Here is a response to questions by Johan and Jim. Darkfield microscopy is accomplished by using a special condenser. The condenser delivers a hollow cone of light with the tip of the cone focused on the specimen. The angle of the light rays is such that all of the light misses the microscope objective, thus causing a dark field of view. However, a specimen can refract or reflect some of the impinging light and divert it into the microscope objective. Therefore, objects in the specimen appear to be bright against a dark background.
With this type of specimen illumination it is possible to detect objects even when they are smaller than the resolution limit of the microscope. However, this does not mean that you can measure their size, at least not directly. A similar situation is known in astronomy. When you look at the stars at night with the naked eye, they appear as pin points of light. Because of their vast distance, the diameters of stars can't be resolved by the naked eye. In fact, even when one observes the stars with the most powerful telescopes on Earth, they still look like pin points of light because their diameters are too small to be resolved!!! Nevertheless, the stars are seen easily, we just can't see their shape or measure their diameters (although astronomers have special techniques to measure the diameter of stars). Here is an example that most of us have seen. Early during the day, or late in the day, when the sun shines through a window, it is easy to see dust floating in the air. These particles are much too small to be resolved by the naked eye, yet we can see them. The operating principle in this case is similar to the darkfield microscope. So the question is, can you measure a smaller particle size with darkfield illumination than brightfield? Perhaps you could push the theoretical resolution limit slightly with darkfield. Suppose you could measure a particle with a 0.1 micron diameter using darkfield compared to 0.2 micron with brightfield. Will this help our research significantly? I don't think so but correct me if I am wrong. But there is one thought I had. Perhaps one could estimate the relative size of CS particles in a darkfield according to how bright they appear. If the amount of light diverted by a CS particle is proportional to its size, then larger particles should appear brighter in the field than smaller particles. To do darkfield work you need to pay attention to the numerical apertures of the condenser and objective. The numerical aperture should be stamped on the body of the condenser and the barrel of the objective. For example, an Abbe condenser (for brightfield work) typically has a numerical aperture of 1.25. This is a measure of the cone of light it will deliver to the specimen. The numerical aperture of the objective is a measure of the cone of light that it will accept from the specimen. A typical achromat objective of 100 power (oil immersion) will have a numerical aperture of 1.25, a high dry objective of 40 power will have a numerical aperture of about 0.65. You also need to have some understanding about the refractive indices of different materials. Air has a refractive index of 1.0, water 1.33, glass 1.5. If you want to use the full numerical aperture of an Abbe condenser, it is necessary to apply immersion oil between the condenser and the bottom of the specimen slide. Without the oil, you limit the numerical aperture to just below 1.0 because the refractive index of air is 1.0 (i.e., light rays of a greater angle will be reflected at the glass to air interface and not proceed into the specimen). My darkfield condenser has a numerical aperture of 1.33. The cutoff for the inner surface of the hollow cone of light it delivers is 1.2 (condenser is stamped with 1.33 - 1.2). Therefore, this condenser would be totally useless if it is not oiled to the slide (or one could use water since it has a refractive index the same as the NA of this condenser). If you don't oil this condenser to the bottom of the slide, all of the light from the condenser will be reflected off the glass-air interface and none will reach the specimen. Now if I want to use my high power objective with this condenser, I have a problem. Some of the light from my condenser will enter the objective because it has a higher NA (1.25) than the inner cutoff of the condenser (1.2). In order to overcome this problem, my objective is fitted with an adjustable iris (like in a 35 mm camera) which can be used to stop down the objective (i.e., reduce the NA). Then the iris is stopped down just until the field becomes dark. At this point I have maximum resolution with a dark background. What if you don't have an iris in your high power immersion objective? If it has a higher NA than the cutoff of your condenser you have a problem. You could try to fashion a stop to insert inside your objective but I would not recommend doing this. In this case you will probably have to settle for using your high dry objective (40 power). Remember that you will probably be looking at particles that are not resolvable with the immersion objective (100 power) so perhaps it does not matter so much if you use 40 power. The only problem will be that the lower NA of your 40 power objective will not gather as much light as a stopped down oil immersion objective and thus the particles will not be as bright. Now on to making a stop for converting your condenser to darkfield. You will have the best results if your condenser has a high numerical aperture, like an Abbe with 1.25. You will need to oil the condenser to the slide with immersion oil. Hopefully you have a filter holder attached to the condenser. If so, you can fashion a disc stop to fit the holder. This works somewhat in reverse of an iris in a lens, where you are stopping (closing) the outer portion of the lens to light. For darkfield you want to stop light from the inner portion of the condenser lens. This can be done by fitting an opaque disc centered on a clear glass disc which will fit the condenser filter holder. You will need to experiment with the diameter of the opaque disc until you find the minimum diameter that will still yield a dark field (i.e., if you make the opaque disc too small, light will enter your objective and the field will not be dark. Of course, the required diameter of the stop will be different for different objectives. If you have a condenser with NA 1.25, there is no use to try and use your oil objective with NA 1.25 unless you can stop it down. So you will need to develop a condenser stop for use with your high dry objective. If you can't obtain a clear glass filter to apply the opaque disc to, another solution is to make a wire frame that will fit into the filter holder. You could bend the wire into a circle and then bend the end to cross the diameter of the circle. Then glue the opaque stop to the part of the wire that crosses the diameter. The stop must be centered in the circle. Hope this information is useful. And please, if anyone finds errors in this explanation, let me know. I am a little rusty on these issues. Jeff La Favre -- The silver-list is a moderated forum for discussion of colloidal silver. To join or quit silver-list or silver-digest send an e-mail message to: silver-list-requ...@eskimo.com -or- silver-digest-requ...@eskimo.com with the word subscribe or unsubscribe in the subject: line. To post, address your message to: silver-list@eskimo.com List maintainer: Mike Devour <mdev...@id.net>
