Optimum Magnification Range

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75RR
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Optimum Magnification Range

#1 Post by 75RR » Wed Jul 01, 2015 5:14 am

This is a quote from Fundamentals of Light Microscopy by Douglas B. Murphy

"Oculars or eyepieces are needed to magnify and view the image produced by the objective.
To make optimal use of the resolution afforded by the objective, an overall magnification equal to 500–1000 times the NA of the objective lens is required.
More magnification than this gives “empty” magnification, and the image appears highly magnified but blurry.
For most applications, 10x eyepieces work well. When higher magnifications are required for a specific objective, a magnifying booster lens (in Zeiss microscopes, an Optovar lens magnification system) can be rotated into the optical path. Alternatively, a different set of higher-magnification eyepieces can be employed."


The reason I looked this up was that I thought that in making this video: viewtopic.php?f=6&t=1171 I might have overdone the magnification and entered into the realm of Empty Magnification.

The objective used was a Zeiss Neofluar 25x/0,60

Upper range = NA 0.60 x 1000 = 600x
Lower range = NA 0.60 x 500 = 300x

I used the objective in conjunction with the 2x of my Optovar (available magnifications are: 1x, 1.25x, 1.6x and 2x) and a 10x eyepiece.

So, 25 x 2 x 10 = 500x

It would seem therefor that I was within the optimum magnification range.

However, the use of Phase Contrast reduces the effective NA (see: http://www.microscopy-uk.org.uk/mag/ind ... -test.html), if the drop were to be to NA 0,50 for example, that would give an upper optimum magnification range of 500x, putting the video on the limit of Empty Magnification.
Similarly in Brighfield one also has to subtract NA due to the action of the Condenser Diaphragm, the use of which entails an inevitable compromise between NA and contrast.
One can see how reaching Empty Magnification under all types of illumination is not hard to do.

We are assuming of course that we are starting out with the full NA of the objective, this assumption rests on the condition of the microscope, the care taken in setting it up, the precision which which we have achieved Köhler and last but not least the condition of the objective.

What I did not know until I read up on this is that if one is below the optimum magnification range of the objective's NA, even though the full resolution and therefor the detail provided by the specific NA of the objective is there, it will not be apparent.
To return to the original example, using just a 10x eyepiece with the Zeiss Neofluar 25x/0,60 gives a total magnification of 250x, that is below the optimum magnification range of 300x - 600x for the objective's NA.
Last edited by 75RR on Wed Jul 01, 2015 2:57 pm, edited 2 times in total.
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Re: Optimum Magnification Range

#2 Post by JimT » Wed Jul 01, 2015 1:26 pm

Most interesting and informative. Thanks for the post.

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Re: Optimum Magnification Range

#3 Post by apochronaut » Wed Jul 01, 2015 2:25 pm

I think the important word in Murphy's text, is optimal. In theorizing about microscope optics, it is easy to forget about the resolution capability of the eye. This is where the lower limit of magnification comes into play. With an objective such as a 25X neofluar,with a potential N.A. of .60, dropping the magnification down to 250X doesn't lower the N.A., or resolving power of the objective, if other factors such as correct condenser adjustment are also factored in but it does lower the magnification sufficiently so, that some critical details would be below the resolving power of the eye. If that scenario is o.k., and 250X is the target then a 25X objective is not needed; something between 10X and 20X would suffice, which has the benefit of a longer working distance. For this reason, using more of an objectives potential magnifying power, by increasing the multiplying factor makes sense, or one could also maintain the status of the magnifying factor and change to a more powerful objective, which almost always raises the N.A. bar too . I don't know the Zeiss 25X NeoFluar well enough to know it's working distance but I have used a Zeiss 25X planapo on an Axiomat and own a Reichert 25X planapo , which I use frequently. Those both have such a small working distance that they have a guard collar surrounding the front lens so it is impossible to hit the coverslip, The Reichert 25X has a shorter working distance than it's cousin 40 X planfluorite..
At the other end, since your objective does have the built in specification so that it can be used as high as about 600X with limited loss in resolution, magnifying near to the theoretical potential as you noted, is only possible if all the other ducks are in a row. The condenser is the most wandering of those ducks.
It is the biggest limiting factor for most microscopes. There is a lot of attention paid to objective N.A. and type but almost none paid to condenser N.A. and type. You can't maximize the resolution of high N.A. fluorite and apochromat, even sophisticated achromat objectives, unless the condenser is giving it up. This almost always mandates something of above an abbe 1.25 category of condenser, kohler illuminated or not. Abbe 1.3, Abbe aspheric,achromat or achromatic aplanatic condensers will all provide an edge with which to reach the maximum N.A. of an objective.
Oiling. If a condenser is marked with an N.A. below 1.0, it is to be used unoiled but it's positioning is very critical in order to hit that mark. Condensers marked with an N.A. above 1.0 must be oiled to achieve that, otherwise they are in the .6 to .9 range, depending on the design and the N.A. as an oiled unit.
As a general rule you can knock off about .50 when no oil is used.

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Re: Optimum Magnification Range

#4 Post by 75RR » Wed Jul 01, 2015 3:05 pm

Hi apochronaut,
I don't know the Zeiss 25X NeoFluar well enough to know it's working distance ...
Working Distance of 0,54 mm. You make a good point it terms of the thought that should go into choosing a particular objective for a given task.
As a general rule you can knock off about .50 when no oil is used.
Does that mean that using a 100x/1,25 objective dry would give the equivalent of a 100/0,75 and if so is that a feasible/practical use of the objective?
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Re: Optimum Magnification Range

#5 Post by 75RR » Wed Jul 01, 2015 3:47 pm

Thanks JimT.
I am only now beginning to get around to reading all the books and articles I have bookmarked and never got around to.
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Re: Optimum Magnification Range

#6 Post by apochronaut » Wed Jul 01, 2015 4:40 pm

As a general rule you can knock off about .50 when no oil is used.
Does that mean that using a 100x/1,25 objective dry would give the equivalent of a 100/0,75 and if so is that a feasible/practical use of the objective?[/quote]
Somehow the response I had intended didn't get into the post.

All objectives are different. I would think some , work better at lower N.A.'s than intended. Here is a little test I did with several condensers, dry and oiled.. All the other factors are equal as much as I could keep them. I did this quickly, photographing afocally because the particular microscope I had that would accept all of the condensers does not have a photo set up, yet.
Objective is a 100X planachro oil immersion , photographed through a 19mm f.o.v. 10X eyepiece. Tungsten illumination.
Attachments
1.25 N.A. abbe dry
1.25 N.A. abbe dry
DSC00654 (1024x575).jpg (117.6 KiB) Viewed 5010 times
1.25 N.A. abbe aspheric dry
1.25 N.A. abbe aspheric dry
DSC00652 (1024x575).jpg (129.23 KiB) Viewed 5010 times
.90 N.A. achromat dry
.90 N.A. achromat dry
DSC00664 (1024x575).jpg (127.05 KiB) Viewed 5010 times
1.3 N.A. achromat dry
1.3 N.A. achromat dry
DSC00649 (1024x575).jpg (125.38 KiB) Viewed 5010 times
Last edited by apochronaut on Thu Jul 02, 2015 5:05 pm, edited 2 times in total.

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Re: Optimum Magnification Range

#7 Post by mrsonchus » Wed Jul 01, 2015 5:57 pm

Extremely interesting post - thanks for posting it. I'm going to have a good look at this subject as soon as I get time - it's obviously very important to get the priorities of all the various factors clear and to attempt to optimize them as much as possible with one's available kit.
:)
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Re: Optimum Magnification Range

#8 Post by apochronaut » Thu Jul 02, 2015 5:20 pm

Here are the same 4 condensers , all other parameters kept equal , except this time they are immersed.

The results of this simple test are pretty much what one would expect. The 1.25 abbe condenser induced the worst chromatic abberration, whether dry or immersed, however there wasn't as much of a difference in it's performance when immersed.
The .90 achromat , which is intended to be not immersed ,performed by far the best as a dry condenser but still didn't attain the level of performance an immersed achromat does.
Chromatic aberration is worse with all un-immersed condensers.
The immersed 1.3 achromat is noticeably the best, in resolution and freedom from chroma.
The 1.25 abbe noticeably the worst.

A big surprise to me is how good the 1.25 abbe aspheric is when immersed and how much immersing the .90 achromat downgraded it's performance
Attachments
1.25 abbe immersed
1.25 abbe immersed
DSC00659 (1024x575).jpg (128.52 KiB) Viewed 5008 times
1.25 abbe aspheric immersed
1.25 abbe aspheric immersed
DSC00658 (1024x575).jpg (111.99 KiB) Viewed 5008 times
.90 achromat immersed
.90 achromat immersed
DSC00657 (1024x575).jpg (122.42 KiB) Viewed 5008 times
1.3 N.A. achromat immersed
1.3 N.A. achromat immersed
DSC00656 (1024x575).jpg (113.95 KiB) Viewed 5008 times

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Re: Optimum Magnification Range

#9 Post by 75RR » Thu Jul 02, 2015 5:57 pm

Wow!
Thanks for all the work apochronaut.
I have gone from assuming that one could not use an oil objective dry to having to examine the images carefully to see the difference. Went so far as to drag the two 1.25 NA Abbe images on to the desktop and then join them in Photoshop in order to make a side by side comparison.
This would appear to establish dual use for oil objectives should circumstances require it.
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Re: Optimum Magnification Range

#10 Post by apochronaut » Thu Jul 02, 2015 6:59 pm

The one parameter I couldn't control was focus, so there may have to be some interpretation around that. The fine lines in the diatoms were very hard to define and there is obviously some drift from image to image to image but the bigger picture is still apparent. The abbe aspheric is a surprise. Very good as an oiled condenser, almost the equal of the achromat.
Hope this is some general help, though.

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Re: Optimum Magnification Range

#11 Post by Charles » Thu Jul 02, 2015 8:25 pm

To clarify your testing, the objective is oiled to the slide in all your shots and it was just the condenser which was not oiled or oiled?

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Re: Optimum Magnification Range

#12 Post by gekko » Thu Jul 02, 2015 8:30 pm

apochronaut: A very nice comparison: thank you!

75RR: No, I don't think you can use an oil-immersion objective dry and expect to get acceptable images (assuming that you can get the object in focus).

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Re: Optimum Magnification Range

#13 Post by 75RR » Thu Jul 02, 2015 8:54 pm

75RR: No, I don't think you can use an oil-immersion objective dry and expect to get acceptable images (assuming that you can get the object in focus).
I thought the test was to show that one could.
Wasn't it in response to the question below?
Does that mean that using a 100x/1,25 objective dry would give the equivalent of a 100/0,75 and if so is that a feasible/practical use of the objective?
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Re: Optimum Magnification Range

#14 Post by gekko » Thu Jul 02, 2015 9:39 pm

75RR wrote:
75RR: No, I don't think you can use an oil-immersion objective dry and expect to get acceptable images (assuming that you can get the object in focus).
I thought the test was to show that one could.
Wasn't it in response to the question below?
Does that mean that using a 100x/1,25 objective dry would give the equivalent of a 100/0,75 and if so is that a feasible/practical use of the objective?
Well, I didn't really understand that part of the discussion :( I understood the test to be a comparison among different classes of condenser, both dry and oiled to the slide using identical conditions to the extent possible, including the same 100x oil-immersion planachromat objective (oil-immersed, of course).

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Re: Optimum Magnification Range

#15 Post by apochronaut » Thu Jul 02, 2015 10:09 pm

Charles wrote:To clarify your testing, the objective is oiled to the slide in all your shots and it was just the condenser which was not oiled or oiled?
Yes; only the oil or no oil between the condenser and slide was altered. Focus of the camera had to be corrected with each shot because I was using the eyepieces between shots. Acouple of times I accidentally nudged the stage controls, changing condensers , so there is a slight change in registration from frame to frame. The set up was fairly crude but the adjustment of the condensers was attended to to get the best image possible from each one in each case.

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Re: Optimum Magnification Range

#16 Post by apochronaut » Thu Jul 02, 2015 10:21 pm

gekko wrote:
75RR wrote:
75RR: No, I don't think you can use an oil-immersion objective dry and expect to get acceptable images (assuming that you can get the object in focus).
I thought the test was to show that one could.
Wasn't it in response to the question below?
Does that mean that using a 100x/1,25 objective dry would give the equivalent of a 100/0,75 and if so is that a feasible/practical use of the objective?
Well, I didn't really understand that part of the discussion :( I understood the test to be a comparison among different classes of condenser, both dry and oiled to the slide using identical conditions to the extent possible, including the same 100x oil-immersion planachromat objective (oil-immersed, of course).
That response was in reference, to my comment that you could knock off about .50 N.A. using a condenser dry, so I assumed 75RR's response to be about condensers. I see now that he was referring to oil immersion objectives( weren't you?). I did deviate from the objective discussion to ,how objectives are seriously impacted by condenser type and set up, so I thought it a good idea to illustrate that.
This was entirely about " having ducks in a row" when assessing an objective and that the N.A of an objective( as well as other corrections) can perform quite below specification with condensers that are poorly corrected.

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Re: Optimum Magnification Range

#17 Post by 75RR » Thu Jul 02, 2015 10:23 pm

Charles wrote:
To clarify your testing, the objective is oiled to the slide in all your shots and it was just the condenser which was not oiled or oiled?
Yes; only the oil or no oil between the condenser and slide was altered.
Glad that was clarified.
So in answer to my question: Does that mean that using a 100x/1,25 objective dry would give the equivalent of a 100/0,75 and if so is that a feasible/practical use of the objective? The answer is no, or should we say not tested.

Perhaps I should modify the question to: Can an oil objective be used dry and if so how would the result compare to its intended use i.e. wet?
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Re: Optimum Magnification Range

#18 Post by lorez » Thu Jul 02, 2015 11:53 pm

Perhaps I should modify the question to: Can an oil objective be used dry and if so how would the result compare to its intended use i.e. wet?
This is a test you can do for yourself. You will have to close the aperture a lot more than usual and focus the 100X a little bit closer (I think it's about .001") to the specimen than the 40X, assuming they are par focal under normal circumstances.

Then do it with the oil objective being used as designed.

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Re: Optimum Magnification Range

#19 Post by 75RR » Fri Jul 03, 2015 12:05 am

This is a test you can do for yourself.
Have been avoiding using oil objectives. Do not even have any oil.
I suppose I will have to give it a go eventually ... if only to confirm that it is messy and finicky.
... and focus the 100X a little bit closer (I think it's about .001") ...
Wiki says that is 0.0254 mm! Case closed!!
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Re: Optimum Magnification Range

#20 Post by lorez » Fri Jul 03, 2015 12:28 am

Well, I guess I could tell you the answer... the 100X objective will make an image, but it is not worth the extra magnification at the expense of everything else. A 60X dry objective would be a much better solution. This is an opinion based upon empirical evidence.

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Re: Optimum Magnification Range

#21 Post by 75RR » Fri Jul 03, 2015 12:39 am

lorez wrote:Well, I guess I could tell you the answer... the 100X objective will make an image, but it is not worth the extra magnification at the expense of everything else. A 60X dry objective would be a much better solution. This is an opinion based upon empirical evidence.

lorex
Many thanks.
I had been thinking of getting a 63x/0.9 Neofluar and boosting it with my Optovar's 1.25x Will start looking!
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Re: Optimum Magnification Range

#22 Post by Charles » Fri Jul 03, 2015 1:25 am

Here is a shot of a diatom I took a while ago with an AO Achro 100X 1.25 NA without oil. I should have taken a photo with oil, but...I didn't want to have to clean up the oily mess at that time. It is lacking in resolution but you can make out the structures and some detail. I need to revisit this and take some comparison photos.
Diatom with AO Achro 100X 1.25 NA No Oil
Diatom with AO Achro 100X 1.25 NA No Oil
AO 20 Diatoms 100X.jpg (82.28 KiB) Viewed 4947 times

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Re: Optimum Magnification Range

#23 Post by 75RR » Fri Jul 03, 2015 2:34 am

Nice closeup shot of a nice looking diatom.
I need to revisit this and take some comparison photos.
Comparison photos would indeed be interesting. Like the comment about the oily mess!
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Re: Optimum Magnification Range

#24 Post by apochronaut » Fri Jul 03, 2015 3:11 am

AO made a 100X .90 Cat. # 1867, dry planachro objective for use uncovered. It works well for quick smears that are being quickly assessed but with a cover it has a very blurry image which is about as good as their coverslip corrected 100x when used without oil. It's spherical aberration that is the trouble in both cases and a drop in N.A.
Lorez is correct, use a high dry objective configured for a cover slip. Some companies did make 80X dry objectives. I have a Hacker 80X .85( 160mm), so it is usable with 10X. B & L made an 80X .90 flat field fluorite but it is an industrial objective and has a large thread. Adapters to R.M.S. are available. It is a no cover objective for a 215mm tube. On the Balplan frame , which has an undisclosed tube length and an odd system of corrections , it has only a small amount of spherical aberration. The image at around 800X is usable and well corrected. Some of these high dry objectives are worth searching out. They usually go very cheap and are user friendly.

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