Am I on the right track to reasonable DIC performance?

enricosavazzi · Post by **enricosavazzi** » Tue May 29, 2018 2:53 am

I am setting up an Olympus BX50 with DIC which has replaced my preceding (and very little used) Zeiss microscope, in large part to keep me busy if I will retire soon as it seems. Since I have little experience with compound microscopes I would like comments/suggestions as to whether I am on the right track or the equipment is substantially misaligned/misused. I think I got right the basic alignments and image quality looks good to me, but I might be missing something obvious.

The following initial tests are from a water sample collected outdoors, not fixed and not stained, imaged with UPlanApo 40x 0.85 (air), U-DP40 condenser DIC prism, U-ANT analyzer DIC prism, direct projection on Micro 4/3 (Olympus E-M1), single image (no stacking) with standard 100W halogen illuminator and built-in daylight compensation filter. I cropped away most of the picture area and reduced to 25% of original size (the 20 Mpixel images contain plenty of empty magnification). The scope is still not set up for immersion and focus stacking, and this is as high as I can go with magnification for now.

The DIC results I am getting with a 10x U-ACH and U-DP10 prism, and a UPlanApo 20x and U-DP20 prism are overall quite similar to these. I also have the U-DP100 100x prism, but it does not seem to give very different results from the 40x prism with the UPlanApo 40x.

Also a question: The U-UCDB-2 condenser has individually centerable prisms/annuli, but I don't see any difference when operating the centering controls with DIC. Is the centering only used with phase annuli?

Pollen with ?diplococci (and air bubbles)

undetermined, not stained, adjusted for maximum DIC pseudo-relief. This was almost invisible in brightfield.

Pau · Post by **Pau** » Tue May 29, 2018 3:26 am

Congrats for your system!

U-ANT analyzer DIC prism

U-ANT is only an analyzer, not a prism, it could be mounted alone in an empty slider or over the U-DICT analyzer prism slider, please clarify if you actually do use an objective prism (U-DICT, U-DICTS....) under the analyzer

If you don't use an objective prism what you're getting is a kind of oblique illumination that someone in the forum called "voila"

Also a question: The U-UCDB-2 condenser has individually centerable prisms/annuli, but I don't see any difference when operating the centering controls with DIC. Is the centering only used with phase annuli?

Look at the prism with the condenser dismounted of the microscope while actuating the centering knobs. In my older Zeiss DIC they move and this does affect the image, but likely in yours it doesn't

To me the images look like DIC but with not high contrast.

enricosavazzi · Post by **enricosavazzi** » Tue May 29, 2018 4:07 am

Pau wrote:Congrats for your system!

U-ANT is only an analyzer, not a prism, it could be mounted alone in an empty slider or over the U-DICT analyzer prism slider, please clarify if you actually do use an objective prism (U-DICT, U-DICTS....) under the analyzer

Correct, the U-ANT is mounted atop a U-DICT.

Also a question: The U-UCDB-2 condenser has individually centerable prisms/annuli, but I don't see any difference when operating the centering controls with DIC. Is the centering only used with phase annuli?
Look at the prism with the condenser dismounted of the microscope while actuating the centering knobs. In my older Zeiss DIC they move and this does affect the image, but likely in yours it doesn't

Also correct, I cannot see any movement when operating the centering "screwdrivers", so they do not seem to engage the prism rings.

To me the images look like DIC but with not high contrast.

I guess I will start by dusting again all optics and realigning. The scope was delivered to me quite full of dust, and I cleaned all the optics I could easily see, but there may still be dust somewhere inside the trinocular head for example. I keep the scope covered, but dust is still collecting on the top lens of the condenser and the exit of the illuminator below the condenser, so I may need better dust protection. The no-name C adapter I am using as photo tube might also need flocking.

Pau · Post by **Pau** » Tue May 29, 2018 4:17 am

Are you able to get black background when playing with the U-DICT knob?
If not, check the polarizer position and the condenser prisms position

Also the condenser height could alter DIC contrast (and the slide and cover thickness and their quality...)

enricosavazzi · Post by **enricosavazzi** » Tue May 29, 2018 5:54 am

Pau wrote:Are you able to get black background when playing with the U-DICT knob?
If not, check the polarizer position and the condenser prisms position

Also the condenser height could alter DIC contrast (and the slide and cover thickness and their quality...)

Not completely black, but after cleaning and realigning I can get a dark gray (darker than the subjects), with the NE and SW edges of the FOV lighter that move in and out of the FOV when turning the U-DICT knob, which I assume is normal.

Below is what I get at 10x on a very poor Chinese slide of Daphnia from eBay, in BF and DIC (it looks like the mountant has dried out and crystallized in virtually all these slides over a time of 3-4 years, making them useless in DIC, although it is not very obvious in BF. The eye spot of the Daphnia is definitely black, so I assume that whatever is causing the loss of contrast in DIC is not in the optics above the analyzer but has something to do with the DIC system.

[/img]

PS - This is as dark a background as I can get at 10x with a slightly cleaner slide:

Pau · Post by **Pau** » Tue May 29, 2018 7:11 am

A very good subject to test DIC are cheek cells: transparent, difficult to see in BF, pretty big and all the time at hand

Your feather image shows a dark band, not a good uniformity background. Maybe the condenser prism doesn't match well the objective.

enricosavazzi · Post by **enricosavazzi** » Wed May 30, 2018 1:12 am

Pau wrote:A very good subject to test DIC are cheek cells: transparent, difficult to see in BF, pretty big and all the time at hand

Your feather image shows a dark band, not a good uniformity background. Maybe the condenser prism doesn't match well the objective.

I think you are right on spot. This objective is an UIS ACH 10x 0.25 infinity/-. I just succeeded in finding a PDF about UIS objectives, and the Ach series is only specified for brightfield and darkfield. With the UPlanApos 20x and 40x and the corresponding DIC prisms the gray band is almost as wide as the field of view.

I am looking at a UPlanFLN 10x 0.30 (from the UIS2 series) as a replacement, but I notice in a UIS2 brochure that there are no UPlanFLN, only UPLFLN. Are they the same thing?

With a phase telescope I can see something that looks like a coarse ground-glass diffuser (or a terrible amount of dust or minute oil droplets/molds) on an optical surface somewhere in the optical path with the 10x, 20x and 40x objectives. With the 4x objective I cannot reach this focus position with the phase telescope. I can see this "diffuser" with all DIC components, polarizer, analyzer, illumination filters and trinocular slider indifferently in or out of the optical path.

It is always the same particle pattern with all three objectives, albeit at different magnifications, and I see this "diffuser" move if I slightly move the top lens of the condenser, so it is somewhere in the condenser or below the condenser. With the phase telescope the "diffuser" is at the same focus position as the illumination filters mounted in the base, which may or may not mean it is physically near the filters.

Pau · Post by **Pau** » Wed May 30, 2018 4:12 am

I am looking at a UPlanFLN 10x 0.30 (from the UIS2 series) as a replacement, but I notice in a UIS2 brochure that there are no UPlanFLN, only UPLFLN. Are they the same thing?

I've emailed you a document with info about Oly DIC components compatibility.

With a phase telescope I can see something that looks like a coarse ground-glass diffuser (or a terrible amount of dust or minute oil droplets/molds) on an optical surface somewhere in the optical path with the 10x, 20x and 40x objectives. With the 4x objective I cannot reach this focus position with the phase telescope. I can see this "diffuser" with all DIC components, polarizer, analyzer, illumination filters and trinocular slider indifferently in or out of the optical path.

Try rotating the condenser, if it rotates it's placed at the condenser, if not it's at the illumination train before it, most likely a diffuser or dirt on a filter or lens surface.

With the UPlanApos 20x and 40x and the corresponding DIC prisms the gray band is almost as wide as the field of view.

If you're not able to get fully uniform background an useful tric is to rotate the camera to better match the good DIC zone, I do it usually as my DIC system is far from perfect

enricosavazzi · Post by **enricosavazzi** » Wed May 30, 2018 5:46 am

Pau wrote:
I am looking at a UPlanFLN 10x 0.30 (from the UIS2 series) as a replacement, but I notice in a UIS2 brochure that there are no UPlanFLN, only UPLFLN. Are they the same thing?
I've emailed you a document with info about Oly DIC components compatibility.

Thanks Pau. According to this document the UPLANFLN require different DIC prisms (U-DIC10 in this case) than the UPLANAPO and UPLANFL series (which use the DP10 prism I already have), so still a somewhat risky match, even though the UPLANFLN are designed for DIC.

With a phase telescope I can see something that looks like a coarse ground-glass diffuser (or a terrible amount of dust or minute oil droplets/molds) on an optical surface somewhere in the optical path with the 10x, 20x and 40x objectives. With the 4x objective I cannot reach this focus position with the phase telescope. I can see this "diffuser" with all DIC components, polarizer, analyzer, illumination filters and trinocular slider indifferently in or out of the optical path.
Try rotating the condenser, if it rotates it's placed at the condenser, if not it's at the illumination train before it, most likely a diffuser or dirt on a filter or lens surface.

Unfortunately my condenser does not rotate, only slides in and out. The "diffuser" moves somewhat when I slide the condenser out, but this may be caused by the optical axis shifting sideways rather than the "diffuser" moving with the condenser.

If you're not able to get fully uniform background an useful tric is to rotate the camera to better match the good DIC zone, I do it usually as my DIC system is far from perfect

This is entirely doable. A small problem I can anticipate is that the DIC "false 3D" illumination direction also rotates. In the BX50 as-is the false-3D effect makes it look like illumination comes in from the top left, which is the most common orientation for oblique illumination in scientific illustrations. Rotating the camera or trinocular head makes it look like light is coming directly from the top. A small tradeoff for getting a more uniform background without a substantially higher cost.