Euglena (images added)

Images made through a microscope. All subject types.

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Pau
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Euglena (images added)

Post by Pau »

Euglena found in a dirty pond (ID species much appreciated).
EOS 7D with my hybrid DIC microscope

1. Video showing its light attraction. Leitz NPL Fluotar 40X 0.70
http://youtu.be/8s7mLePIDCA

2. Flattened under the coverslip. Zeiss Planapo 63X 1.4, more or less cropped:
Image

Image

Image

Hope you like it
Last edited by Pau on Sat Jan 04, 2014 1:08 pm, edited 1 time in total.
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arturoag75
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Post by arturoag75 »

Your plan apo 63x works very good, i'm looking for a nice one but it seems very hard to find!
nice job :)

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Post by Pau »

Thanks, Arturo

The Planapo 63 1.4 160/- is BTW a good objective but not an easy one: its working distance is extremely short, it just can focus under the coverglass, when trying to focus at more deep yo are pressing the coverglass with the objetive tip (I've displaced this way diatoms shells in permanent slides :( ). This is why I needed to let almost dry the poor Euglenas to picture them with this objective.
In plus it isn't fully parfocal with my other objectives, making it even more difficult to use. Not a very satisfactory experience. If I ever find a Leitz equivalent at a reasonable price I would be happy to sell it to you :)
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Post by arturoag75 »

Hi
Did you tried also certified coverslips n.1? I had same problems but with this size all is better.
I had in last 6 months two pl apo leitz 63x 1, 40 160mm and i sold them becouse images were not good ( some blue fringes around samples) ; this not with zeiss if it is in good optic conditions....
I suggest you to keep your zeiss 63x. :wink:
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Post by Cactusdave »

I agree with Arturo. High quality number 1 coverslips are essential to get the best (or even to reach focus on occasions, as you have found) out of this objective. Diatom fanatics of my acquaintance check their coverslips with a micrometer and even resort to thinner (no.0) coverslips at times, though these are very fragile. I'd be surprised if the same requirement wasn't true of the corresponding Leitz objective. I think all planapo objectives of magnification X10 and above need the best no.1 coverslips you can get hold of. Marienfeld are the best I have found, but I'm sure there are others as good.
Leitz Ortholux 1, Zeiss standard, Nikon Diaphot inverted, Canon photographic gear

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Post by Pau »

Thank you Arturo and Dave for your coverglass suggestion. I'm using Nahita covers marked 0.13-0.17 , no standard number marked, but this may be between 1 and 1.5 (am I right?). Those aren't expensive nor clean, just schooll stuff. I haven't a caliper to measure them.

The problem may be that dry objectives are corrected for 0.17 and one will need to know what objective to use before making the slide.

Arturo, I'm intrigued with your bad experience with Leitz Pl Apo 63X. I was thinking on them for two reasons: most of my objectives and eyepieces are from Leitz and because its front lens is much wider than the Zeiss one, I think that the working distance will be too (what matters is the entrance angle...).
Did you used Leitz eyepices?, The blue halos are longitudinal CA?...I will like to see some pictures taken with them (and I would like to test one but...)
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Post by Cactusdave »

I really do recommend getting some high quality no. 1 coverslips. If you can't find Marienfeld, Chance Propper and VWR are good. I used both in my professional microscopy days, and they are available on Ebay at reasonable prices. I honestly don't recommend generic Chinese coverslips for working with highly corrected, tiny working distance Planapo objectives. :wink:
Leitz Ortholux 1, Zeiss standard, Nikon Diaphot inverted, Canon photographic gear

arturoag75
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Post by arturoag75 »

Pau wrote:Arturo, I'm intrigued with your bad experience with Leitz Pl Apo 63X. I was thinking on them for two reasons: most of my objectives and eyepieces are from Leitz and because its front lens is much wider than the Zeiss one, I think that the working distance will be too (what matters is the entrance angle...).
Did you used Leitz eyepices?, The blue halos are longitudinal CA?...I will like to see some pictures taken with them (and I would like to test one but...)
Pau i noticed this problem in an old topic ..you can see here:
http://www.photomacrography.net/forum/v ... highlight=

below you find a pic in brightfield i got some time ago by using a leitz periplan eyepiece, but also with Zeiss kpl 10x 8x and projective nikon 2,5 i had same result....you can see disturbing fringes blu and Yellow. :x :x
I have also a nice Zeiss plapo40x 1.0 with absolutely no delaminations but it has also this problem; i have to close internal diafraghm a little to clean these fringes..
this is a little frustrating becouse lenses perfect in appearance, are really poor. :cry:
hope this help and i'll be free to hear other experiences about.
Arturo
Image

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Post by Pau »

Thanks again, Arturo, for all that info.
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Post by Ichthyophthirius »

The correct cover glass thickness should be No 1.5 (0.16-0.19 mm), not No 1 (0.13-0.16 mm). 0.17 mm is the reference cover glass thickness for all major manufacturers. That's why they also sell No 1.5H cover glasses (0.17 +/- 0.005 mm; actually for high NA immersion objectives; see below).

For dry objectives, any diversion from the 0.17 mm thickness results in spherical aberration. That aberration is worse for cover glass that are too thin, i.e. a thickness of -0.02 is much worse than a thickness +0.02 http://www.olympusmicro.com/primer/java ... spherical/ last figure.

The Zeiss Planapo 63/1.4 Oil is marked "160/-". The "-" does not mean "tolerant to variation of coverglass thickness from 0.17" but "tolerant to variation of coverglass thickness from 0.17 taking into account NA and magnification".

Modern oil immersion objectives don't use homogenous immersion any more. The refractive indices of cover glass, oil and front lens are all different. Therefore, cover glass thickness matters for high NA objectives, regardless of whether the object is embedded in water or resin.

For objects embedded in resin (like histological sections; diatoms), variations of No 1-1.5 cover glass thickness don't matter (range 0.13-0.19 is well tolerated) with objectives up to NA 1.3, as far as I know. For NA 1.4 objectives like the Planapo 63/1.4, the tolerance is only 0.17 +/- 0.01 mm for best results.

Using a No 1 coverglass (0.13-0.16) in order to increase the working distance is OK, but you have to keep in mind that this is a compromise. Sometimes a Planapo 40/1.0 or Planapo 100/1.3 will give better images for objects embedded in water.

If you want to investigate diatoms embedded in resin on antique slides, you could consider the old Leitz Pl Apo 100/1.32 170/0.17 objectives (the old 170mm, not the new 160mm) which have an incredible working distance of 0.24-0.25 mm (vs. 0.09 mm in Zeiss)!

Ichthy

Pau wrote: The Planapo 63 1.4 160/- is BTW a good objective but not an easy one.

Ichthyophthirius
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Post by Ichthyophthirius »

arturoag75 wrote:...you can see disturbing fringes blu and Yellow.
It's not necessarity a problem with the lens. The diatom sceleton also acts like a (silica) lens and can cause colour fringes that cannot be corrected by the objective. You can use green light and grayscale your image to get around that.

To get peace of mind, test the objective with an object that has zero thickness and is black and white, like an object micrometer. If you still see fringes, the problem could be: objective, tube length, eyepiece or camera adapation.

Regards,

Ichthy

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Post by Pau »

Ichthy, many thanks for your skilled comments,
Ichthyophthirius wrote:The correct cover glass thickness should be No 1.5 (0.16-0.19 mm), not No 1 (0.13-0.16 mm). 0.17 mm is the reference cover glass thickness for all major manufacturers. That's why they also sell No 1.5H cover glasses (0.17 +/- 0.005 mm; actually for high NA immersion objectives; see below).
-I didn't know about those 1.5H cover glasses
Ichthyophthirius wrote: For dry objectives, any diversion from the 0.17 mm thickness results in spherical aberration. That aberration is worse for cover glass that are too thin, i.e. a thickness of -0.02 is much worse than a thickness +0.02 http://www.olympusmicro.com/primer/java ... spherical/ last figure.
The Zeiss Planapo 63/1.4 Oil is marked "160/-". The "-" does not mean "tolerant to variation of coverglass thickness from 0.17" but "tolerant to variation of coverglass thickness from 0.17 taking into account NA and magnification".
Modern oil immersion objectives don't use homogenous immersion any more. The refractive indices of cover glass, oil and front lens are all different. Therefore, cover glass thickness matters for high NA objectives, regardless of whether the object is embedded in water or resin.
- Well, mine is an old 160 corrected objective and according to Zeiss literature from its age /- means to be used both with and without cover glass, so I assume that it is for homogeneus immersion ( but in the some booklet the 63/1.4 is marked as designed for 0.17 cover :?: )
Ichthyophthirius wrote:For objects embedded in resin (like histological sections; diatoms), variations of No 1-1.5 cover glass thickness don't matter (range 0.13-0.19 is well tolerated) with objectives up to NA 1.3, as far as I know. For NA 1.4 objectives like the Planapo 63/1.4, the tolerance is only 0.17 +/- 0.01 mm for best results.
0.01 is really small!, beyond the precision of most makers :shock:
Ichthyophthirius wrote:Using a No 1 coverglass (0.13-0.16) in order to increase the working distance is OK, but you have to keep in mind that this is a compromise. Sometimes a Planapo 40/1.0 or Planapo 100/1.3 will give better images for objects embedded in water.
I'll post Euglena images taken with the NPL Fluotar 50/1.00. They have good contrast and are easier to take, but not better resolution
Ichthyophthirius wrote:If you want to investigate diatoms embedded in resin on antique slides, you could consider the old Leitz Pl Apo 100/1.32 170/0.17 objectives (the old 170mm, not the new 160mm) which have an incredible working distance of 0.24-0.25 mm (vs. 0.09 mm in Zeiss)!
- I have one of these Pl Apo 100/1.32 170/0.17 but I can't get DIC with it. In fact I don't find it superior to the Leitz PL Fluotar 100/1.32 160/0.17 (again unable to get DIC in my system) :?:
Pau

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Post by Pau »

More images of the same Euglena rich sample. Some motion blur, as the algae were moving and the exposure time was long. Now I used a coverglass from a different maker and focusing was easier, but still with the Planapo touching the coverglass. Images only a bit cropped.
8X Zeiss KPL eyepiece with 0.25X camera lens adapter.

1. Leitz NPL Fluotar 50X 1.00 oel:
Image

2. Zeiss Planapo 63/1.4
Image

3. Togheter with the former specie, I found a lot of chloroplast free smaller forms. Zeiss Planapo 63/1.4
Image

Any help for species ID will be much appreciated
Pau

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Post by Ichthyophthirius »

Hi Pau, First of all, great photos! Amazing detail and contrast.
Pau wrote:
-I didn't know about those 1.5H cover glasses

- Well, mine is an old 160 corrected objective and according to Zeiss literature from its age /- means to be used both with and without cover glass, so I assume that it is for homogeneus immersion ( but in the some booklet the 63/1.4 is marked as designed for 0.17 cover

- I have one of these Pl Apo 100/1.32 170/0.17 but I can't get DIC with it. In fact I don't find it superior to the Leitz PL Fluotar 100/1.32 160/0.17 (again unable to get DIC in my system)


You can "make" the 1.5H coverglasses yourself using a micrometer gauge! :D http://en.wikipedia.org/wiki/Micrometer This was commonly done 100 years ago when variation in thickness was much higher than today.

Sadly I cannot get DIC with the Pl Apo 100/1.32 170/0.17 either (exit pupil too low?). But it's handy for invesigating antique slides (in brightfield) because of the long working distance.

The 63/1.4 is indeed designed for 0.17; the "-" should be read "taking into account the NA and magnification" which means that no modern biological NA 1.4 objective can be used with and without coverglass.

I wish I could find the paper that compared the effect of coverglass thickness on NA 1.3 and NA 1.4 objectives; maybe later.

It's important that there is an NA mismatch between coverglass (1.522) and oil (1.518). Homogenous immersion was abandoned more than 50 years ago; maybe it would be better to call it oil immersion, as there is a DIN/ISO standard for the oil. Modern oil immersions with NA >1.40 even have correction rings to compensate for coverglass thickness. NA 1.4 is right on the edge when it comes to thickness tolerance (as I said; no problems with NA 1.3 lenses).

This is from the Zeiss Publication that also includes the Planapo 63/1.4 (Optical Systems of the Microscope): "Objectives designed for homogeneous immersion may be used with covered or uncovered specimens. The cover-glass thickness is naturally of no importance with these systems. lt is different with present-day [1980s] immersion objectives, in which the principle of homogeneity has been abandoned. [...] While the slight degradation of the image thus produced may still be tolerable for routine work, the trouble of covering the specimen should definitely not be shunned in critical work where full use is made of the high performance of the objective."

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Post by Pau »

Thanks again, Ichthy

I have a PDF copy of that Zeiss booklet, but I didn't spotted the importance of the paragraph you quoted.
But I still find the info unclear: The Planapo 63/1.4 appears as designed for 0.17, but mine -and all I've seen- are marked 160/-, and seem to have trouble with the coverglass thickness and minute working distance combination.
Pau

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