Dark Field for Higher Magnifications

[JimT]

I use a filter with a dark spot about 3/4" (2 CM's) for the 4x and 10x obj's. Works great. Should I go larger or smaller for the40x obj? So far nothing has worked. Would appreciate suggestions. Can one use a dark spot filter on the 40x?

Thanks. JimT

[gekko]

I use a filter with a dark spot about 3/4" (2 CM's) for the 4x and 10x obj's. Works great. Should I go larger or smaller for the40x obj?

Larger.

In an attempt at answering the remaining part of your question, here are my (rather inordinately long) 2-cents' worth:

1. Important concepts that need to be kept in mind. (a) The objective aperture is a conjugate of the condenser aperture when the microscope (focus of objective and condenser) is adjusted correctly. Therefore, an image of the condenser iris diaphragm is formed at the back focal plane of the objective.
(b) For darkfield, the direct ("background", "zeroth order") light from the condenser is blocked by the darkfield stop, and so the image is formed by the light scattered by the object on a dark background.
(c) In order for the direct light to be blocked, the size of the darkfield stop must be such that its image at the back focal plane of the objective completely blocks the objective aperture.
(d) In order for a darkfield image to be formed with the darkfield stop (as described in c above) in place, the (hollow) cone of light provided by the condenser must be wide enough to illuminate the object while the direct light is blocked. In other words, the working numerical aperture (NA) of the condenser must be larger than that of the objective.

2. How to determine the size of a darkfield stop using a brute-force method: make a number of stops of different sizes and try them. In each case start with the condenser iris fully open, then close it for best image. For larger NA objectives (say, NA of 0.5 or larger), but only if you are using a condenser with aperture of 1.2 or larger, try using immersion oil between the condenser and the slide to increase the working NA of the condenser. Do not use oil on condensers that are not meant to be used with oil (some low NA of 0.9 or so, or large working distance condensers are not designed to be used with oil, and may get damaged if oil is allowed to seep into them); in any case using oil will not help with condensers of NA less than 1.0.

3. How to determine the size of a darkfield stop using the concepts I tried to describe in 1 above: With the microscope correctly adjusted (objective focused on an object, and condenser height adjusted properly), remove the eyepiece and, while looking down at the back focal plane of the objective, close the condenser iris diaphragm (it should be seen to be in focus) then open it only enough to just completely uncover the objective aperture, then keep it there. The condenser aperture is now set to equal the objective aperture. Without changing this setting, remove the condenser and measure the diameter of the iris diaphragm opening. This is the diameter needed for the darkfield stop for that particular objective. As a practical matter (to allow for light diffracted from the edge of the stop, for imperfect centering of the stop, and for the stop not being exactly in the plane of the condenser diaphragm), a stop with a very slightly larger diameter would probably be necessary. If the stop is too large (such that it blocks all light from reaching the object), then clearly an image will not be obtained.

4. If the condenser is not capable of providing a large enough cone of light to illuminate the object with the correct size darkfield stop in place, then either a larger NA condenser (but, even with oiling the condenser to the slide, the max NA for a condenser would be about 1.2 to 1.4), or a smaller aperture objective will be needed. Some older high NA objectives (40x and up) may be supplied with a cone that is placed into the back of the objective to reduce its NA and so allow their use with a darkfield stop. Some newer objectives are equipped with an iris diaphragm that serves the same purpose.

5. There are specialized darkfield condensers that can be used with high power objectives, but I don't know anything about those.

I hope that this is both reasonably correct and helpful rather than merely confused and confusing .

[microgreen]

Point 3 that gecko made is the best method by far, and the easiest.

If you'll forgive the pun - it's spot on

Tony
East Riding of Yorkshire(UK)

[75RR]

I tried gekko's recommended scientific approach (#3), ended up using the brute force method (# 2) :)
I would have thought that 20mm would be too large for the 10x and perhaps even for the 40x.

Link is for two pdf files (to be printed on acetate) that combined will give you a good range of Darkfield Stop sizes for 32mm filter holders.

viewtopic.php?f=12&t=134

[JimT]

Thanks to all. #3 made my head hurt I'll go with #2.

BTW, I rest the filter on the lamp. That way I can move the filter to ensure the dark spot is centered.

[The QCC]

Resting the darkfield stop on the lamp is not the place to put it.
The stop, to be effective, must be immediately below or in the condenser.

The following stop sizes are suggested by the Olympus microscope resource site.
See Table 1 - Approximate Field Stop Diameter Size

[gekko]

Resting the darkfield stop on the lamp is not the place to put it.
The stop, to be effective, must be immediately below or in the condenser.

I fully agree.

The following stop sizes are suggested by the Olympus microscope resource site.
See Table 1 - Approximate Field Stop Diameter Size

A great link: thank you! Much better and clearer than what I wrote (of course ) Yet I think that a basic understanding of the first part of what I wrote above (the concepts) would be helpful when using any kind of stop (darkfield, oblique, Rheinberg, etc.) I am sure that there are much better elucidations of those concepts somewhere (I'll try to post a link here if I come across any).

[JimT]

QCC and Gekko, thanks for all the good input and the site of stop sizes.

How does one position the stop in the exact center of the clear acetate? That has been my problem and that is why I went to resting the filter on the lamp. Works for the 4x and 10x but no matter what size, not too well for the 40x. I guess I will have to get more precise and use my dividers.

Thanks again. JimT

[gekko]

If you've not seen it, this thread might be helpful (what applies to Rheinberg filters applies, with obvious modifications, to darkfield stops):
viewtopic.php?f=5&t=57&hilit=stop
If you have a laser printer, printing them on laser printer transparency paper, as per Oliver's suggestion may be a good way to make them. Do you have a filter holder as part of the condenser housing below the condenser iris diaphragm? Usually that is the most convenient place close to the condenser aperture. And the stop or filter, if made to the right diameter, will be centered automatically in the holder. Or is what I'm saying not applicable to your microscope? (With mine, there is no good place to put a darkfield stop or Rheinberg filter when using the bright field condenser, but fortunately, my turret phase contrast condenser has a built-in DF stop that is coplanar with the condenser diaphragm, and it works well with objectives upto the 40x, NA=0.70).

[gekko]

I just found an excellent, extremely lucid, illustrated guide to making darkfield stops and Rheinberg filters (along the lines of what I tried to do above but done far better):
http://www.quekett.org/resources/making ... tion-discs
I will also post the link in the "Resources" section to make it easy to refer to.

[Crater Eddie]

I have been following this thread with interest. I went to Staple's office supply to get a packet of laser printer transparency acetate last weekend... $69 for a packet of 50 sheets. Yikes! I tried a sheet of regular clear sheets and it just smudged.

[The QCC]

Crater Eddir:
If you are not 110% committed to making your own dark stops, these are the stops I purchased on eBay.
Darkfield stops

And the Rheinberg stops.