Dark field difficulties

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matt123
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Dark field difficulties

#1 Post by matt123 » Thu Dec 21, 2017 2:34 pm

Hello,

I'm relatively new to microscopy and am using an Amscope T490B compound microscope. I'm an experienced macro photographer (http://www.mattcolephotography.co.uk) and so my main interest is in photo microscopy but I've got a lot to learn when it comes to microscopy. I've been experimenting with different lighting techniques since getting my microscope.

One difficulty I'm having is creating a dark field that is truly dark. I'm using a glass filter with a black circular stopper made out of insulation tape which sits beneath the condenser and I've experimented with a variety of different sized stoppers. To keep things simple I've been mainly using the 10x objective. It seems that no matter what sized stopper I use I can never get the background to be darker than a dark gray colour. Yet I regularly see photos and videos taken using dark field in which the background is jet black. Is this jet black a result of post processing in Photoshop or should I be able to create a jet black background using the microscope alone?

My backgrounds are also full of specks and dots of dust and imperfections within the slides/cover slips etc but I realise that is a separate problem that's difficult to avoid.

I think I understand the basic theory behind dark field so my gray background obviously suggests the stop is not effectively blocking the light but no matter what I try I can't seem to solve it. Does anyone have any suggestions? Many thanks.

Matt

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mrsonchus
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Re: Dark field difficulties

#2 Post by mrsonchus » Thu Dec 21, 2017 3:45 pm

Hello Matt,
I've just tried darkfield too, and the result was quite good, with a jet-black background. Have a look at my post from yesterday here --> rough & ready darkfield

Here I used a ground-glass 'filter' in my condenser's filter-holder with the stop made from a piece of 'Blu-Tack' simply squashed to the right diameter (i.e. the stop) to block all direct light.

Field-iris full open, move the condenser up & down until two conditions are met;
1) the edges of the 'stop' whatever you use to make it, are in focus at the same time as the specimen is..
2) the stop is of a size that exactly (within reason of course) matches the field of view of the objective - that is to say blocks all light coming 'straight through' the condenser - the rest of the light will 'come from around' the stop and illuminate the specimen 'sideways' whilst the background will be black (as no light is getting through from below) - the so-called 'dark-field'.

Your too-light background is very likely due to the stop being insufficiently opaque and not stopping all light completely - the 'Blu-Tack' stops all light and is easy to improvise to the right size to get started with...

My first result seen in the post is with a x2.5 objective, I've yet to try it out with the others such as the x10 for starters...

John B. :)

p.s. welcome.
John B

matt123
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Re: Dark field difficulties

#3 Post by matt123 » Thu Dec 21, 2017 7:19 pm

Many thanks John and also for the welcome.

I was fairly confident that my stops were blocking the light but I've just tried using blu tack and I still have exactly the same problem - a grey background. The dark field effect is working i.e. the subject is illuminated by side lighting but I cannot get a black background.

I've varied the stop size and on each occasion the only point I get the dark field effect is when the condenser is in its highest position almost touching the underneath of the slide. The only exception is if I make the stop too large and then the subject is still in darkness even when the condenser is in its highest position. So I'm not actually sure how to do your points (1) and (2) above as there is only 1 point at which I get the dark field effect (and that is when the condenser is in its highest position).

I don't know what else to try or what I could be doing wrong...

Matt

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mrsonchus
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Re: Dark field difficulties

#4 Post by mrsonchus » Thu Dec 21, 2017 7:31 pm

Hi Matt, hmm, which objective are you using, a x4 or x10 maybe? A picture of your 'scope's stage and condenser may be useful. In the meantime I'll run through it again and take some images to try to give a far better idea of what I did - back hopefully later tonight with an update....

John B. :)
John B

matt123
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Re: Dark field difficulties

#5 Post by matt123 » Thu Dec 21, 2017 8:08 pm

thanks John. I have the same problem whether I use the 4x or the 10x objectives.

I'm not yet sure how you post photos but the following page on Amazon shows a number of images of my microscope including the stage and condenser

https://www.amazon.co.uk/AmScope-T490B- ... ords=t490b

Matt

MicroBob
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Re: Dark field difficulties

#6 Post by MicroBob » Thu Dec 21, 2017 8:45 pm

Hi Matt,
your grey background - does it occur together with a verly light object? The metering system of a camera is adjusted to give images of medium brightness. It tends to get exposure wrong in dark field microscopy.
Dark field enhances all dirt in the slide and in the light path. When you see a really clean dark field image it usually has been cleaned with an image editing program.
Does your microscope have a field diaphragm and is the lamp movable?

When you have an immersion condensor (n.A. over 1) you might try to put a drop of demineralized water between Condensor to lens and slide.

Getting really good micro images is not easy and first you have to know enough about microscopy itself. So don't let these difficulties disencourage you.

Bob

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Re: Dark field difficulties

#7 Post by apochronaut » Thu Dec 21, 2017 8:57 pm

The condenser does usually need to be at it's highest possible N.A. to get the best DF. Is there some reason why you don't want to use the condenser at it's highest elevation?

If the condenser is too low, you will need a very large stop in order to ensure that you are only getting light that originates outside the N.A. of the objective. If any light originates from an N.A. lower than that of the objective, it will cause a gray image.

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Re: Dark field difficulties

#8 Post by MicroBob » Thu Dec 21, 2017 9:09 pm

Many microscopes have a stop that limits the movement of the condensor. It might be adjusted a bit conservative so that you can't turn the condensor high enough. Normaly it should be able to just touch the slide.

matt123
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Re: Dark field difficulties

#9 Post by matt123 » Thu Dec 21, 2017 10:35 pm

thanks for the further help and advice.

Microbob - the grey background isn't an exposure issue unfortunately as I'm see it through the eyepieces as well as in the photo. I'm quite experienced in terms of photography and post processing so can create a darker background using the camera/software but I suspect I should be able to generate a darker background using the microscope alone. My microscope doesn't have a field diaphragm (only a diaphragm below the condenser) and the light isn't moveable. I won't let this discourage me. I must admit I'm already quite pleased with some of the images I've got so far.

apochronaut - it's not a problem that the condenser is always at its highest point but I mentioned it as it seemed to contrast with John's advice to move the condenser up and down until 2 conditions are met.

Thanks again. I'll keep experimenting.

Matt

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mrsonchus
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Re: Dark field difficulties

#10 Post by mrsonchus » Thu Dec 21, 2017 11:08 pm

Hi Matt, yes that's the way I managed to do it with my 'scope and a x2.5 objective, which isn't a common objective either.
I tried to 'do it' with my x10 objective and failed miserably - hence the lack of a follow-up image.... :oops:

I would definitely suggest that Apo's advice will be the more valuable to you as mine is very much geared to my particular setup whereas Apo's will be very sound advice 'from the ground up' as it were. My experience and knowledge of microscopy and in particular the methods re many types of 'scope comes nowhere near Apo's level of expertise, knowledge & experience.

It was very useful to me also to read the comment re the attainment of maximum n.a. (of the condenser) as I'm very new indeed to darkfield of any type also. For now though I'm really pleased with the x2.5 objective's result - I'll work-on from there too.

John B. :)
Last edited by mrsonchus on Thu Dec 21, 2017 11:34 pm, edited 1 time in total.
John B

apochronaut
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Re: Dark field difficulties

#11 Post by apochronaut » Thu Dec 21, 2017 11:17 pm

The stop is smaller , the farther from the objective it gets, so both the maximum and minimum N.A.'s of the condenser reduce, when compared to the same stop, raised higher. It sounds like you are getting some direct light that is creeping past the stop into the objective. Raising the stop, will effectively make it larger, relative to the aperture of the objective and ensure that the only light entering the system, is outside the aperture of the objective and thus ensuring a dark background.

There is also a requirement that the DF stop be absolutely centered, otherwise direct light can creep in from one side only and foul the works.

DF tends to work best, if you maximize the aperture of the condenser.

John; are you using an aux. condensr lens with the 2.5X and not with the 10X?

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mrsonchus
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Re: Dark field difficulties

#12 Post by mrsonchus » Thu Dec 21, 2017 11:33 pm

Hi Apo' I used the condenser with the top element swung-out for the x2.5, with the condenser well below it's maximum travel. The bottom lens of the 600 series is I think n.a. 0.25 or thereabouts.
I tried both with the top lens (1.25 n.a.) in and out with the x10 and with the condenser up in it's usual (almost max height) position as when focusing the field-iris leaves in the image plane with the specimen.

It really was a rushed job though and I'll take another look tomorrow with a far more careful and accurate approach and see what I can get with the x10. The x2.5 gives me the nice darkfield rather easily, unlike the x10 so far at least.

Thanks for the pointers, they really are useful snippets that help a lot.

John B. :)

p.s. perhaps I should try the 0.90 top-lens condenser with the x10 - I've both the 1.25 and the 0.90 versions to choose from.
John B

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Re: Dark field difficulties

#13 Post by JimT » Thu Dec 21, 2017 11:45 pm

Matt123, here is info about stop sizes based on the obj. being used. Hope this is a help.

Magnification Numerical
Aperture Stop Size (mm)
1X 0.03 25-30
2x 0.05 8-11
4X 0.10 8-14
10x 0.25 16-18
20X 0.40 18-20
20x 0.65 20-22
40X 0.65 22-24
I must admit I'm already quite pleased with some of the images I've got so far.
We'll be looking forward to seeing some.

JimT

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lorez
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Re: Dark field difficulties

#14 Post by lorez » Fri Dec 22, 2017 3:07 am

One thing that is often overlooked is the quality of the lenses in the system. I have conducted workshops where each participant provided their own microscope and even though the accessories being used were identical the results were not. All the technique in the world cannot produce a quality image from a "budget" microscope.

lorez

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Re: Dark field difficulties

#15 Post by Suphot » Fri Dec 22, 2017 9:05 am

Hi matt123

May I suggest you to test your set up in the Dark field like this;



- Cut a normal photocopy paper in the size of standard microscope slide (around 1 x 3 inch I think).

- Use your Prepared Microscope slide and set up your Dark field to the best result as you can. (Cross section of any wood stem is a good to try).

- Take Prepared Microscope slide out of the microscope stage, replace it with normal photocopy paper that you have cut.

- In this step, when you look at the microscope stage, you should see Bright dot of illumination from the condenser focus on photocopy paper. The focus of bright dot should be very small. When you look into microscope eyepieces, you can see the magnified image of photocopy paper. It will not show Dark field image because all of the photocopy paper was illuminated no dark background can be show.

- Now use your hand to move the photocopy paper out of the stage completely. Looking into eyepieces, your microscope should show complete black image. If it show grey image, take eyepiece out and looking into the microscope. You should see the back of microscope objective, it should be dark, no illumination from the condenser can be seen.

- If the back of Objective was illuminated, the Dark field can not be done. I hope you can see by yourself that where the light came from. You may see that the field stop is not large enough, or the field stop is not exactly center or adjusting condenser up or down. You may try to fixed this until the back of Objective lens is dark.

- Please note that, material that you use to make a Dark filed stop should be completely opaque. The illumination from condenser was very high intensity, I usually stack multiple sheet of black tape together to make it completely black.


I hope this may help


Suphot

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Re: Dark field difficulties

#16 Post by photomicro » Fri Dec 22, 2017 10:16 am

Isn't this model supposed to come with a dedicated dark-field condenser? Presumably this comes with ready made stops off the right size...

Not sure therefore why you are making your own.

Hobbyst46
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Re: Dark field difficulties

#17 Post by Hobbyst46 » Fri Dec 22, 2017 10:36 am

Hi Matt

You already got many expert advices that in total explain the optics. Let me add an example.

I use a Zeiss GFL with very good optics (original Zeiss and Olympus, they all yield tge same DF moreor less). I have a phase condenser. Zeiss instructs to use the Ph2 or Ph3 phase stops of the condenser for DF wth low mag objectives. So I do and get good DF with 6.3x-16x-25x. It is seldom pitch black, often very dark gray, but the diatoms are brightly illuminated and shine against the background. So do the dust particles of course.

Reading your post I took a CD plastic package box, you know it consists of a black plate and a transparent cover. Took it apart. The center of the black plate has three annular section openings that form a non-continuous annular "window". Surrounded by a "ridge" - a raised part, like a ring. I cut it out with a sharp knife. The improvised stop, diameter about 30mm, is shown below.

I placed it inside the filter holder that resides below the condenser. Focused on a diatom slide (home made and fairly poor quality). Created Kohler illumination with the 25x/0.45 objective. The condenser is near the slide, raised as far as it goes. Brightfield position. Then open the iris to maximum. Use strong illumination - I have a 10W LED lamp. Then lowered the condenser slowly until a DF is obtained - and the stop is fairly in focus. Again, not pitch black but dark.

I believe that there is nothing wrong with your optics and the insulation black band is also adequate. The mechanical and optical optimization is the challenge here and it is trial and error.

Incidentally, I think that AmScope markets DF condensers as well.

Happy Holidays.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Hobbyst46
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Re: Dark field difficulties

#18 Post by Hobbyst46 » Fri Dec 22, 2017 10:38 am

Sorry - here is the picture of my improvized darkfield stop.
Attachments
DF stop from CD box-1.jpg
DF stop from CD box-1.jpg (17.68 KiB) Viewed 3953 times
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

matt123
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Re: Dark field difficulties

#19 Post by matt123 » Fri Dec 22, 2017 10:49 am

many thanks for the further help.

Suphot - I will definitely try going through those steps to work out where the apparent light leak is coming from.

Amscope do produce a dark field condenser and I did actually purchase one. However, frustratingly it is slightly too wide to fit into the condenser ring. The condenser that the scope came with is already very tight and the DF condenser is very slightly wider. I got onto Amscope who apologised and said it was a result of a slight redesign! Hence why I've been making home made stops.

Matt

Hobbyst46
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Re: Dark field difficulties

#20 Post by Hobbyst46 » Fri Dec 22, 2017 10:53 am

Just to note: Of course the diameter of the active part of this piece is that inside the three windows, 11-12mm. A Common CD box. Opaque black plastic.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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Re: Dark field difficulties

#21 Post by Micro-Bob » Fri Dec 22, 2017 11:13 am

Hi Matt,
I think Suphots method will help to show here problems are.
Microscope illumination with low power objectives is often only improvised to get along with the standard condenser, so dark field methods will be different in this case.
If you are interested in the theory behind it you might habve a look at Köhler illumination. Köhler worked out a theory for setting up the microscope for optimized resolution and contrast. True Köhler illumination is not so often found in microscopes. In many cases there is a frosted lens or filter somewhere in the light path that makes setting up easier an the light more uniform, but giving away a little contrast.
You microscope is not made to offer this adjustability, but you may be able to see wheter there is room for optimisation. You could e.g. use a diy field diaphragm to close out unneeded light.
Your objectives are no plan apos for shure, but I think that contrast won't be their weak point. Whith many microscopes it is fairly easy to get a good image with the 4x and 10x objective. With the 40x and 100x it is more demanding, a good slide, good optics and an experienced user is needed.
So it is probably best to get it right for the lower magnifications first.

Bob

matt123
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Re: Dark field difficulties

#22 Post by matt123 » Fri Dec 22, 2017 11:37 am

thanks Bob. As you note, I'm not able to use Kohler illumination because of a lack of a field diaphragm but I have ordered a cheap iris diaphragm to see if I can improvise a little with the light source.

Last night I did try something that seemed to darken the background slightly. Although I don't have a field aperture I artificially reduced the diameter of my light source by placing a black ring of metal on the lamp which reduced the 'aperture' of the lamp to around 15-20mm. I then reduced the size of my blu tack stop accordingly. As I say, this did seem to help. I don't really see why it did help as I presumably should be able to use the full aperture of the lamp with a larger stop and create the same dark background. But I wonder if my lack of a field diaphragm (and my light source being too wide?) is part of the problem.

I have upgraded my 10x objective with an Amscope plan achromat but I must admit I can't see a great deal of difference, if any. It was something of an experiment but maybe I'd need to spend more to see an improvement. I'm very familiar with macro lenses for cameras where that is certainly true. However, I must say that I have been quite impressed with the quality of the Amscope objectives but that may reflect my lack of experience of different objectives.

Matt

Micro-Bob
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Re: Dark field difficulties

#23 Post by Micro-Bob » Fri Dec 22, 2017 1:33 pm

For dark field you want a hollow cone of light. The outer angle so that the back lens of the objective is fully used.
Your dark field spot has to fit to the objective, it is not influenced by the field diaphragm.
One part of setting up a Köhler illumination is focusing the condenser on the field iris and closing the field iris so it just moves outside the visible image circle. You might be able to do something like this with a diaphragm or cardboard stop on top of the light opening.

manu de hanoi
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Re: Dark field difficulties

#24 Post by manu de hanoi » Thu May 03, 2018 9:19 am

here are some last resort tips:
-lower the iso if you're on a digital camera
-make sure the iris, the stop, output of the condenser, objective are properly aligned (there are some tutorials and tips above)
-make sure you dont have ambiant light parasiting !
-a good way to debug darkfield is to insert a sheet of white paper instead of the slide, you should see the dark core and bright ring when moving the condenser up/down (and possibly light leaks), but you want them to be nearly merged for observation

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