PZO PhZ question(s)

Smokedaddy · Post by **Smokedaddy** » Thu May 09, 2019 2:16 pm

Looking for some help for those PZO users that may know what my problem is and/or how to solve it. PLUS, what is PhZ is supposed to actually look like. So I've been trying to setup PhZ on my PZO Biolar and running into a few problems. I've reached out to a couple of other PZO individuals privately but haven't resolved the issue. I realized the stacked image of the Diatom below isn't spot on and needs some work but that isn't the problem. I can fix that in Photoshop. The PZO's stock slide in factory bulb illuminator isn't that great to begin with but that's what I have. I have had good results with it so I'm not complaining. PZO did make a PZO LH100, 12V 100W halogen lamphouse but I don't have that accessory. Anyway, in the 'very large' diatom image below you can see a dark shadow on the left side of the diatom which must be the annulus/phase ring. If I move the adjustment screws on the PhZ condenser and don't align the phase rings the spot moves, so that must be the issue. I centered the phase rings and moved the large diatom out of my camera's view so you can clearly see the dark spot on the camera screen. If I slide the PZO light source in or out, the spot stays there and is the same size but it's simply darker or lighter.

So I can easily live with this image. Once stacked properly and manipulated in Photoshop it would be fine.

Now the image below is a different story. Obviously it's not one large diatom that fills the viewing area like the previous image. You can clearly see what the issue is, meaning that dark spot. It's just a single shot image, no stacking and no Photoshoping.

It's also not clear to me HOW PhZ is supposed to actually look? Part A or part B?

Now below image ... here's what the dark spot (phase ring etc.) looks like if I move the stage and therefore move the diatom out of the field of view. Note the spot on the camera's screen. That's the dark area you see on the left size of the diatom in the first image I posted. I was able to get the color banding rings out of the image by placing a diffuser on top of the PZO field lens.

Below is a cell phone picture looking through the bertrand lens at the phase ring alignment. Just an example, I have it aligned perfectly for the images I've posted above.

Below is a picture with the phase rings not centered.

-JW:

JohnyM · Post by **JohnyM** » Thu May 09, 2019 11:39 pm

How is your polarizer set?

Smokedaddy · Post by **Smokedaddy** » Fri May 10, 2019 12:26 am

In that particular image or I should say images that I posted the polarizer was removed from the bottom of the condenser. I had the polarizer installed previously while experimenting but saw no real noticeable difference when rotated.

zed · Post by **zed** » Fri May 10, 2019 5:55 am

Do you still see this spot if you put your condenser into a Brightfield position?

It looks to me like you are picking up the edges of the condenser phase annulus - which can happen if your condenser is out of focus. I would try to align your transmitted path for Köhler illumination and then switch back to your phase setting to see what happens.

zzffnn · Post by **zzffnn** » Fri May 10, 2019 6:31 am

JW,

Good phase contrast should look closer to B in your photo#2.

I agree with Zed and am guessing that your condenser is slightly too high or too low.

If you return to photo#2 and adjust your condenser height (vertically) very slightly while observing through camera or eyepiece, do you see A goes away or expand?

Your bertrand lens (alignment) image still seems slightly off (in terms of condenser height). You can see alternating light/dark parts within both objective phase rings, which I am not sure is optimal (which should not reveal that much with single phase ring rigs). Also there seems to be some light spillage around phase rings (which may be changed by adjusting condenser height).

Smokedaddy · Post by **Smokedaddy** » Fri May 10, 2019 12:55 pm

zed wrote:Do you still see this spot if you put your condenser into a Brightfield position?

No

Ichthyophthirius · Post by **Ichthyophthirius** » Fri May 10, 2019 1:12 pm

Smokedaddy wrote:It's also not clear to me HOW PhZ is supposed to actually look? Part A or part B?

It should look like either of them.

B: negative phase contrast
A: positive phase contrast ("normal phase contrast)

With the system you're supposed to switch between positive and negative contrast by turning the rotatable polariser by 90 degrees. Normally it should be homogenous either A or B across the entire field.

The two rings diaphragms in the condenser are covered by polariser film. That film is often delaminated. Yours doesn't look good in the pictures. Can you confirm that they are OK?

Regards, Ichty

Smokedaddy · Post by **Smokedaddy** » Fri May 10, 2019 1:28 pm

I've been working on this for a few days now, long hours too (8 to 10 hours). I've had help off line as well but no solutions. I also can't seen to get consistent 'results' even staying with the same objective. Of course I've tried other magnifications as well, even the PhZ 100x. Yes I've tried raising and lowering the condenser slightly and haven't noticed any changes. Adding the polarizer on the bottom of the condenser, rotating it and even completely removing it. In some cases I haven't been able to perfectly center the phase rings at some magnifications using the adjustment screws. However I was able to rotate the condenser ever so slightly to accomplish the alignment at some magnifications.

I followed the procedure from a PZO PhZ document I have for Köhler illumination ...... set condenser to zero (BF),which is always done first. Close down the field lens and condenser aperture, center everything, open field lens to fill the view, open condenser etc., ... rotate condenser to the proper objective to be used, insert Bertrand lens, center phase rings, remove and insert objective.

A while ago I gave it another shot with a 10x PhZ objective. I know you're not supposed to place the Bertrand lens where I have it in the photo below but I simply wanted to see if the phase rings looked the same as they do via the eyepiece tube. It's difficult 'for me' holding the cell phone up to the Bertrand lens. I also realized I don't have the Bertrand in focus.

... and below is what the view looks like through the Bertrand lens when placed in the proper place, meaning the eyepiece tube.

... and below is what it looks like through the eyepiece with the smartphone (same setup).

... and lastly, what the camera screen looks like.

-JW:

Smokedaddy · Post by **Smokedaddy** » Fri May 10, 2019 1:31 pm

Early this morning I got the results shown below with the same procedure. I haven't been able to duplicate that. I am totally confused what's going on.

Ichthyophthirius · Post by **Ichthyophthirius** » Fri May 10, 2019 1:39 pm

Hi,

And what does it look like when you turn the polariser by 90 degrees?

Smokedaddy · Post by **Smokedaddy** » Fri May 10, 2019 1:41 pm

Ichthyophthirius wrote:
Smokedaddy wrote:It's also not clear to me HOW PhZ is supposed to actually look? Part A or part B?
It should look like either of them.

B: negative phase contrast
A: positive phase contrast ("normal phase contrast)

With the system you're supposed to switch between positive and negative contrast by turning the rotatable polariser by 90 degrees. Normally it should be homogenous either A or B across the entire field.

The two rings diaphragms in the condenser are covered by polariser film. That film is often delaminated. Yours doesn't look good in the pictures. Can you confirm that they are OK?

Regards, Ichty

Thanks ... I remember reading about the polariser in the document I have (rotating it to achieve negative and positive phase). When I saw no difference in the actual image I was even more confused.

So phase rings inside the objectives are fine, so you are saying the phase rings and/or polarizer film inside the condenser are history?

I will confirm and post images. I wonder if the film can be replaced by end-user.

-JW:

Ichthyophthirius · Post by **Ichthyophthirius** » Fri May 10, 2019 3:19 pm

Smokedaddy wrote:so you are saying the phase rings and/or polarizer film inside the condenser are history?

There should be polariser films over the light rings in the condenser. The two rings should come in and out alternately if you rotate the condenser in front of an LCD screen or pol filter (remove the front lens to see them better).

Regards, Ichty

Smokedaddy · Post by **Smokedaddy** » Fri May 10, 2019 4:52 pm

Ichthyophthirius wrote:Hi,

And what does it look like when you turn the polariser by 90 degrees?

Here's the setup and the video. This is the 20x slot. Not what I expected to see. I don't recall seeing anything like that in the Bertrand lens while viewing through it.

https://www.youtube.com/watch?v=au2uS_c ... e=youtu.be

-JW:

Ichthyophthirius · Post by **Ichthyophthirius** » Fri May 10, 2019 5:12 pm

Thanks

That looks OK to me. The pol filters seem to be working.

Now I don't have many more ideas what could be wrong. You said you set up Koehler illumination and you also tried to adjust the condenser focus without success.

The front lens and the condenser optics are OK? The front lens is NA 1.2 and should be used with immersion (normally oil but glycerol or destilled water will also work). See if that makes a difference. Water immersion helps with PZO's DIC condenser (KPI).

Your last photos with the PhZ 10 look good - that's about what my own PZO phase contrast looked like (PhA). Brown colour and strong halos are normal.

The images you took though the phase telescope - it looks like there is a little bit of direct light coming though on the edge. That would be a problem. All the direct light must go through the phase rings. Is your condenser fitting working alright (keeping the condenser perfectly horizontal)?

Are you using the condenser iris in any way?

Regards, Ichty

Smokedaddy · Post by **Smokedaddy** » Fri May 10, 2019 6:02 pm

Ichthyophthirius wrote:The front lens and the condenser optics are OK? The front lens is NA 1.2 and should be used with immersion (normally oil but glycerol or destilled water will also work). See if that makes a difference. Water immersion helps with PZO's DIC condenser (KPI).

The lens (a 20/0,4) looks fine to me including the phase ring. The Abbe(sp) lens looks fine too.

Are you serious about using oil etc.? Sure don't want to ruin anything.

Ichthyophthirius wrote:The images you took though the phase telescope - it looks like there is a little bit of direct light coming though on the edge. That would be a problem. All the direct light must go through the phase rings. Is your condenser fitting working alright (keeping the condenser perfectly horizontal)?

Are you using the condenser iris in any way?

The condenser seems to be seated fine to me. I have it pushed up flush with the bore that is in the stage. I have do idea where the direct light is coming from that you mentioned. I'm not an expert with all this and haven't even thought about where the light might be coming from. Once I do the Köhler illumination routine I open the condenser Iris all the way (wide open).

BTW, this is what my field lens opening looks like when I have it closed down all the way. I can't get it in perfect 'sharp' focus on the blade edges.

-JW:

PZO PhZ question(s)

PZO PhZ question(s)

Re: PZO PhZ question(s)

Re: PZO PhZ question(s)

Re: PZO PhZ question(s)