Algae and filters

[Crater Eddie]

Brightfield (obviously) 10x objective



Brightfield 10x objective




Darkfield 10x objective




Darkfield 10x objective



Rheinberg 10x objective

The Rheinberg shot didn't work very well, but I thought it was an interesting comparison.

[75RR]

Interesting algae. What magnification was the objective that you used with Darkfield?
If it is a 25x or 40x do tell us how you managed it. I can't get past 16x.

[gekko]

Very nice. You might try different colors for the Rheinberg, as not all combinations work equally well esthetcially. I tried a different Rheinberg filter yesterday with some prepared slides and was not happy with the color combination. Darkfield is very nice, and may I suggest you try also polarization-- I suspect you will get lovely results with those algae, especially if you use some plastic as retarders or waveplates.

[JimT]

Like the first dark field image and agree with gecko that polarized light might also be neat.

[Crater Eddie]

These were all taken with the 10x objective. Sorry I forgot to add that in the descriptions, I'll edit it in later if I remember.
I printed out several simple Rheinberg filters on a sheet of clear film, this one was Green center stop with Red annulus. It just happened to be the first one I cut out of the sheet, so it's the one I used. It wasn't really suitable for this subject, but since it was my first Rheinberg shot I thought I would include it.
These darkfield shots are very challenging for me. They look great through the eyepiece, but I have difficulty adjusting my camera to get a good image. The second DF image above was amazing through the eyepiece. The bright areas that are overexposed in the image were stunning visually.
I do have some polarizing material, I just haven't played with it yet. So much to do in such a short time.
CE

[75RR]

These darkfield shots are very challenging for me. They look great through the eyepiece, but I have difficulty adjusting my camera to get a good image.

Are you using a tripod? DF images look slightly out of focus.

[gekko]

Interesting algae. What magnification was the objective that you used with Darkfield?
If it is a 25x or 40x do tell us how you managed it. I can't get past 16x.

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.

[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.

[Crater Eddie]

Are you using a tripod? DF images look slightly out of focus.

No, I am using the AmScope 3mp USB eyepiece camera in one port of my binocular head. I am only now experimenting with camera settings for DF imaging. I was focusing while observing the DF image on the screen and juggling camera adjustments. What I should have done was to focus first using the brightfield image, then switch over to DF but I didn't think of that at the time. Hopefully this will all be a little easier once I get the trinoc head (which was recently scanned in New York by the way!).
I believe that you have, or had, the 5mp version of this camera? I would welcome your suggestions as far as settings and usage.
CE

[75RR]

Hopefully this will all be a little easier once I get the trinoc head (which was recently scanned in New York by the way!).

Good news on the trinoc! Will get a lot easier (well more comfortable anyway).
What you should try to achieve is parfocality with the camera tube and the binocular - that way you focus through the eyepiece.

I believe that you have, or had, the 5mp version of this camera?

No, just an Olympus E-p2 which I use with legacy Olympus OM lenses.

[gekko]

If I may butt in, and I may well be wrong, but I believe what 75RR is describing as out of focus may be areas that are overexposed resulting in loss of detail, thus giving the impression of being out of focus.

[75RR]

If I may butt in, and I may well be wrong, but I believe what 75RR is describing as out of focus may be areas that are overexposed resulting in loss of detail, thus giving the impression of being out of focus.

Will split the difference: 50/50

[Crater Eddie]

Ah, I was thinking of someone else then, obviously.
You are both correct, the focus is off a bit and the images do suffer from... shall we say... less than optimum exposure. Also, it was a pretty deep field, so only a small part of it could be in focus at one time anyway. I need to work on keeping a thinner water film under the cover slip.
Achieving parfocality between the camera and the remaining eyepiece is a challenge. The nose of the camera fits into the eyepiece tube with merely a friction fit, and one must slide it in and out while watching the screen and the remaining eyepiece simultaneously trying to match the focus... another "less than optimum" situation.

[gekko]

Would it be possible to focus the microscope through the eyepiece at a prepared slide, or a stage micrometer, etc. and say to yourself, "this is the position that should also give an in-focus image by the camera", so, without readjusting the microscope focus, simply move the camera in or out of the other tube until the camera image is sharp (marking the exact position so you can fix it there)? Parfocality between eyepiece and camera is more than a convenience issue. If you have to move the microscope focus more than a very small distance (of the order of micrometers) to change the focus between eyepiece and camera, your camera image (but not visual image) will suffer from increased spherical aberration. The larger the focus discrepancy and the higher the NA of the objective in use, the worse the aberration becomes; such non-parfocality is tantamount to having the wrong tube length or wrong cover glass thickness as far as the camera us concerned.

[Crater Eddie]

I appreciate the suggestions.
I understand that this is what I should do, indeed I had planned to do just that not long ago. I have a piece of pipe the proper diameter to make a spacer, I had planned to shave it down on the lathe a few thousandths at a time until I achieved proper focus... then that trinoc popped up on Ebay and it all seemed too much trouble. Now I have preliminarily designed a couple of adapters to fit the AmScope camera to the trinoc, one to connect the AmScope relay lens directly to the trinoc, another to connect the camera to the LOMO relay lens bypassing the AmScope relay lens. We will see which works best. Of course the ultimate goal is to mount the Canon XTi camera to the trinoc via the LOMO relay lens, but that is another thread.
By the way, the trinoc was scanned again just moments ago as leaving the sorting facility in New York, so hopefully I'll have it in a few days.

[microgreen]

CE
Whatever - in my view the darkfield IS darkfield and the Rheinberg IS Rheinberg(the focus may be a tad out but they are still good images)
Tony
East Riding of Yorkshire(UK)

[Crater Eddie]

Thanks Tony, I appreciate that.

[75RR]

Whatever - in my view the darkfield IS darkfield and the Rheinberg IS Rheinberg(the focus may be a tad out but they are still good images)

I do not think anyone was criticizing Eddie's images as such. Constructive criticism if anything.

They look great through the eyepiece, but I have difficulty adjusting my camera to get a good image.

Just trying to help Eddie capture what he sees though the oculars.

[Crater Eddie]

All comments and suggestions are appreciated, I assure you.

By the way, I was making the focusing bit much harder than it needed to be. The camera, when fully seated into the occular tube, was pretty close to parfocal with the remaining (left) eyepiece, but not quite. So instead of fiddling with the camera, it finally occurred to me to simply adjust the diopter on the left occular.
Shazam! Parfocality!
I am quite embarrased that it took me so long to realize this so simple solution.

[75RR]

Shazam! Parfocality!

Personally, I love simple solutions, even if they are (in hindsight), very simple. Well done!

[gekko]

Great! So the discrepancy had not been that large anyway, it seems.

[Crater Eddie]

Getting back to the subject of the algae, take a look at these photos: 40x objective





See the tiny "hairs" growing out from the sides of the cell walls? Are these bacteria growing on the cell walls, or part of the algae itself? I know the focus isn't great here, I was trying to focus on the "hairs", not the cells.

[Crater Eddie]

I'll throw in this image just because I like it. 10x objective

[75RR]

I'll throw in this image just because I like it. 10x objective

I like it too. I have no idea about the "hairs"

[gekko]

Those are beautiful (both the algae and the images)! The last one may be an ostracod (subject to verification).

[75RR]

Getting back to the subject of the algae, take a look at these photos: 40x objective

Lots of interesting things inside that alga - have you tried stacking?
http://www.microbehunter.com/forum/phot ... -practice/

[Crater Eddie]

I am not sure stacking would work very well, as the contents of the cells move around quite a bit. Either cell circulation or Brownian motion, possibly a bit of both. I think this would interfere with the stacking. Some cells do not exhibit this motion though, so I guess it would work for them.
Thanks for the link.