MicrobeHunter.com Microscopy Forum

A while ago, Rod Nabbolz ("rnabholz") posted a darkfield image that I erroneously thought was illuminated off-center. That gave me the idea of trying to use an off-center stop to obtain oblique darkfield illumination. More recently, John B ("mrsonchus") posted what he called "Dark-field of a sort" ( viewtopic.php?f=6&t=3300 ) that I think, from his description and from the appearance of the image, corresponds to what I've called oblique darkfield illumination. Below are image pairs taken from student-grade commercial slides comparing darkfield images with those taken using oblique darkfield illumination. The oblique darkfield images were obtained by de-centering the darkfield stop which was large enough to still block the direct illumination, as represented in the diagram below:
                  
where the green circle represents the darkfield stop**, and the condenser iris** is shown fully open.
______________________________
** as seen at the back focal plane of the objective.

Another way may be to use a centered darkfield stop, but block the illumination from one side (or, using a darkfield condenser, block the illumination reaching the condenser from one side). I've not tried this yet, but I think that should work as well (except perhaps would give less light).

Descriptions were copied from the slide label. The left image was taken using regular oblique illumination, and the right oblique darkfield. All images were taken with a 10x objective, and the DF position of the Nikon Phase Contrast condenser was used as a darkfield stop (and shifted slightly to produce oblique darkfield).

__________________________ Darkfield ____________________ ___________________ Oblique Darkfield _________________

"Silver berry scaly hair"


"Angora rabbit hair"


"Frog blood"


"Nymphaea of aqustio stem"


"Diatom"

Bonjour gekko
Des fois pour certain sujets je fait comme vous, fond noir décentré et je trouve que c'st plus spectaculaire et très facile à mètre en oeuvre.
Car ça donne du bon relief à image.
Voici une photo avec la même technique que vous utiliser.
Cordialement seb

Great work, gekko.

I think you meant the left is regular darkfield.

I am guessing for "oblique darkfield" to work well, subject would have to be thick, translucent and have many planes of focus depth. And probably objective NA at 0.4 or less.

From your photos, I personally prefer oblique darkfield for #1 (due to enhanced depth) and regular darkfield for #2, #4 and #5 (due to more even lighting). My eyes are not good enough to see differrnce in #3.

Works very well. Thanks for posting this.

vasselle: your example is excellent.

Very nice technique, very well explained and demonstrated.

The effect on the Diatom is quite pronounced and interesting.

I guess I will be sticking more things into my condenser again...



Rod

Thanks. Something else to experiment with. Good explanation and examples.

seb, thank you very much for contributing your comment (which I totally agree with) and, expecially, your superb example (much better than my attempts).

zzffnn, thank you! Yes, I meant the left to be "regular" (concentric) darkfield. I think you are right: I think the same criteria that apply to objects useful for "regular" oblique illumination also apply to oblique darkfield. I may be wrong, but I don't think special NA limitations apply; I think whenever one can get concentric darkfield, one can get oblique darkfield (either by extending the darkfield stop in one direction or by blocking the light coming from one direction. I would be interested if you would like to try covering about half the bottom of your darkfield condenser and confirm this or prove my statement wrong. I'm not 100% sure it would work as I've not tried it, but I can't think of why it wouldn't. It would reduce the light by about one half, but apart from that I think it should work.

75RR, many thanks!

Rod, thank you very much. I chose to use a diatom as an example on purpose . I look forward to see how well this would work with your diatoms and darkfield condenser.

JimT, many thanks: yes I thought you would like to experiment with stuff like that .

Thanks to all.

gekko wrote: zzffnn, thank you! Yes, I meant the left to be "regular" (concentric) darkfield. I think you are right: I think the same criteria that apply to objects useful for "regular" oblique illumination also apply to oblique darkfield. I may be wrong, but I don't think special NA limitations apply; I think whenever one can get concentric darkfield, one can get oblique darkfield (either by extending the darkfield stop in one direction or by blocking the light coming from one direction. I would be interested if you would like to try covering about half the bottom of your darkfield condenser and confirm this or prove my statement wrong. I'm not 100% sure it would work as I've not tried it, but I can't think of why it wouldn't. It would reduce the light by about one half, but apart from that I think it should work.

gekko,

I did try covering up half of my darkfield condensers, three times, with your B&L paraboloid, an AO 214F and a LOMO OI-13 cardioid. None worked for most high magnification subjects that I tried. Part of the subjects just went dark, even though my 4000 lumen LED was used at max. For very low magnification, it may work, since light is ample.

I guess in practice, high NA high mag darkfield needs lots of light, very precise alignment and optical correction, which may not go well with the way oblique darkfield works. I would be interested to see if anyone can get good oblique darkfield at 40x NA 0.65 or above. I couldn't for sure.

zzffnn, thank you so much for taking the time and trouble to do the test so quickly. So that seems to rule out high magnification oblique darkfield. I have a 40x/.70 objective with which I can barely get darkfield, so I will test it and see what will happen. I may not be able to get to try it until Sunday, but I'll report what I find. Thank you again.

I did those experiments a few months ago. Phil holds the opinion that "oblique darkfield" generally does not work well. I did not believe him first, but then after doing my own experiments, I think it is indeed the case at or above 40x NA 0.65.

At NA 0.4 or lower, the darkfield illumination cone lies far outside of objective edge NA, so one would have some room for error/playing around.

I just found out that there there is an article by Thomas L Webster published in Micscape Magazine in 2005 entitled "Oblique Darkfield Illumination",
http://www.microscopy-uk.org.uk/mag/ind ... lique.html
with the same idea. Instead of a darkfield stop, Webster uses a condenser phase annulus but the idea is the same. I was not aware of that paper when I posted this thread, and I apologize for not citing his work at the time.

I was not aware of that paper when I posted this thread, and I apologize for not citing his work at the time.

You should be a little easier on gekko. Bit unfair to expect him to be all knowing! ;)

i.e. Don't be so hard on yourself.

Thank you, 75RR, but I was only giving credit where credit is due.

Hi Gekko,

I just stumbled upon this.. Your photos are superb!!... Some of the best darkfield illumination I have ever seen...

BillT

Thank you, BillT for your much-too-kind comment. I think what I've done was to use the easiest route to DF, low magnification and prepared slides. The really challenging DF is what Rod and others do, with 100x objectives!
Despite zzffnn's warning, I was trying this morning to get some darkfield and oblique darkfield images with my 40x/0.8-1.3 oil-immersion objective just as a challenge (I don't even dare try the 100x), but I haven't looked at the pictures yet to see if I have succeeded or not (hard to tell by looking through the eyepiece) I have acquired new respect for the difficulties involved in darkfield imaging with a darkfield condenser where you are working literally in the dark and see nothing until everything is adjusted (so easy to poke the slide with the objective or condenser). And that is not even with a 100x objective...

Well, I think you did a great job!...
BillT

Thank you, BillT. I will post my attempts at DF and Oblique DF using my 40x objective and hope to get some critical comments (positive or negative).

Those really stood out Gekko, nice work, trying to get my eyes adjusted now.

Rodney

Rodney wrote:Those really stood out Gekko, nice work, trying to get my eyes adjusted now.
Rodney

Thanks Rodney. And sorry if the images got your eyes out of adjustment .