Problems with my Oblique Lighting

[Micro_UTRG]

Hi everyone.

I have been enjoying my Olympus BX50. The U-PCD is a very good condenser and with one wheel I can change between Brightfield and Darkfield, but I like my Oblique better and it is my most used technique. However, it works best with my 10x objective only, the 4x can be used, but only a small portion of the field will have the relief and glow. My 40x objective is my second most used objective and I would like to use the Oblique lighting with it, but the Oblique doesn´t work as well.

I add here the images with the 10x objective. All of them are straight out of the camera, except for a bit of cropping and equalizing brightness.

I start rotating the filter wheel from brightfield to whichever direction and the filter holder starts blocking a greater part of the incoming light.

N71_4563.jpg (32.18 KiB) Viewed 5308 times

N71_4564.jpg (35.71 KiB) Viewed 5308 times

N71_4565.jpg (36.05 KiB) Viewed 5308 times

N71_4566.jpg (37.61 KiB) Viewed 5308 times

N71_4567.jpg (41.45 KiB) Viewed 5308 times

During the first part, the most noticeable difference is the introduction of contrast and a bit of relief. The Diatom chain starts looking 3D.
You can also see a bit of chromatic aberration as fringing around the edges.

1/3

[Micro_UTRG]

2/3

Here is the second part of images with the 10x objective.

N71_4568.jpg (44.1 KiB) Viewed 5306 times

N71_4569.jpg (46.74 KiB) Viewed 5306 times

N71_4570.jpg (50.61 KiB) Viewed 5306 times

N71_4571.jpg (43.19 KiB) Viewed 5306 times

The 3D effect is even greater; the cell walls start glowing and the chromatic aberration is more noticeable. At some point it can be removed with Lightroom, but at the end it is just greater than the yellow orange colours and if I remove it the image looks bad. Nevertheless, there is a sweetspot and I generally stay there.

Beyond a certain point, where the blocking part can be seen in the frame, I basically get darkfield.

[Micro_UTRG]

3/3

These next images were made with my 40x objective.

N71_4572.jpg (42.04 KiB) Viewed 5303 times

N71_4573.jpg (43.24 KiB) Viewed 5303 times

N71_4574.jpg (45.37 KiB) Viewed 5303 times

N71_4575.jpg (48.8 KiB) Viewed 5303 times

N71_4576.jpg (57.09 KiB) Viewed 5303 times

This configuration offers much less flexibility. The relief effect can be noticed, but it is not nearly as pronounced, the cell also never starts glowing and I certainly don't reach darkfield.
Chromatic aberration is more noticeable without the relief and glow being equally visible. I also start to notice halos or double images forming.
The 100x objective also suffers from this, but the 40x objective images are good example of what happens with the 100x objective as well.

I have seen sample thickness is a very important factor and perhaps this quick experiment had a thick slide, but the idea remains. I haven't got much better results with thinner samples.
I have also tried oiling the condenser, changing the filter shapes, moving the condenser up, down, left , right, etc. Nothing seems to work.

At last the Questions:
What can be done to improve the image at higher magnifications and get the effects that the 10x obj. produces?
Higher NA condensers? Objectives?

Thanks for all of your help.

Kind regards
Uriel

[Greg Howald]

Your quest is a typical quest. All of us would love to achieve better dark field or oblique lighting at 40x, and with standard optics this is generally impossible. It is possible for me to achieve dark field at 100x because I have a 100x objective with a built in iris diaphragm allowing me to adjust the NA of the objective. Such objectives can most likely be purchased in 40x but are usually very expensive and hard to locate. The 100x objective I have has a cost of $400. I don't use it often and I sometimes wonder if it was worth the price paid for it.
I am assuming that you are trying to do this with a 40x objective and a 10x eyepiece resulting in a 400x image.
The easiest solution for 400x darkfield or oblique lighting at 400x is to use a 20x objective and 20x eyepieces.

The 20x objective seems to be a natural or ordinary threshold for dark field and oblique lighting using items placed in the condenser's filter holder. Those filters you use at 10x will often produce good results at 20x without great difficulty. Sometimes I have had to close the condenser diaphragm slightly to achieve desired results or you may have to make new filters for best performance, but that is the easiest solution I have found.

Others on the forum have vastly more experience with this problem than I do, and they may offer solutions that produce terrific results, with a good deal of technical input,, but with a 40x objective there is no easy way out and don't forget you wallet.

Good luck with it.
Greg

[Hobbyst46]

Uriel,
Although I never saw the U-PCD condenser in person, its specs as seen on the net suggest, that it should provide decent to excellent DF with typical 40X objectives of NAs of 0.6-0.8.
The posted diatom images seem to show non-optimized brightness of field.
I ignore for the sake of the argument the chromatic effects, diatoms are wonderful glass bodies that produce rich and variable light color effects. Oblique and DF of images diatoms are often colored like that.
I assume that the 10X eyepieces are used and would recommend to stick with them, rather than 20X-s.
The trick for Oblique is first to set up Kohler illumination, then rotate the condenser turret until satisfactory (highly subjective!) oblique is achieved. Then the iris can be also adjusted.
The trick for DF is to set up Kohler illumination and verify centration of the condenser. Then switch the turret to the DF position, open the field aperture, put a drop of immersion oil on the condenser's top lens, then slowly raise the condenser until it nearly touches the slide. Looking from the side at the stage and slide, you will see a sudden flash of light when contact via the oil drop is obtained. If necessary, optimize centration of the condenser.
For DF, always set the maximum brightness of the illuminator.
Remember that the NA is the determining factor, not the magnification.
If, as I see, the condenser's NA is 1.1 (or maybe 1.25), there is no chance to achieve true DF with a 100X objective, unless the latter includes an iris to reduce the NA, as commented above. You might achieve nice oblique ("COL") under the above conditions.

Edit: the DF position of the condenser does not need any additional filter or light-stop to yield DF.

[zzffnn]

It is a trial-and-error process at 40x objective and above, depending on lots of factors. Stop size / shape / position, sample thickness / purity shape, Kohler/ light train alignment all play roles.

You would want to look at objective back focal plane (remove eyepiece and peek down the eye tube to see shadow shapes) and try different arrangements with a specific subject.

[Micro_UTRG]

The posted diatom images seem to show non-optimized brightness of field.

Thanks for your reply Hobbyst46.

That's caused by the way I'm taking the photographs.

I used to do an afocal projection onto my DSLR with a 50mm lens, but I kept seeing tons of dust spots, so now I do a "direct" projection. I have a binocular head, so I take out the eyepieces, the lens of the camera and use a tripod to keep everything in place. That way, when I misalign the camera, the border of the eyepiece holder starts to show. Otherwise, I always center the condenser and keep an even illuminated field.

I am also very happy with the Darkfield at 40x, I just noted that with the 10x, I can achieve DF by going too far with the light blocking because the incoming rays illuminating the specimen are all coming from the side and not from the , but I cannot with the 40x obj.

Do you think Oblique is just too difficult with the 40x obj?
Would a Hoffmann Modulation Contrast, PC or DIC make more sense?

Thanks

[Hobbyst46]

...I am also very happy with the Darkfield at 40x, I just noted that with the 10x, I can achieve DF by going too far with the light blocking because the incoming rays illuminating the specimen are all coming from the side and not from the , but I cannot with the 40x obj.

Do you think Oblique is just too difficult with the 40x obj?
Would a Hoffmann Modulation Contrast, PC or DIC make more sense?

Sorry, but I am a bit confused.

Picture no 4571 is a fine DF at 10X.
One other way (out of several) to achieve DF at 10X is by using a non-phase contrast 10X objective and one of the higher-magnification phase positions of the condenser - say, Ph3 or Ph4. WITHOUT oil. It works fine on my microscope and turret condenser, should be possible on others too.

Pictures no. 4572 through 4576 are, IMHO, oblique, but under too low contrast illumination, so I suggested above setting up the illumination, in analogy with how I set up my Zeiss turret condensers and achieve acceptable results. Which of the pictures you posted was
supposed to be DF at 40x ?

Oblique is not difficult, but it is very sensitive to the fine details of the setup, and the results are more subjective than, say, phase contrast. Again, it should start with bright and uniform illumination of the field of view in brightfield, just before you tweak the condenser to reach oblique. The initial illumination requirements are mandatory (for good performance) in all modes of contrast. PC, DIC, whatever. AFAIK.

DIC might yield nicer views than oblique but the equipment is VERY expensive.
PC yields contrasty views but without relief, very different from oblique and DIC. PC is much less expensive than DIC though.

[DonSchaeffer]

The problem is narrow depth of field. Points more distant from the lens are out of focus. Try closing the condencer iris down a touch to increase depth of field.

[Rossf]

Hello Micro_UTRG -I’ve seen some really impressive results of oblique with quite low N/A inverted objectives (Leitz)-like 20x .30 and 40x .50 (!) I agree with Don -I think the lower depth of field of a standard 40x (.65 etc) makes darkfield and oblique a situation of diminishing returns-but don’t let that stop you experimenting! There are so many ways of achieving oblique and oblique adjacent techniques (VAC filters-radial oblique-gradient stick-all somewhere on this forum in detail)
Good luck
Regards ross

[Chas]

This discussion has been really useful for me, I hadn't previously considered what objectives give the effect and why !

I also tried direct projection using a camera body (without my usual lens-less adapter ) and for some reason I also put a set extension tubes on the front of the camera and mounted the camera on a tripod (not something I would ever do).
The really surprising thing is how little off-axis light gets into the camera ... I had an air gap of 8 inches between the microscope tube and the front of the extension tubes and whilst some light got in, it wasn't enough to (completely) blow out the image
So it is a way to get a bit of a sense of what a higher powered eyepiece might show.

As reference pic from the 20x objective; this is with the camera lens bashing against the 10x eyepiece:

Directly through 10x eyepiece.jpg (23.65 KiB) Viewed 4979 times

This is the photo taken with the 8 inch air gap, adjusted a bit for exposure :

Air gap (2).jpg (53.64 KiB) Viewed 4979 times

In my 'strange eyepiece' box I had a 10x projection lens, so I stuck that on the front ..it seemed to work too, but with a 4 inch air gap:

10x projection eyepiece.jpg (20.91 KiB) Viewed 4979 times

These pics dont have the strong 3d effect of your off-centered condenser (they are from twizzling the mirror) but I think that they show that 'over' magnifying the view of a 20x objective does not destroy the depth effect.

[DonSchaeffer]

I bought some light conducting cable where light comes out of the side of the cable. I threaded it through the filter holder off center. It puts a little off axis light from the main light source to the specimen, functioning almost like an off axis block filter.

[Chas]

I am guessing that the relief effect in the above pics comes from using divergent light (fully open condenser lowered below focus of an LED light panel).
-It seems easier to adjust the lighting with the concave side of the mirror than with the flat side.
One potential benefit compared to the results I have had from an off-centered condenser is that the views look like they might be stackable (they dont smear off to one side).

In the 'strange eyepiece' box was a Watson 20x ...on a monocular microscope, at least, the view is fabulous ..you are really in amongst the thing's innards.
Maybe just get one and pretend that your microscope came with only one eyepiece tube?!;-)

Don..what sort of 'light conducting cable' (electric powered?????)

[Hobbyst46]

-It seems easier to adjust the lighting with the concave side of the mirror than with the flat side.

Which mirror ?

[Chas]

Hobbyst46, sorry I should have explained a bit better,
I use either either a seperate illuminator (like this one https://www.quekett.org/resources/kohler) or an LED light panel... so the mirror I am talking about is the one underneath the condenser.
On quite a few microscopes with Kohler illumination built into the base there looks to be enough room to put a mirror above the existing illuminator.

Out of curiosity, why do you not like your 20x eyepieces?

[Hobbyst46]

Out of curiosity, why do you not like your 20x eyepieces?

1. My experience with 20x eyepieces is that they cover a relatively small part of the field of view
2. The ones I know have a low eyepoint, which makes it uncomfortable for eyeglass wearers
3. They are only valuable for low magnifications. Example: for the 40x0.65 objective and 20x eyepiece, 40*20=800, i.e. >650 (1000*0.65).
for the 25x0.45 objective and 20x eyepiece, 25*20=500, i.e. >450 (1000*0.45). The above means empty magnification.

[Chas]

1] Yes, it is a bit like being trapped in a broom cupboard with whatever you are looking at.
2] Definitely no spectacles .. the diameter of the glass on the top of the Watson x20 is ~5mm
I think that you are being a bit tight re. N.A.s of 20x objectives for the 20x 20x solution/ approach that was suggested.... I have 2 or 3 old 20x's, nothing special, they are 0.5.
However using the eyepiece does feel a bit like peering into a rat's eyeball .. I would hope that modern 20x 's would have a lot more glass on top!!

P.S. The LED panel-concave mirror images do seem to stack, not too badly :
(Baker x40, cropped):

Bakerx40 on Series IV stack.jpg (42.01 KiB) Viewed 4791 times