Zeiss Neofluar 40x0.75 objectives: phase vs non-phase performance in BF, Oblique, DF

[Hobbyst46]

A recent collaborative inspection of the same species of Wakura Beds (Japan) diatom, in essentially identical mounts, by Microbob and myself, has pointed out a possible difference in performance between a Leitz 100X objective and a phase contrast Zeiss 100X objective (both 160mm TL). The Leitz appeared to be better. The exact reason for the difference was not identified, but it motivated the comparison of phase-contrast and "plain vanilla" objectives, especially when the objectives are otherwise identical to each other in specs. In addition, such comparison might contribute info about the performance level of phase contrast objectives under non-phase illuminations.

My only pair of objectives that meets this condition is the Zeiss 40X0.75 Neofluars. Both are optically clean. Both are 160/0.17. The older one bears the (serial?) number 4814106, is a Ph2 phase contrast and will be named "Ph2" here. The newer one bears Zeiss catalog number 460720, is non-phase and is named "plain" here. Zeiss often states that their objectives have been "recalculated" (relative to previous series or batches), so perhaps the plain is better than the Ph2, in some unknown respect.

I tested the two objectives, side by side, with Petroneis Humerosa marine diatoms (PetrHum), that I had collected from the Mediterranean Sea, cleaned with mild chemicals, isolated one by one and mounted in Pleurax. They are 60 um long. According to SEM images in the literature, stria density is 14/10um and Puncta along a line are spaced at 0.5 um intervals. Incidentally, this species is found along UK coast lines as well.

Illumination was cold LED light filtered through a KR-12 amber filter ("white light") and, in some tests, a green interference filter ("green light"). Brightfield (BF), oblique (OL), phase contrast (PC) and darkfield (DF) were used. DF was obtained with the Ultracondenser (oil). All images are cropped by the same factor.Please note that the exact focus point is not always the same for both objectives. So, my conclusions rest on the resolution of stria and puncta alone.
The test also covered diatom 2 (perhaps a Diploneis sp.) and diatom 3, of approximately the same length as PetrHum.

Image 1 is the PetrHum diatom under PC.

1- Ph2, PC, green light - PetrHum.JPG (81.95 KiB) Viewed 1838 times

Images 2 and 3 show that, the resolution of OL (right column, puncta are visible)) is better than that of BF with both objectives to about the same extent.

2- Ph2.jpg (73.08 KiB) Viewed 1838 times

3- Plain.jpg (68.74 KiB) Viewed 1838 times

Image 4 is B&W versions of OL panels of images 2 and 3, a more pleasant view.

4- Ph2 and Plain.jpg (63.21 KiB) Viewed 1838 times

[Hobbyst46]

(cont'd)
Images 5 and 6 yield the same results as images 2 and 3 above, for diatoms 2 and 3, respectively. In image 6, the stria under Oblique are visible, although poorly.

5- Ph2 and plain.jpg (93.16 KiB) Viewed 1837 times

6- Ph2 and plain.jpg (44.31 KiB) Viewed 1837 times

Image 7 shows, using PetrHum as example, that the two objectives give very similar darkfields.

7- Ph2 and plain.jpg (52.85 KiB) Viewed 1837 times

All comments are welcome.
Note: Clearly, the scattered/diffracted/transmitted colors of the diatom depend on the illumination mode (BF vs OL). The situation is complex, however. Recent studies show that diatoms can act like nano-scale lenses, that selectively concentrate red light (favored for photosynthesis) into the chloroplasts. Not just gems - smart gems!

[hans]

Interesting comparisons. It looks like there is contrast inversion of some features comparing between the plain/PH2 and BF/OL images of PetrHum. Any thoughts whether that would just be due to variation in focus point you mentioned, or could be related to phase ring in the PH2 objective? Are the images with the same objective but changing from BF to OL expected to have the same focus point, or were you refocusing for each individual image?

[MicroBob]

Hi Doron,
interesting comparison!
From the images I would say the Ph2 is not worse then the bright field, possibly even better. I have the phase version and like the phase image a lot.

I think the potential for usability of phase objectives in bright field might depend on the size of the phase ring. Ph2 is used for the 40:1 Neofluar with 0,75 aperture but also for weaker objectives, 25:1 achromat and 16:1 Neofluar I think. This probably means that phase contrast and usability in bright field will be different from stronger to weaker objectives. The biggest problem should be a phase ring in the outer area of the aperture, less so when it is located more towards the middle.
I'm a bit busy right now but I can try to add a comparison between 100:1 Plan Ph3 and bright field, lets see if I will find the time.

Bob

[Hobbyst46]

Interesting comparisons. It looks like there is contrast inversion of some features comparing between the plain/PH2 and BF/OL images of PetrHum. Any thoughts whether that would just be due to variation in focus point you mentioned, or could be related to phase ring in the PH2 objective? Are the images with the same objective but changing from BF to OL expected to have the same focus point, or were you refocusing for each individual image?

Thanks for the comment.
The two objectives are parfocal within a few micrometers. I usually tweak to optimize the focus.
The protocol was as follows:

1. Set brightfield. Focus on the diatom with the Ph2 objective. Acquire image.
2. Switch to plain objective. Refocus to optimize view. Acquire image.
3. Switch back to Ph2 objective.
4. Set oblique (rotate the condenser turret slightly) and refocus. Acquire image.
5. Return to BF without touching the focus, inspect image. Return to OL.
6. Switch to plain objective. Tweak and refocus to optimize view. Acquire image.
7. Repeat step 5.

Steps 5 and 7 verified the expected resolution improvement upon BF->OL.
I believe that this (expected) improvement demonstrates proper functioning of the microscope setup, especially the objective and focusing. Naturally, each focus point depends on the viewer. My aim was to compare between the two objectives where each of them is aligned and focused to the best judgment of the user. Besides the appearance or disappearance of the "secondary" structure features (dots or stria), there was no other change in the image as I lowered or raised the objective.

[Hobbyst46]

Hi Doron,
interesting comparison!
From the images I would say the Ph2 is not worse then the bright field, possibly even better. I have the phase version and like the phase image a lot.

I think the potential for usability of phase objectives in bright field might depend on the size of the phase ring. Ph2 is used for the 40:1 Neofluar with 0,75 aperture but also for weaker objectives, 25:1 achromat and 16:1 Neofluar I think. This probably means that phase contrast and usability in bright field will be different from stronger to weaker objectives. The biggest problem should be a phase ring in the outer area of the aperture, less so when it is located more towards the middle.
I'm a bit busy right now but I can try to add a comparison between 100:1 Plan Ph3 and bright field, lets see if I will find the time.

Bob

Thanks for the comment, Bob.
Yes, the Ph2 phase setting applies to the 25X and 16X as well. I have a 25x0.45 Ph2 achromat and a 25x0.45 Planachromat, so they are less the same than the 40x I tested. Likewise, I have a 16x0.40 Ph2 Neofluar (delaminated...) and a 16x0.32 Planachromat - again, different from each other in more than one feature. And most of my diatoms are too small for 25x and 16x anyway.

Even with the 40x objective, the 60um Petroneis is fairly small on the screen. I use the 10X digital zoom of the camera to focus on it.