Nuts and Bolts of Darkfield

[75RR]

Having problems getting Darkfield with objectives above 16x i.e. 25x and 40x

[gekko]

If I remember correctly, your 16x is fluorite with a rather large NA. The 40x also has a rather large NA (0.65 or so). For darkfield (also Rheinberg), with the darkfield stop in place, the condenser needs to be able to provide a cone of light that will illuminate the object sufficiently to obtain an image. If your condenser has a numerical aperture greater then 1, then you could try oiling to condenser to the slide (but do not oil a condenser having a NA of less than 1). In the old days, manufacturers sometimes provided a cone that would be placed into the objective to reduce its NA for darkfield use. Modern objectives of high NA designed for darkfield have a built-in iris diaphragm to reduce its NA.

[moved from another thread]

[75RR]

If your condenser has a numerical aperture greater then 1...

Had not considered this. Could that be the problem?
My Abbe condenser is 0,9
Not sure about my Phase condenser probably 1,3 or 1,4
My objectives:
2.5x/0,08
6.3x/0,20
10x/0,25
16x/0,35
25x/0,60
40x/0,65

[gekko]

If your condenser has a numerical aperture greater then 1...

Had not considered this. Could that be the problem?
My Abbe condenser is 0,9
Not sure about my Phase condenser probably 1,3 or 1,4
My objectives:
2.5x/0,08
6.3x/0,20
10x/0,25
16x/0,35
25x/0,60
40x/0,65

My 2-cent's worth: Yes, if your Abbe condenser is 0.9, then I think you may not be able to use it for DF with your 25x or 40x objective. Is it marked "Abbe"? If not, it could be achromatic (Abbe is not corrected). I've not seen Abbe condensers with 0.9 apertures. If your phase condenser has a BF setting (I think they all do?) with NA of 1.25 or lager, then, if oiled to the slide, it may well work nicely with your 25x and 40x objectives.

[75RR]

I've not seen Abbe condensers with 0.9 apertures.

0,9 Zeiss Flip-Top

[The QCC]

Success with Darkfield stops depends primarily on the objective and a stop of the appropriate size.
Your condenser is NA= 0.9 with the top flipped out and NA=1.5 with it in. The flip out is NA=0.25.
The flip out top should be out for obj. <= 6x and in for obj. => 7x.
For example.
2.5x obj. needs a small stop of about 8mm - 11mm.
6.3X 10-14 mm
10x 16-18 mm
16X 18-20 mm
25x 20-22 mm
40X 22-24 mm
The above are guidelines, but I have had good results using stops of that size.
These are the stops I use for the B&L microscope.

P1030593.jpg (128.22 KiB) Viewed 7343 times

[gekko]

I may well be wrong, but my reading of the Zeiss condenser is that the NA is 0.9 with the top lens flipped in, and a much smaller NA (for very low power objectives as The QCC indicated) with it flipped out (Zeiss manual should say what the NA would be). But I don't see that it is designated as Abbe, and my guess is that it is optically corrected (achromat?) which an Abbe isn't. As I said, at least as far as I know, Abbe condensers seem to always have a nominal NA of 1.25 or so.

I take back what I said above: My 2-cent's worth: Yes, if your Abbe condenser is 0.9, then I think you may not be able to use it for DF with your 25x or 40x objective. The QCC (thank you) made me rethink as follows:

Yes, you may be able to get DF even with the 40x since the NA of the condenser is 0.9 and of the objective 0.65, but you have to select the diameter of the DF stop precisely (not much leeway in size or centering error-- if a little too large then not enough light will get to the object), but it would be much easier and less critical if you had an oil-immersion condenser with a wider cone of light. I can get a good DF image with my 40x, 0.7 NA objective using my phase condenser dry using its DF position, which, without oil, will have a maximum NA of about 0.9 to 0.95. In conclusion, I agree with The QCC that you probably can get DF images using your 0.9 NA condenser, but it will require careful determination of the stop size, and condenser height adjustment may also be rather critical to get an optimal image.

[75RR]

Thanks The QCC and gekko.

Just been rereading some old posts on my Phase condenser in the photomicrography.net forum.
It would seem that I would have to oil the slide to achieve Darkfield with my 25x and 40x.
Was hoping to avoid the messy stuff! Had thought/hoped it would be easier.

OK. It's a regular phase contrast condenser then. According to a later catalogue:
"Darkfield with oil-immerged front lens 1.4 in position D for objectives NA 0.65 - 1 with maximum slide thickness of 1.2 mm;
Darkfield with front lens 1.4 in position 3 for objective NA up to 0.32"

So for the lower magnification (10, 16) you can use the ring aperture "3", for higher magnification (40) you use D, for objective 100, you need an objective with iris or field stop to reduce the NA down to 1.0.

The nominal max with my 0,9 Flip Top would therefore seem to be 16x as well.
I have however from time to time managed to get something from the 25x with the Standard Junior but not consistently.
Hope the Standard RA will be more productive.
Will try with more accurate stops - 1mm diameter steps and careful condenser height adjustment and centering.

[einman]

I am guessing your 20X is a flourite or apo? the NA of 0.60 seems high for a regular objective. oops that is a 25X not 20X. My mistake. The general rule of thumb is dry condensers can be used for objectives having an na of 0.65 or less. If you wish to go above that you would be advised to obtain a cardioid or paraboloid condenser which usually requires oiling.

A very good article on making stops etc can be found here:

http://www.olympusmicro.com/primer/tech ... field.html

I have both types of condensers for my B&L's. I have a paraboloid for my Dynazoom/Dynoptics and a Cardioid for my Balpan. I will be setting the Balplan up soon. I have a planachromat 100X with iris for that scope for the purpose of turning down the NA as the highest recommended objective na with the cardioid condenser is 1.05.

Interestingly if you have a phase condenser and offset the annulus and objective you can get a darkfield effect. I did this quite by accident. start at 1:35. I made this using a phase condenser..looks more like darkfield doesn't it?

https://www.youtube.com/watch?v=NZp9VVugY9k

[75RR]

Have a NeoFluar Pol 25x/0,60 and recently picked up a Ph2 NeoFluar 25x/0,60

Interestingly if you have a phase condenser and offset the annulus...

Previous image is of my Phase condenser.

What objective were you using when you achieved the Darkfield effect at 1:35 ?

Nice video and Vorticella colony. Did you keep them long?

[einman]

Have a NeoFluar Pol 25x/0,60 and recently picked up a Ph2 NeoFluar 25x/0,60

Interestingly if you have a phase condenser and offset the annulus...

Previous image is of my Phase condenser.

What objective were you using when you achieved the Darkfield effect at 1:35 ?

Nice video and Vorticella colony. Did you keep them long?

Honestly I cant recall with any accuracy what combination I was using. I thought at the time I had the correct annulus and objective combination. Then I found that by not centering the annulus exactly beneath the objective the result was a "darkfield" effect. Try viewing in the eyepiece as you slowly change the annulus. I think the mag was 40X. I had just started making videos so these were my first. After video taping the Vorticella I returned them to a shallow pan I was using to maintain lake water. They were still alive up until the fire so about a year. Though I can't say it was the same colony as I added additional water from the lake regularly. I initially located the colony using a stereoscope. Extracted them and placed them onto a regular slide. At the time I had multiple projects going on ie. several colonies of ants, collembola, pond life, seed shrimp (ostracods), water fleas (daphnia).

[75RR]

I thought at the time I had the correct annulus and objective combination.

So it was a phase objective.
And either correct annulus and not centered correctly or wrong annulus correctly centered or wrong annulus and not centered correctly.
Will play around and see what I get.

[gekko]

What I think (for what it is worth), but I'm not sure if I'm addressing any of the discussion above: The difference between a phase condenser annulus and a darkfield stop of the same size as the phase annulus center is merely the size of the "open" area: one can close the condenser diaphragm when using the DF stop to obtain a "gap" or "annulus" the same size as the corresponding phase annulus. In fact, theoretically one can use such an arrangement with a phase objective instead of a phase condenser for phase contrast, but the challenge would be to get the size and centering exactly right, which, considering the size of the phase ring in the objective, is not trivial from a practical point of view. Where the center stop of a phase condenser annulus is the right size for the objective NA in use, one can easily use a phase condenser for DF (and the objective can be a regular or a phase objective-- makes no difference, but, of course, with a phase objective you will be using a higher-NA phase position for dark field). The disadvantage is that the relatively narrow width of the phase annulus allows less light to pass to form the DF image (which, therefore, will be somewhat darker).

[75RR]

Hi gekko,
I can achieve Darkfield with my 6.3x, my 10x and my 16x in Phase 3 position. Easy as pie.
In theory I understand it should be possible to achieve Darkfield with my 25x and my 40x in D position.
No luck so far. Will try with oil at some stage. Do not have any oil yet. Will try to obtain some.

[einman]

Yes Gekko,
that is exactly what I believe happens resulting in a "darkfield" image rather than a phase-contrast image. If you examine a darkfield stop and a phase annulus they are in reality the same basic design it is how you pair them to the objective etc.
That is what I believe happened in the video I had made. I just could not recall what the pairing was at the time. I was actually just fooling around at the time. I now record settings when making observations.