Diatom photo test (was: Finally my old_new Olympus BH2 is refurbished)

[deBult]

New (CH2-TR45 Trino plus a rigid stand: Camera no longer coupled to the microscope)

Old (BH2-TR30 Tino): with the Camera directly coupled to te microscope.

[deBult]

Hello, Thank you for the feed-back (keep them coming please) The next version: using a separate stand for the camera: aim is to reduce the un-sharpness caused by the camera shutter.

Data:
* Olympus BH2-BHT stand, NFK 2.5 projective, Adapter L, SPlan Apo 20* na 0.7
* Olympus PL6 M4/3 camera, JPEG all auto settings, approx. 1/400 second
* Bright field (no phase)
* Picolay stack (7 pictures) with standard settings (I'm new to P.)
* White balance correction in LR6 and output sharpening set to "Screen".

Best, Maarten

[deBult]

Hello, Thank you for the feed-back (keep them coming please) The next version: using a separate stand for the camera: aim is to reduce the un-sharpness caused by the camera shutter.

Data:
* Olympus BH2-BHT stand, NFK 2.5 projective, Adapter L, SPlan Apo 20* na 0.7
* Olympus PL6 M4/3 camera, JPEG all auto settings, approx. 1/400 second
* Bright field (no phase)
* Picolay stack (7 pictures) with standard settings (I'm new to P.)
* White balance correction in LR6 and output sharpening set to "Screen".

Observation: I think the sharpness improved, so step by step we are getting somewhere.

Please feed-back on improvement options !!

Best, Maarten

[rhamvossen]

Hallo Maarten,

That's a big improvement and a world of difference from your first image. I am still curious how a single, unstacked image would look. You make one with the Splanapo and also the Splan? Best,

Rolf

[deBult]

I am still curious how a single, unstacked image would look. You make one with the Splanapo and also the Splan?
Best, Rolf

SPlan Apo 20* na 0.55
Splan 20* na 0.46
Splan 20* na 0.46 with IF550 green filter and converted to B&W (all channels)


Observation 1: Each change of objective required refocussing (they were on different exchangeable revolvers). So focus point is not identical. Depth of Filed is best at low na.
Plus each change of objective required tuning the condenser Köhler settings.

Observation 2: the one with the green filter looses sharpness: will need to check this as i only added the filter in the light path (and did not intentionally change focussing).

Feed Back appreciated,

Best Regards, Maarten

[rhamvossen]

Hallo Maarten,

The image from the Splan really looks good. There are some parts of the diatom that are not completely in focus yet but with some oblique illumination you will be able to get everything sharp in a single image, without stacking. The residual blue color (because it's an achromat) I don't find disturbing at all, on the contrary, it brings some liveliness in the image. Best,

Rolf

[deBult]

Rolf: thanks for feedback

As I'm using an older model BH2 Phase Contrast condenser (the first photo in this thread you commented on was a phase contrast one)  Oblique lightning is a bit complicated (I have a 3D printed insert I can try).

I'm working on a new batch of pictures: with NH and PLL phase contrast. Same diatom.

Best, Maarten

[deBult]

3D printed condenser oblique inserts used for the next experiment.

Olympus BH2-BHT, Phase Condenser, SPlan 20* objective, NFK 2.5 projective
Olympus E-PL6 m 4/3 camera on separate stand (to reduce shutter vibrations)

[deBult]

 1) Left oblique insert used
2) Right oblique insert used
3) Wedge used

Observation 1: the "wedge" inserts gives the best overal sharpness impression (but the previous shown stacked images have better overall sharpness)

Observations 2: I still cannot match Rolf's technique or his Leitz optics   

Feed back appreciated,

Best Maarten

[rhamvossen]

Hallo Maarten,

That second image is excellent. If you want more depht of field, you could try a more 'agressive' oblique illumination.

I still cannot match Rolf's technique or his Leitz optics

I don't agree, your second image is as good as mine.  And remember that I used a better corrected objective.

I have a BH2 myself (that I funnily enough rarely use.....) that also has a plan 20 on it. I will do some tests with it next week so that we can compare our experiments in a better way. The reason I used the image of Arachnoidiscus taken with the Leitz objective was because I already had it. Best,

Rolf

[deBult]

Thanks Rolf,

Eagerly awaiting your results.

Best, Maarten

[rhamvossen]

Hallo Maarten,

I did a few tests with my BH2 and a Splan20 objective. The projection eyepiece I have is unfortunately the NFK5xL, so with the OM photo adapter, the image gets too big. I improvised a bit to shorten the distance between the projective and the sensor of my Olympus PEN E-PL (first image). Only this way I could get a complete image of the Arachnoidiscus. I realise that this setup may not be optimal because the projective is calculated for a longer distance. The first image of Arachnoidiscus is taken this way. For the second image, I used a Sigma 30 mm lens as relais optics. With a projective, this is of course not needed but the image looks a bit sharper to me. Both images are single images taken in normal brightfield. I performed a whitebalance correction and a little contrast enhancement. Best,

Rolf

[deBult]

Thank you Rolf for doing this experiment

This sets a reference point: your entire diatom is sharp: so more depth of field (or a more flat specimen ?)

Do you have an idea why mine is not ? Tips to try out?

Our equipment is nearly identical?

Will give it another try this weekend using another scope, plus I'll use a test slide with defined flat surface (silver on a cover slide) kindly send to me by Wolfgang. The latter to validate I did not make an error when rebuilding this scope.

Best, Maarten

[rhamvossen]

Dag Maarten,

your entire diatom is sharp: so more depth of field (or a more flat specimen ?)

To really compare, we should have exactly the same specimen. I can sent you the slide, but don't break it If you agree, sent me your adress via personal message.

Do you have an idea why mine is not ? Tips to try out?

See above.

Our equipment is nearly identical?

Not identical but at least highly comparable. Major differences are the projection eyepiece (NFK 5x versus NFK2.5x) and the camera.

Best,

Rolf

[anne]

Hallo Maarten,
diese Species hat eine flache Seite und eine Seite die geformt ist wie eine flache Schale. Je nachdem welche Seite am Deckglas liegt musst Du mehr oder weniger "tief" vom Rand aus in die Mitte der Diatomee fokussieren. Auf Deinem Präparat könnte ich die Schalen mal von der einen und der anderen Seite gelegt haben. Vielleicht hilft das.
lg
Anne