Darkfield - is resolution lost?

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Sauerkraut
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Darkfield - is resolution lost?

#1 Post by Sauerkraut » Sun Jun 30, 2019 5:26 pm

So I finally obtained the darkfield condenser specific for my scope. First observation is that the quality of workmanship for the dark patch is poor. You can see spots that are not completely black and there are a few places along the edges that are scalloped - like little bite marks - and therefore not dark. Not impressed.

But the bigger issue is that the depth of field of the pond sample specimens is considerably less than with bright field. So the sample will look mostly out of focus (reminds me of the loss of depth of field observed at higher power magnifications). Is this normal?

The condenser is 1.25 NA. I tried with and without oil (it works better with oil so I assume it is an oil condenser). I followed procedures to center the condenser and the darkfield itself looks quite nice, but have used it up to 20x power with poor visual results. Given that the workmanship quality of the condenser is poor, I'm wondering if it is just poorly designed and executed or if these losses are normal with darkfield. Or could I be overlooking something?

As always thanks for any insights.

Heather

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Re: Darkfield - is resolution lost?

#2 Post by PeteM » Sun Jun 30, 2019 5:55 pm

My understanding is that resolution is increased.

You'll likely have to spend more time preparing then sections, making sure the slides and most everything else are very clean, and may want some more light.

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Re: Darkfield - is resolution lost?

#3 Post by Hobbyst46 » Sun Jun 30, 2019 7:26 pm

Sauerkraut wrote:...
Welcome back.

One of the easiest static qualitative test samples for DF is air bubbles in a viscous liquid, say pure glycerin, or a dilute dolution of methyl cellulose in water, or a syrupy solution of sugar in water, or even nail polish. Tiny bubbles, in an otherwise clean liquid, spread normally as a thin layer on the slide under the coverslip, and shine brightly on a dark background if the DF setup is working. Static bubbles (in a hardened or very viscous mountant) are easy to see at 10X-40X.

In contrast to BF, the light seen in DF is mostly diffracted or scattered, and its intensity is very sensitive to the angle of illumination. Hence the alignment of the condenser is important and can be difficult. You might find the following link relevant:
viewtopic.php?f=5&t=7677
Good luck.
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Re: Darkfield - is resolution lost?

#4 Post by apochronaut » Sun Jun 30, 2019 10:44 pm

Could you post pictures of your condenser from a few angles. Some of the details you mention are a bit odd for a DF condenser, so I wonder about it's design.

One does not necessarily need a DF condenser "for their microscope", especially if the condenser mount is a sleeve type, that allows for the mounting of any condenser that fits the sleeve or can be adapted. Sometimes therefore, one can bypass the purchase of a new DF condenser that might not be the best from their microscope's supplier and opt instead for a superior quality used condenser from a better maker.

The two outstanding qualities that DF can contribute to microscopy are

1) enhanced resolution. This is typically only true with oil DF condensers of very high N.A., often 1.40 or higher.

2) increased depth of field. I have yet to figure out why this is the case but it is. Even using an oil immersion objective nominally rated for 1.32 N.A., I have had astounding depth of field using DF.

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Re: Darkfield - is resolution lost?

#5 Post by carlh6902 » Mon Jul 01, 2019 1:38 am

Darkfield is inherently lower resolution than brightfield, and also is not a faithful representation of the specimen image, due to the loss of the zero-order light in the fourier-reconstructed (via interference) final image.

At highest power, the objective NA must in practice be limited either by an internal iris or funnel stop, to exclude all of the zer-order light from the DF condenser. So even though the NA of the condenser can theoreticcaly reach perhaps 1.5 (unless the specimen is mounted in low refractive index medium, such as water), the overall NA is still limited.

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Last edited by carlh6902 on Mon Jul 01, 2019 2:09 am, edited 1 time in total.
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Re: Darkfield - is resolution lost?

#6 Post by zzffnn » Mon Jul 01, 2019 1:42 am

In my humble opinion, darkfield offers lower resolution (inside a structure like a diatom) and less DoF, but make it up with much better edge/border contrast than bright field, so you may perceive it as having higher resolution and deeper DoF. Some people like it and some don't. For hobbyists, it is up to the user.

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Re: Darkfield - is resolution lost?

#7 Post by PeteM » Mon Jul 01, 2019 6:29 am

So in this article: https://www.ruf.rice.edu/~bioslabs/meth ... opy/dfield.
. . . when the author says "Better resolution can be obtained using dark field as opposed to bright field viewing" what's meant is the greater contrast and edge definition??

And this case made for better resolution with oblique? https://micro.magnet.fsu.edu/primer/tec ... intro.html

My personal experience is that moving a darkfield annulus to one side (thus somewhat oblique) often gives at least a subjective appearance of greater resolution. I suppose there's a human factors element to this, since our vision systems are at least partially wired for edge detection.

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Re: Darkfield - is resolution lost?

#8 Post by MichaelG. » Mon Jul 01, 2019 6:44 am

PeteM wrote:[...] often gives at least a subjective appearance of greater resolution. I suppose there's a human factors element to this, since our vision systems are at least partially wired for edge detection.
I believe your analysis is spot-on, and nicely succinct.

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Re: Darkfield - is resolution lost?

#9 Post by Hobbyst46 » Mon Jul 01, 2019 9:12 am

MichaelG. wrote:
PeteM wrote:[...] often gives at least a subjective appearance of greater resolution. I suppose there's a human factors element to this, since our vision systems are at least partially wired for edge detection.
I believe your analysis is spot-on, and nicely succinct.MichaelG.
Seeing it from another angle (pun). Darkfield arises from higher order diffraction bands, hence it allows us to see objects that are smaller than the classical resolution of the microscope (Raleigh criterion). Tiny invisible details within the protist/diatom, become visible in DF, hence contrast and resolution are improved, relative to BF, at least theoretically.
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Re: Darkfield - is resolution lost?

#10 Post by MichaelG. » Mon Jul 01, 2019 11:57 am

Hobbyst46 wrote:... Tiny invisible details within the protist/diatom, become visible in DF, hence contrast and resolution are improved, relative to BF, at least theoretically.
I would be much more comfortable if you had written "contrast and visibility"
The problem being that some of those 'tiny invisible details' may not be real.

MichaelG.

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Edit: extract from https://micro.magnet.fsu.edu/primer/tec ... intro.html
Oblique illumination techniques are ideal for imaging a wide variety of unstained objects such as living cells, crystals, diatoms, and similar transparent or semi-transparent specimens. However, the resulting images must be viewed and interpreted with caution because the diffracted orders from one side have not contributed to the image formation. Potential false structures appearing in the image can seriously limit the usefulness of oblique light to examine and quantitatively describe previously unobserved specimen detail. This fact should always be taken into consideration when observing specimens with oblique illumination.
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Re: Darkfield - is resolution lost?

#11 Post by 75RR » Mon Jul 01, 2019 1:39 pm

... Potential false structures appearing in the image can seriously limit the usefulness of oblique light to examine and quantitatively describe previously unobserved specimen detail. This fact should always be taken into consideration when observing specimens with oblique illumination.
It would be interesting to know if this is a serious problem (i.e. invalidating observations) or merely a 'potential' one.
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Re: Darkfield - is resolution lost?

#12 Post by MichaelG. » Mon Jul 01, 2019 2:22 pm

75RR wrote: It would be interesting to know if this is a serious problem (i.e. invalidating observations) or merely a 'potential' one.
I think the fact that artificial/fictitious bas relief effects are frequently created by these techniques is probably sufficient proof.

MichaelG.
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Edit: stretching the topic a little ... this is worth a look:
http://www.microscopist.co.uk/wp-conten ... nfocal.pdf
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Re: Darkfield - is resolution lost?

#13 Post by 75RR » Mon Jul 01, 2019 2:34 pm

I think the fact that artificial/fictitious bas relief effects are frequently created by these techniques is probably sufficient proof.
I imagine that it would depend on how much of what one is viewing is fictitious, a little on the outer edge of a structure for example can be allowed for or even ignored.

It must also be pointed out the the amount of oblique applied can be varied.
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Re: Darkfield - is resolution lost?

#14 Post by apochronaut » Mon Jul 01, 2019 3:16 pm

In theory, neither DF or BF can have better resolution than the other, if one uses simple formula(e) to determine resolution. Microscopy however, is a group of applications of theory, all brought together to created an ultimate image realized in the viewer's brain. Everything, including the individual viewer's eyes and brain influence the quality of that image. So it is, that theory of resolution becomes the important factor in much writing about the topic, when in fact it is the perception of resolution that is the important factor........ and diatoms are not the only subject viewed through a microscope. Yes, they can be a valuable tool in the determination of resolution but they are much less valuable in a determination of contrast and not much value at all in a determination of condenser colour correction. Contrast, needs to be evaluated in very low contrast subjects and condenser colour correction with subjects that produce no intrinsic ca; something diatoms are not the best at providing.

Both BF and DF systems can be amended to ameliorate some of their less desirable attributes and therefore improve their perceived resolution. One of the great improvements in the case of BF, has been the enhancement of contrast, something that does little to enhance a DF image, since it's forte is in fact ultra contrast. What has enhanced DF the most is condenser colour correction. In this regard, mirror condensers such as oil immersion cardioids spew little ca, while the best commonly available condenser for BF is an achromat aplanat, which still has some defects in the area of ca, something that can reduce apparent resolution.

Transmitted illumination has an inherent problem of diffraction, something that DF eliminates partially, since the illumination source for DF is in fact tir. This is why a super critical factor in creating a fine DF image is the slide thickness and cleanliness of the system. By limiting the sample thickness, further control of diffraction can take place; allowing the natural contrast of the system to shine , so to speak.

Depth of Field. I don't know how a lens that has a partially closed iris diaphragm can have less depth of field than the same lens with the iris diaphragm wide open. DF has enhanced depth of field. I have witnessed the effect so many times, that I couldn't count them all. Even using a 1.32 N.A. objective perfectly stopped down to a black background, and a deliberate flow established in a teeming live aquatic sample, the depth of focus can be set to view organism after organism in a well focused vertical column, rolling and tumbling above a focused slide surface, crowded with a community of bottom dwellers. It is more like viewing a tiny aquarium, than a simple two dimensional pond sample and provides a unique window into behaviour, rather than just structure.

It is true that DF gives a somewhat skewed negative image but in doing so, it often provides imaging of structures that otherwise wouldn't be seen at all in BF. I often use both, as well as 3 or 4 types of phase, staining and oblique. Sometimes with DF, as with no other method, details all of a sudden are liberated.

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Re: Darkfield - is resolution lost?

#15 Post by Hobbyst46 » Mon Jul 01, 2019 3:27 pm

Just to note, that "even" DIC image is not a totally reliable representation of the true structure of the object. How I wish I owned such...
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Re: Darkfield - is resolution lost?

#16 Post by 75RR » Mon Jul 01, 2019 3:40 pm

Hobbyst46 wrote:Just to note, that "even" DIC image is not a totally reliable representation of the true structure of the object. How I wish I owned such...
I wonder if SEMs are more accurate ...
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Re: Darkfield - is resolution lost?

#17 Post by Sauerkraut » Mon Jul 01, 2019 3:46 pm

PeteM wrote:My understanding is that resolution is increased.

You'll likely have to spend more time preparing then sections, making sure the slides and most everything else are very clean, and may want some more light.
Thank you for this suggestion. This might at least be part of the issue as I didn't realized how important sample preparation might be. I had the rather romantic idea that a regular drop of pond water might yield amazing results with little thought or effort. Perhaps that is not so. As for more light. The 6V 30W halogen bulb is cranked all the way up. Do you use more light intensity than that?

Hobbyst46 has a good suggestion which is to create a sample of small bubbles that could at least be used as a baseline for comparisons. That might show me if there is lint on the coverslip, for example, or how the more uniform/controlled images compare - BF vs DF.

At this point I'm just trying to figure out if the condenser is ok and functional because it's in the return window. But ultimately I'll also need to figure out how to improve the images too.

Heather

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Re: Darkfield - is resolution lost?

#18 Post by Sauerkraut » Mon Jul 01, 2019 3:55 pm

apochronaut wrote:Could you post pictures of your condenser from a few angles. Some of the details you mention are a bit odd for a DF condenser, so I wonder about it's design.

One does not necessarily need a DF condenser "for their microscope", especially if the condenser mount is a sleeve type, that allows for the mounting of any condenser that fits the sleeve or can be adapted. Sometimes therefore, one can bypass the purchase of a new DF condenser that might not be the best from their microscope's supplier and opt instead for a superior quality used condenser from a better maker.

The two outstanding qualities that DF can contribute to microscopy are

1) enhanced resolution. This is typically only true with oil DF condensers of very high N.A., often 1.40 or higher.

2) increased depth of field. I have yet to figure out why this is the case but it is. Even using an oil immersion objective nominally rated for 1.32 N.A., I have had astounding depth of field using DF.
This is the condenser. On the inside bottom piece of glass is the black dot. And yes, $95 was steep for this but, well, sometimes we must take desperate measures for our hobbies:

https://www.microscope.com/accessories/ ... enser.html

I would like to try nicer condensers and shall keep an eye on eBay. The ones from Amscope didn't seem to work well and my attempts at using patches did not yield good results. I have a feeling that some of you are so advanced and skilled at microscopy that you could get amazing images out of the most terrible microscope, and that must also factor into your excellent DF results.

Heather

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Re: Darkfield - is resolution lost?

#19 Post by Sauerkraut » Mon Jul 01, 2019 4:03 pm

apochronaut wrote: Depth of Field. I don't know how a lens that has a partially closed iris diaphragm can have less depth of field than the same lens with the iris diaphragm wide open. DF has enhanced depth of field. I have witnessed the effect so many times, that I couldn't count them all. Even using a 1.32 N.A. objective perfectly stopped down to a black background, and a deliberate flow established in a teeming live aquatic sample, the depth of focus can be set to view organism after organism in a well focused vertical column, rolling and tumbling above a focused slide surface, crowded with a community of bottom dwellers. It is more like viewing a tiny aquarium, than a simple two dimensional pond sample and provides a unique window into behaviour, rather than just structure.
This sounds dreamy. I have a long way to go then. A very long way.

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Re: Darkfield - is resolution lost?

#20 Post by Hobbyst46 » Mon Jul 01, 2019 4:33 pm

Other easy test specimens, besides bubbles, I have found are the white fibers of pappus of certain flowers (Sonchus and similar family members). Just mount them in glycerin, liquid mount. Or single fibers of cotton wool fibers.

Place the slide on the stage and focus with the 10X objective, using the Abbe (or other brightfield) condenser. Adjust Kohler, centration etc. Lower the condenser rack, replace with DF condenser. Put a drop of immersion oil on top and raise the condenser. Look at the condenser, not through the eyepieces. When the condenser oil drop touches the slide and spreads, you will see a flash of light at the contact point. Then, start viewing through the eyepieces. Very gradually, adjust condenser height for best background.

If it works, shoud give good results up to the 40X0.65 (dry!) objective - at least.
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Re: Darkfield - is resolution lost?

#21 Post by apochronaut » Mon Jul 01, 2019 5:11 pm

Sauerkraut wrote:
apochronaut wrote:Could you post pictures of your condenser from a few angles. Some of the details you mention are a bit odd for a DF condenser, so I wonder about it's design.
This is the condenser. On the inside bottom piece of glass is the black dot. And yes, $95 was steep for this but, well, sometimes we must take desperate measures for our hobbies:

https://www.microscope.com/accessories/ ... enser.html

I would like to try nicer condensers and shall keep an eye on eBay. The ones from Amscope didn't seem to work well and my attempts at using patches did not yield good results. I have a feeling that some of you are so advanced and skilled at microscopy that you could get amazing images out of the most terrible microscope, and that must also factor into your excellent DF results.

Heather
The Omano condenser seems a bit odd. Usually DF condensers have a specification that covers two N.A.s, an upper limit and a lower limit. Some don't have an N.A. but give a measurement of the slide thickness range that is correct for the condenser. This is just another way of stating the N.A. range.
An example would be 1.2-1.4 What this means is that the illuminating disc covers an N.A. of 1.2 up to 1.4.
Your Omano, giving a BF type condenser version of the N.A. ( 1.25), kind of implies that it is simply a BF condenser that has a stop fashioned inside. Perhaps the maximum N.A. is 1.25 but you kind of need to know the lower limit too. Maybe that is the lower limit but both are usually given. It also states that it is a cardioid type. If this were true, you would see a small convex mirror up inside when you look from the bottom. If you see a stop, then it isn't a cardioid; it may be a paraboloid or I am thinking it is a more simple type with a stop installed.

Whatever it's type, in order for it to work properly, the objective you are using it with will need to be functioning at around .80 N.A. or lower, so your 20X should have worked well.

I would be inclined to return the condenser for a refund. You have a good case. It appears to have mfg. defects and it sounds like it has been falsely advertised. I cannot see that it is a cardioid.

You could then start over with a better condenser of known performance.
I will look around and see which inexpensive condensers have a 37mm sleeve.

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Re: Darkfield - is resolution lost?

#22 Post by Sauerkraut » Mon Jul 01, 2019 5:47 pm

apochronaut wrote:
Sauerkraut wrote:
apochronaut wrote:Could you post pictures of your condenser from a few angles. Some of the details you mention are a bit odd for a DF condenser, so I wonder about it's design.
This is the condenser. On the inside bottom piece of glass is the black dot. And yes, $95 was steep for this but, well, sometimes we must take desperate measures for our hobbies:

https://www.microscope.com/accessories/ ... enser.html

I would like to try nicer condensers and shall keep an eye on eBay. The ones from Amscope didn't seem to work well and my attempts at using patches did not yield good results. I have a feeling that some of you are so advanced and skilled at microscopy that you could get amazing images out of the most terrible microscope, and that must also factor into your excellent DF results.

Heather
The Omano condenser seems a bit odd...giving a BF type condenser version of the N.A. ( 1.25), kind of implies that it is simply a BF condenser that has a stop fashioned inside. Perhaps the maximum N.A. is 1.25 but you kind of need to know the lower limit too. Maybe that is the lower limit but both are usually given. It also states that it is a cardioid type. If this were true, you would see a small convex mirror up inside when you look from the bottom. If you see a stop, then it isn't a cardioid; it may be a paraboloid or I am thinking it is a more simple type with a stop installed.

Whatever it's type, in order for it to work properly, the objective you are using it with will need to be functioning at around .80 N.A. or lower, so your 20X should have worked well.

I would be inclined to return the condenser for a refund. You have a good case. It appears to have mfg. defects and it sounds like it has been falsely advertised. I cannot see that it is a cardioid.

You could then start over with a better condenser of known performance.
I will look around and see which inexpensive condensers have a 37mm sleeve.
I think you are onto something. It does look just like a regular condenser with a dark patch colored (with marker? Grrr) into the lower lens. There is no mirror. That said, the darkfield is quite decent 4x-20x, just with lower resolution than BF, tho I may also need to reconsider sample preparation, esp. where globs of algae are concerned. I finally have time today to play around with it a bit more extensively so am going to do some testing, but it's probably going back, if only, as you note, because of defects and false advertising. I did specifically purchase it thinking it was a cardioid even without fully understanding what that meant.

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Re: Darkfield - is resolution lost?

#23 Post by 75RR » Mon Jul 01, 2019 6:10 pm

I finally have time today to play around with it a bit more extensively so am going to do some testing, but it's probably going back, if only, as you note, because of defects and false advertising. I did specifically purchase it thinking it was a cardioid even without fully understanding what that meant.
Ad does say Cardioid - perhaps you can take some photos of the one you have?
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Re: Darkfield - is resolution lost?

#24 Post by Sauerkraut » Mon Jul 01, 2019 8:05 pm

75RR, I'm not even sure how to upload images here. I tried testing with photobucket but was not happy with the way it linked to the pics (with ads and other issues). Can I just upload them directly without a third party like Flickr or PB?

Also no real need to send a pic of the condenser. It looks just like the pic you included. From the underside, one can see that there is just lenses and a poorly colored black dot. Not acceptable at that price given what it is. I'm in the process of returning it and starting from scratch.

And thank you all for each response to this post. Much appreciated.

Heather

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Re: Darkfield - is resolution lost?

#25 Post by 75RR » Mon Jul 01, 2019 8:17 pm

Sauerkraut wrote: Can I just upload them directly without a third party like Flickr or PB?
There is a limit of 5 pictures per post, each picture can not be larger than 512KB and dimensions must not exceed 1024x1024. Just click on Browse (bottom of post) find image and click ok.
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Re: Darkfield - is resolution lost?

#26 Post by Sauerkraut » Mon Jul 01, 2019 8:30 pm

Thank you. Good to know.

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Re: Darkfield - is resolution lost?

#27 Post by Roldorf » Mon Jul 01, 2019 9:17 pm

You can also upload you pictures to Google Drive (15GB free) or Dropbox basic (2GB free) and provide the link to it in your post.
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Re: Darkfield - is resolution lost?

#28 Post by apochronaut » Mon Jul 01, 2019 11:27 pm

Heather, I had in mind that you had mentioned it was for a 37mm sleeve, somewhere. Is that correct? The bottom of the picture of the condenser looks a bit dovetailish but that is probably an artifact of the way the image was created.

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Re: Darkfield - is resolution lost?

#29 Post by Sauerkraut » Tue Jul 02, 2019 12:17 am

apochronaut wrote:Heather, I had in mind that you had mentioned it was for a 37mm sleeve, somewhere. Is that correct? The bottom of the picture of the condenser looks a bit dovetailish but that is probably an artifact of the way the image was created.
The skinniest part of the dovetail looks to be about 39mm so 37 would fit, and the fattest part of the dovetail (the base) is about 46mm. I can fabricate adapters out of wood and plastic also.

Thank you for your help.

Heather

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Re: Darkfield - is resolution lost?

#30 Post by abednego1995 » Tue Jul 02, 2019 1:10 am

carlh6902 wrote:Darkfield is inherently lower resolution than brightfield, and also is not a faithful representation of the specimen image, due to the loss of the zero-order light in the fourier-reconstructed (via interference) final image.

Carl
Partly agree, partly not.
Please correct me if I'm not understanding correctly...Doesn't higher order rays carry higher frequency information?
I think that COL for utmost resolution in diatom imaging is an implementation of this. (Yes, COL allows for the zero-order ray to enter the objective, but If you have at least the first order and second order rays entering as in DF, that will still form an image. Wouldn't the image represent finer structures that would be otherwise be drowned out in zero/n-order interference background? Again, this still won't break the Abbe resolution limit.)

Cheers,
John

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