the size doubling found in some diffraction images.

Thus my position is that DLC should be considered for its contrast properties. Those interested in viewing protists should try it out as a supplement to phase contrast and dark field. Also note that for more critical uses DLC uses the full aperture of the objective (as does DIC) and lacks halo rings. Against that, as in all oblique lighting (including DIC) must be weighed the need to rotate the specimen in order to avoid false conclusions about structure.

Regards, Selwyn

I agree that the tremendous claims in resolution enhancements with DLC are very questionable. The lack of reproducible results obtained by qualified microscopists does not at all support any of the claimed wonders of what is referred to as "DLC". For me, DLC works not as well as COL or DF to resolve subresolution particles. (I refer to the term subresolution in LM as resolution beyond the diffraction limit.)

I would like to add this additional note. As it has already been known by many microscopists since the beginning of last century and published by Wilhelm Kaiser in 1906, the resolution obtained with oblique illumination with diatoms strongly depends on the angle between oblique illumination and orientation of frustules. This explains why fiddling around with the "Mathias arrow" or DLC can strongly impact resolution when looking at ordered structures (e.g. diatom frustules). But other effects (e.g. effect of doubling lines [2]) cause structures to appear in form of patterns that confuse the observer.

Last but not least, the proclaimed ten times increase in resolution of DLC, as claimed in a recently published paper in Microscopy Today [3], is scientifically not supported. Such a claim cannot even be supported with all the data published in the Micscape Magazine [4] and, of course, in [3]. Additionally, the irreproducibility of the results, which could claim a 1.5 to 2x increase in resolution, provides strong enough evidence that DLC does not offer any resolution enhancement beyond existing oblique illumination techniques, such as COL or DF. At best, it is a contrast enhancing technique, which should not be confused with a resolution enhancing technique.

I am looking forward to read a more complete article about DLC, which should be published in Micscape Magazine containing some photographs of the setup that is used by the inventor. Maybe such an article could shed some light into the reasoning of the author's claims.

Cheers, Gregor

[1] W. Kaiser, Die Technik des modernen Mikroskops, Verlag von Moritz Perles, k. u. k. Hofbuchhandlung, Wien (1906). (See http://caliban.mpiz-koeln.mpg.de/~stueber/kaiser/index.html for a copy of this book. The people working for Google's book scanning service could learn a lot from the folks contributing to http://www.biolib.de/)

[2] G. H. Needham, p. 285, The Practical Use of The Microscope, Including Photomicrography, Charles C Thomas Publisher, Illinois, USA (1958). (Reference obtained from Ted Clarke.)

[3] W. B. Piekos, Diffraced Light Contrast: Improving the Resolution of a Basic Light Microscope by an Order of Magnitude, November issue, Microscopy Today (2006).

[4] The Miscape Magazine, section "In Focus", December 2006 and January 2007.

Dear Selwyn,
Putting aside the claims of super resolution the lure of Piekos' work to me is the ease and low cost to implement it. I believe the standard to judge oblique illumination and even more complex methods of lighting against today is Circularly Oblique Illumination or COL. Paul James describes it well in a three part work in Micscape:
  Link : www.microscopy-uk.org.uk/mag/artdec02/pjcol.html.

Gregor Overney shows it will do a very good job at high resolution:
  Link : www.microscopy-uk.org.uk/mag/artmar04/godarkfield.html

Ted Clarke shows how build the stop, how to use it and what it will do:
  Link : www.microscopy-uk.org.uk/mag/artmar03/tcnaturescope.html

and another piece in Modern Microscopy (L),  'Evaluation of a PrototypeBF-DF-Oblique-Circular Oblique Lighting (BF-DF-Obl-COL) Condenser':
  Link : www.modernmicroscopy.com/main.asp?article=53

In my experiments with Piekos's stop I made a COL stop from a large Sequin and used the iris of the field condenser for the annular stop. The COL image was always equal or better than the image I could get with Zeiss Pol 40x 0.85NA, using a 12.5x Zeiss Kpl W Pol eyepiece and a 2x optivar that formed a fully corrected 1000x image.

I am not [satisfied] that either of the stops are a good as they can be. In experimenting with stops the reflectivity of the stop and the sharpness of the edge seem to matter a good deal.

On the matter of resolution beyond what Ernst Abbe postulated, even the author Barry Piekos [obviously] had questions that caused him to send the negatives to Kodak to see if they thought they could be artifacts. One paper showing what may be resolution beyond Abbe's postulates, without any theory put forth to explain it, is interesting and got a lot of people to try to duplicate the work. Hopefully some will be more successful than me. A technique that easy and inexpensive that extends the range of the light microscope even 10% would be very welcome and sell a lot of microscopes. Thirty or forty percent would make a new industry and improving it an order of magnitude is a revolution in physics. While I doubt that something that simple would be overlooked for so long it surely is not impossible. I am an eternal optimist. The glass is 0.01% full not 99.9% empty, unless I have to bet money on the deal then I go with the odds.

Exploring how well stops of all kinds work at the field condenser and how badly the condenser can be out focus and if putting any kind of stop at the field diaphragm compensates for condenser of lesser quality are questions I hope to answer for myself. If the results are interesting I'll share them. So far my results have not been very interesting.

Gordon

Hi Keith,
Thanks for the reply. I have just posted four photos under a folder name called "DuaneFrybarger" showing recent images taken using the "Mathias arrow". These images were captured using an old Olympus C-460 Zoom digicam held up to the eyepiece and then edited with Picture Window Pro. As is often the case, the image seen through the eyepiece shows more contrast and 3D effect than the photos. I experimented quite a bit with the DLC plate I made yesterday and couldn't come up with a better view than what I get with the arrow.

I generally move between dark field and oblique by pivoting the arrow in the filter holder. My biolam has no built in illumination so I am using an AO 735 illuminator and a frosted glass filter in my filter holder. I switch between a clear filter and a blue filter for the oblique illumination.

Regards, Duane

Hi Selwyn,
Thanks for the reply. I would love to have DIC but that is not an option for me. I agree that the DLC setup I tried showed much greater contrast than more traditional oblique illumination methods (short of a dedicated condenser), however, as I stated in my original post, my "Mathias arrow" works great for me in moving swiftly between bright field, dark field and oblique illumination and so far, I can't get better contrast with my DLC setup than with the Mathias arrow. I posted a few photos under a folder called "DuaneFrybarger" showing images taken with an old Olympus C-460 Zoom digicam held up to the eyepiece using the Mathias arrow.

I have been doing microscopy for about six years and I only view pond water life and don't like anything "fiddly". For me, the Mathias arrow is a great invention. Also, on his website Erhard Mathias has a great method for quickly estimating the size of an object in the field of view. I have been using this method for several years. I recently purchased a micrometer eyepiece and found that the method that Erhard invented is much quicker with as much accuracy as I need for identification purposes.

Erhard Mathias web site...
  Link : www.schwaben.de/home/mathias/english.html

Regards, Duane

Hello all,
I have lost track of whom started this thread on DLC, so I address the group. I have a Lomo trinoc with an Oblique light condenser. See pictures in my photo section here:
  Link: http://tech.ph.groups.yahoo.com/group/Microscope/photos/..

Ian Walker did an article for Micscape here:
  Link : http://www.microscopy-uk.org.uk/mag/artmar06/iw-lomooblique.html

The results I get with this condenser, I think, surpasses anything I have seen to date with DLC or for that matter Hoffman Modulation Contrast. Of course it doesn't compare with DIC. I know this is an expensive condenser, aroung $275.00 US, but I think it is well worth it.

I would be happy to post or send pictures if anyone is interested.

I guess there just isn't any 'free lunch'. You get what you pay for especially when it comes to light manipulation.

Dan

Can I interpret your statement "Of course it doesn't compare with DIC." as "DIC outperforms COL with regard to contrast or even resolution"?

If yes, I want to comment a little about this common misunderstanding, which is often, and falsely, communicated by the sales force of big microscope manufacturers. Why sell something for $100 if one can convince the customer to pay $6000 for all these DIC sliders and special condensers?

COL vs. DIC, the myth:
1) DIC provides a better contrast than COL. - Wrong. A DIC image just looks different with its pseudo 3D effect. Applying certain post- processing functions, which are available in most graphics programs, to a good COL image can produce a very similar faked 3D effect. But I doubt that someone would mutilate his COL images with such a filter.
2) DIC provides a better resolution than COL. - Wrong. Actually COL is capable in detecting more subresolution particles than DIC. COL rivals DF. DIC does not.

For most biologists, DIC is "easier" to use than COL. Why? – Because, for DIC, they can buy all this fancy hardware and take expensive courses. DIC is even shipping with manuals. However, only reasonably experienced microscopists can setup COL. It needs experience to convert a BF setup into a well working setup using COL. The same applies to DF. COL and DF are extremely powerful methods that require care. DIC and Phase is the stuff that works right out of the box. But it is an expensive box.

Cheers, Gregor

Well regardless of all the fancy names and devices to get these effects, us old timers, I am 76, have always used the cheapest method to obtain a diffracted light contrast and or oblique lighting simply by moving your finger or pen or pencil through the light beam before the condenser.I also use the edge of the filter holder for the same purpose.

There is nothing new in these methods, they just get regurgitated from time to time using circles, arrows and bits of wire.

Cheers, Ross.

Ross,
I have a blank filter ring that I could put a solid blanking plate in. I take it this may allow me to improve a view/resolution under certain conditions by blanking off asymetrically part of the light.

How important is the plane that the blanking plate is placed? Under the condenser is default, as that's where the rings attach, but would figuring out how to get a mask up close to the iris be worth it?

I guess I could try (and probably will) unless the view's not worth the climb.

By the way, it's very helpful to me when folks like you share their "tricks", lesson's learned, and knowledge. Especially good when that knowledge has been refined for a significant portion of 76 years. Ageing works for cognac, too! Which gives me an idea...

Thanks for sharing this.
Best, Jim

Very good point. When playing with "DLC", I noticed that moving the blade of a small knife edge in front of the condenser can produce very similar effects. As a matter of fact, when I was a kid without too much money for toys, I used all kinds of tricks to increase contrast while retaining resolution. - Money spoils. Soon I will buy a scope with DIC. ;-)

Cheers, Gregor

"Ross Ferris" wrote:
> ...There is nothing new in these methods, they just get regurgitated
> from time to time using circles, arrows and bits of wire.

Hi Duane,
Thank you very much for posting the photos. These are very good images given the "handheld digicam" procedure - it is so easy to spend way too much time trying for a "perfect" camera setup rather than getting on with observations and recording interesting images.

Your comment re the quality of the image presented to the eye versus what is recorded in a photo describes what is probably the most frustrating aspect of photomicroscopy!

I would appreciate being able to contact you offline for some additional info to be included in the Group archive, so that we don't bore too many people with posts related to details.

Regards, Keith

"duanefrybarger" wrote:
> ...I have just posted four photos under a folder name called "DuaneFrybarger"
> showing recent images taken using the "Mathias arrow". These images were captured using an old Olympus
> C-460 Zoom digicam held up to the eyepiece...

Hello again Dan,
You wrote:
> I would be happy to post or send pictures if anyone is interested.
Yes indeed. In trying to present information that may be useful to a hobbyist microscopist, the most difficult to find in the public domain is comparative images with an accurate description of the optical/illumination and photography setups.

If you could obtain a series of images for a particular specimen of interest under different illumination setups it would be very much appreciated.

But thinking about this for a sec, it may not be so easy if the specimen is running around on your slide? Maybe a video is required - or a shot of retardent?

Thanks, Keith

Dear Gordon,
Thanks for the links.

I suppose I have played around with COL in a small way since I have noticed that I sometimes get a greater clarity of fine detail with the Zeiss oil DF, so-called, "ultracondenser" when I vertically adjust the condenser out of optimal DF. The issue of getting optimum clarity/resolution seems to involve pragmatism (c.f waving a finger under the condenser), the art of microscopy?, rather than adherence to rigid rules. That said, it is well to be aware of practices that induce artifacts rather than enlightenment; I hope that those group members systematically trying out these new (or, rather, ancient?) techniques will document the downsides too.

Incidentally, in testing the supposed super-resolution of DLC it seems best that people use specimens of known light microscope structure viewed with objectives of lesser NA than conventionally reveal the detail. Given the known sizes of structures, the wavelength of the light employed and the NA of objectives it should be possible to agree combinations of higher and lower NA objectives for testing putative resolution improvements of 2x, 4x, 8x etc.

In view of comments made elsewhere it may be sensible to eschew subjects, such as diatoms, with periodicity. Which leaves the question of what would be suitable subjects which are easily available and metrically consistent among examples. I think some the above points have been mentioned elsewhere but bear reiteration.

Regards, Selwyn

Would be happy to Keith. I dismantle movie clips into frames with ImageJ a lot so that may be the way to go.

I will contact you next week sometime to arrange for a screening of the pictures.

Dan

"scitech200" wrote:
> ...If you could obtain a series of images for a particular specimen of interest
> under different illumination setups it would be very much appreciated.

Dan,
You wrote:
> I dismantle movie clips into frames with ImageJ a lot so that may be the way to go.
Thanks for the response - I look forward to see what we can accomplish re comparative images.

I have to also go back to some videos Don Williams made available, for A.pellucida, to see if selected frames demonstrate significant differences in resolution and contrast with varying illumination mode (but see below).

Selwyn has brought up the concept of trying to organize some systematic testing specific to DLC. It's certainly refreshing to see another post suggesting that perhaps a scientific approach (versus an ad hoc set of unrelated experiments and images by Group members) may produce some meaningful results.

He also makes an interesting observation that the periodic structure of specimens, such as diatoms, may generate artifacts that could be construed to demonstrate "super-resolution". I seem to recall a diatom image from Don showing a distinct Moire pattern and I have seen similar effects from high-density integrated circuits, via reflected light microscopy.

Regards, Keith

No problem. Talk to you next week.

Dan

Hi again Duane,
I wrote:
> I would appreciate being able to contact you offline for some additional info to be included
> in the Group archive, so that we don't bore too many people with posts related to details.

Thank you for the offline contact.
In the meantime, I have posted a new Archive page:
  http://www.kscitech.com/MicGroup/Msg_07.1.htm
for the latest DLC thread.

Although most operations for the prep of these webpages are automated via PC software; I still have to edit, resize images, etc. So I'll be playing catchup this weekend to get associated pages finished up!

Regards, Keith

You could try K3CCDTools (software) to dismantle the movie

David

"Daniel Holloway" wrote:
> ...I dismantle movie clips into frames with ImageJ