1. A small diatom. Left: darkfield, right: oblique darkfield.
2. Same as 1 above, but image converted to grey scale.
3. Nymphaea of aqustio stem (center: DF; left: right side of light blocked; right, left side blocked).
Many thanks.
Attempts at DF and Oblique DF imaging using a DF condenser
Attempts at DF and Oblique DF imaging using a DF condenser
I have presented some images using oblique darkfield lighting using a darkfield stop earlier (viewtopic.php?f=28&t=3326). Here I attempted to do the same but using an old B&L darkfield condenser and a Nikon 40x/0.8-1.2 oil-immersion objective (the iris was set at about 1.0 to 1.1 NA). Both examples are from commercially prepared slides. Oblique DF was obtained by blocking about half the area illuminated using a black, opaque piece of paper immediately below the condenser. I would appreciate comments (did it work, and if so, worth pursuing?)
1. A small diatom. Left: darkfield, right: oblique darkfield.
2. Same as 1 above, but image converted to grey scale.
3. Nymphaea of aqustio stem (center: DF; left: right side of light blocked; right, left side blocked).
Many thanks.
1. A small diatom. Left: darkfield, right: oblique darkfield.
2. Same as 1 above, but image converted to grey scale.
3. Nymphaea of aqustio stem (center: DF; left: right side of light blocked; right, left side blocked).
Many thanks.
Re: Attempts at DF and Oblique DF imaging using a DF condenser
Well there is certainly a difference but speaking personally I am not quite sure what I am seeing.
Would it be possible to photograph the same diatom in brightfield and oblique in order to have a baseline?
Would it be possible to photograph the same diatom in brightfield and oblique in order to have a baseline?
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
Olympus E-P2 (Micro Four Thirds Camera)
Olympus E-P2 (Micro Four Thirds Camera)
Re: Attempts at DF and Oblique DF imaging using a DF condenser
As 75RR mentioned, it might be nice to have a brightfield "baseline" to get some bearings. But it certainly appears to to be providing better detail.
Re: Attempts at DF and Oblique DF imaging using a DF condenser
Interesting! Thank you for your work.
#1: grey scale seems to work. Oblique darkfield seems to have more depth, which makes sense. Without grey scale conversion, there is more color fringing with oblique darkfield.
Personally, most of my subjects have some colors, so unless color fringing can be addressed without grey scale conversion, it won't work well for my purpose.
#2 does not work well, due to uneven shadow. Shadows correspond exactly to blocked side.
The NA 0.65 limit that I mentioned is mostly for darkfield mask (and offsetting it thereof) by the way. If you use a wide cone darkfield condenser (and block some of its bottom light entrance), then major concerns are uneven shadow and color fringing. I could not tolerate those in my own application, but I guess someone else could, with careful sample selection and processing.
I guess narrow cone darkfield condenser or cardioid condenser would tolerate "oblique darkfield" less well than wider cone condenser or paraboloid condenser. For example, B&L paraboloid would work better than AO 214F cardioid.
#1: grey scale seems to work. Oblique darkfield seems to have more depth, which makes sense. Without grey scale conversion, there is more color fringing with oblique darkfield.
Personally, most of my subjects have some colors, so unless color fringing can be addressed without grey scale conversion, it won't work well for my purpose.
#2 does not work well, due to uneven shadow. Shadows correspond exactly to blocked side.
The NA 0.65 limit that I mentioned is mostly for darkfield mask (and offsetting it thereof) by the way. If you use a wide cone darkfield condenser (and block some of its bottom light entrance), then major concerns are uneven shadow and color fringing. I could not tolerate those in my own application, but I guess someone else could, with careful sample selection and processing.
I guess narrow cone darkfield condenser or cardioid condenser would tolerate "oblique darkfield" less well than wider cone condenser or paraboloid condenser. For example, B&L paraboloid would work better than AO 214F cardioid.
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apochronaut
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Re: Attempts at DF and Oblique DF imaging using a DF condenser
Oblique DF is a difficult term to use, since by it's nature DF is already has aspects of circular oblique illumination built in. In order for DF to function correctly it needs to be centered as much as possible, otherwise the resolution increase that would be expected is diminished by diffraction and likely unsymmetrical chromatic aberration.
Perhaps a better term would be Offset DF.
Looks like you have both of those happening in the offset image.
Perhaps a better term would be Offset DF.
Looks like you have both of those happening in the offset image.
Re: Attempts at DF and Oblique DF imaging using a DF condenser
75RR wrote:Well there is certainly a difference but speaking personally I am not quite sure what I am seeing.
Would it be possible to photograph the same diatom in brightfield and oblique in order to have a baseline?
c-krebs wrote:As 75RR mentioned, it might be nice to have a brightfield "baseline" to get some bearings. But it certainly appears to to be providing better detail.[/qyote]
75RR and Charles, thank you very much for your comments and suggestions. I tried to find the same diatom on the slide (strewn diatoms) to get a brightfield baseline, but couldn't really be sure which one it was. I tried to repeat the exercise with a similar diatom using brtightfield and darkfield, but the results were even worse than the images here. My purpose here was very limited: to see whether blocking the light from one side of the condenser would result in a shadowing (3D) effect. I would try to get a good DF image if only I could (I don't know how Rod, zzffnn, and apochronaut do it). Thank you again.
Re: Attempts at DF and Oblique DF imaging using a DF condenser
zzffnn, many thanks for your comments. I guess the blocking of shadows in #2 might have been due to the stain not being sufficiently transparent? The whole "project" was triggered by one of John B's darkfield images that showed very nice shadowing effects and I wanted to explore it a bit.
apochronaut, thank you for your comments. Your point about "oblique" illumination is well taken, but I think that this objection would also apply to regular brightfield (except with parallel light). You are exactly right about centering. I had a great deal of difficulty with that, and finally called it "centered" when the image did not shift laterally as I focused the microscope. I have a suspicion that there might be some alignment problem with the microscope illumination that is not corrected by simply centering and focusing the filament and centering the condenser. I also agree offset DF (and offset illumination for brightfield oblique) sounds like a good, accurate, and descriptive term.
apochronaut, thank you for your comments. Your point about "oblique" illumination is well taken, but I think that this objection would also apply to regular brightfield (except with parallel light). You are exactly right about centering. I had a great deal of difficulty with that, and finally called it "centered" when the image did not shift laterally as I focused the microscope. I have a suspicion that there might be some alignment problem with the microscope illumination that is not corrected by simply centering and focusing the filament and centering the condenser. I also agree offset DF (and offset illumination for brightfield oblique) sounds like a good, accurate, and descriptive term.