Nikon Fluophot Test Shots

Images made through a microscope. All subject types.

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Mitch640
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Nikon Fluophot Test Shots

Post by Mitch640 »

The new scope is here, cleaned up, set up and working. Aside from it looking almost brand new, it does have some old lenses that came with it. It has a 6 hole nose turret with only 3 lenses delivered with it. I have put 3 other lenses I bought for the other scope in those holes as dust plugs. :)

So here are some shots I took today, using the same general editing of the RAW files as I have always used. A little crop where needed, resized to 900x600, some Levels Adjustment and a little sharpening to the color channels. I have not messed with the hue and saturation, to show the CA and Fringing. Camera used is the new Canon T1i, with a Nikon projector lens @ 10x, shooting tethered to my PC through EOS Utility.

A Rotifer, showing the Mastax...
Image

A Euglena or some kind of plant?
Image

These next three are the same subject, using all three of the Nikon lenses, to show their quality.

Some kind of plant filament... 4x Objective
Image

Some kind of plant filament... 10x Objective
Image

Some kind of plant filament... 40x Objective
Image
Last edited by Mitch640 on Fri Nov 19, 2010 6:32 pm, edited 2 times in total.

ChrisLilley
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Re: Nikon Fluophot Test Shots

Post by ChrisLilley »

Mitch640 wrote: So here are some shots I took today, using the same general editing of the RAW files as I have always used. A little crop where needed, resized to 900x600, some Levels Adjustment and a little sharpening to the color channels. I have not messed with the hue and saturation, to show the CA and Fringing.
I'm less familiar with Canon raw converters than with Nikon ones, but does your raw converter offer a way to scale the red and blue channels relative to the green, to compensate for radial CA?

I appreciate that you wanted to show how bad it was, and it may be possible to correct it optically, but I wondered to what extent it could be corrected in postprocessing as well.

Mitch640
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Post by Mitch640 »

Chris, I use CS4, so I can edit the RAW's in ACR [Adobe Camera RAW] and then convert them out to 16 bit tiff's. Those I can open in CS4 to correct any CA before saving out as jpg's. I do it that way cause CS4 has better controls than ACR for the color corretion. Or maybe I just know how to work CS4 better. :)

I do hope to be getting newer lenses soon and a better projector lens, so maybe the CA will go away. I'm hoping anyway. LOL

rjlittlefield
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Post by rjlittlefield »

Mitch, your images are showing at least two different types of color fringes.

One is due to the kind of CA that's correctable in Photoshop, where the lens essentially has different magnification for different colors. That results in the classic appearance where you get color fringes in directions that are radial with respect to the center of the image. In your third and fourth images, this is probably what's responsible for the red-inside, blue-outside fringes around the black blobs at upper right.

The other type is seen in your first image, the rotifer. Notice in that case that obvious fringes are radial with respect to the subject and not the image. I've never been quite sure what causes those, but certainly it's not correctable in Photoshop.

If it'll make you feel any better, thick subjects like these are among the hardest things to get clean pictures of. Don't stop trying, though!

--Rik

Franz Neidl
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Post by Franz Neidl »

Hello Mitch,

I see you know a lot of computer (Photoshop, RAW ecc.), but from my own experience I know that for a beginner in microscopy it would be better to have a book (with practical exercises) as introduction into microscopy - with subjects for example: what is a Köhler illumination, the handling of specimens, use of the field iris diaphragma, use of the aperture iris diaphragma, ecc.
Do you have a good book? Computer is not so important...

Franz

Mitch640
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Post by Mitch640 »

Rik, I always learn something from every one of your posts. I saw the purple fringing, and thought it was just from the old lenses. I didn't know there were two kinds with different causes, but now I can see the difference. The problem with cleaning it up in PS is, it makes the images look flat and colorless. Maybe newer and better quality objectives will prevent some of that, although diffraction seems to be the biggest problem.

Franz, I do not have any books about microscopes yet. All I have is a lot of collected bookmark pages from websites that I have been reading about the subject. I have read about most of what you mentioned. Unfortunately, they are more just descriptions and less detailed explanations of how to use them.

Your mention of field iris diaphragm and use of the aperture iris diaphragm is interesting, cause I just yesterday figured out what those are and how to use them. A fairly good explanation of them is given in the PDF owners manual for the Nikon Fluophot that I got from Nikon. Tells how to use them, and what they do, but again, not the finer details of how best to use them. That's what all these websites are missing. Do you know a good book that describes everything?

NikonUser
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Post by NikonUser »

Franz:
I am in a similar situation as Mitch, perhaps worse - I know even less about computers.
I have no books on microscopy, just about everything I have learned has been via PMG.net.
I love books but why can't the subjects on microscopy techniques be broached here in the forums?
Either in the FAQs or Beginner's Micro. A good start for me would be the question:
What is a 'field iris diaphragm' and an 'aperture iris diaphragm' and how should they be used?
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

Pau
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Post by Pau »

Mitch, surely you know it, if not take a look at: http://www.zeiss.de/C1256B5E0047FF3F/?Open

You can download Microscopy from the very beginning, not a true detailed book but very clear.
Pau

NikonUser
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Post by NikonUser »

Wow, an instant reply.
Pau: Thanks for the reference, I did not know of it.
Guess that negates my questions.
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

Franz Neidl
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Post by Franz Neidl »

for Mitch:
I dont know a good book in English. Most of my books are in German... Maybe somebody from this Forum can give you a good advice. The book should not be to theoretical with many formula ecc., but more practical.
The most important thing is the right illumination: In good microscopes is this the Koehler illumination. I found a pdf about this kind of illumination. Although the instruction is for a Zeiss Axiostar it is valid also for other microscopes: http://www.emlab.ubc.ca/pdf/BIFprotocol ... nation.pdf

for NikonUser:
the field diaphragm is the iris which controls the area of the specimen which is illuminated. This iris has to be adapted to each objective.
the aperture diaphragm is the iris in the condenser. (in most of the cases it has to be - about - half open).

Franz

Mitch640
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Post by Mitch640 »

Pau, surely I didn't know about that one, but I have bookmarked the site and downloaded the book. Thanks for the link.

NU, we have the sections for those questions, but I generally forget they are there or to use them. I need to remember to actually check them every day for new posts. That's how forums work, people have to post and not just read. If you have questions, ask, if you know the answers, post. :)

As far as the question;
What is a 'field iris diaphragm' and an 'aperture iris diaphragm' and how should they be used?
Both of these adjustments are on the Fluophot. The Field Diaphragm is a dial on the right hand side of the base of the scope, and it adjusts the diaphragm for the light coming through from the bright-field illuminator at the rear of the scope. It adjusts from almost black to bright enough to make you see spots all day.

The aperture iris is the leaf iris on the condenser.

The Fluophot manual has instructions on how and when to use them, to the extent of a single short paragraph. What would really be nice, is if there were a small chapter on just how, when and what settings to use them at, with pictures, rather than spending weeks of trial and error and not really ever understanding them fully. LOL

Franz, thanks for that link, I have downloaded it and read it. All of these things add to the total of my growing understanding. If anyone wants the Fluophot or Optiphot manual, let me know. It has pictures and a slightly more voluble explanation of how they work, how to set them and what they do.
Last edited by Mitch640 on Sat Nov 20, 2010 7:50 am, edited 1 time in total.

Franz Neidl
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Post by Franz Neidl »

Meanwhile I found a better instruction for the Koehler illumination:

http://swehsc.pharmacy.arizona.edu/expp ... gnment.pdf

Franz

Mitch640
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Post by Mitch640 »

My library of PDF's is growing. Thans Franz, another good instruction tutorial. I forgot you should adjust the condenser every time.

rjlittlefield
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Post by rjlittlefield »

Mitch, see also the books by D.J.Jackson, described HERE.

Regarding your current investigations...
Mitch640 wrote:The Field Diaphragm is a dial on the right hand side of the base of the scope, and it adjusts the diaphragm for the light coming through from the bright-field illuminator at the rear of the scope. It adjusts from almost black to bright enough to make you see spots all day.
Is that control actually labeled "Field Diaphragm"??

In any case, if that's what it does then it's not the same as the field diaphragm described in connection with Köhler illumination.

Let's see if I can quickly summarize. See Visualizing the effect of a microscope's condenser aperture. That cone of light passing through the specimen and spreading out toward the objective has four important properties. (There are others, not listed.)
  • Intensity -- how bright is the light.
  • Angular width -- how broad is the cone.
  • Footprint -- what is the cross-section at the subject focus plane.
  • Focus -- where a real image of the light source would be formed.
Each of these properties has a clearly separate effect on the image that you see through the eyepieces.
  • Intensity changes brightness, and nothing else.
  • Angular width essentially stops down the objective -- a narrower cone will make the image less bright while increasing DOF and diffraction. As with normal photographic lenses, stopping down a bit is likely to increase contrast by reducing certain aberrations in the objective.
  • Footprint at the subject plane is almost like a lens hood for normal photography -- it has no effect on the image except to reduce flare caused by light entering the lens from outside the image area.
  • Focus of the condenser changes how uniform the light is, when it passes through the subject.
In some ideal world, each of these properties would have its own separate control, with zero interactions between those controls. Unfortunately, in most microscopes they do interact, sometimes quite strongly. One of my scopes has one physical diaphragm in the base, another in the condenser, and a flip-out lens for use with high versus low magnification objectives. With the flip-out lens set one way, diaphragm A mostly controls intensity while diaphragm B controls angular width. But with the flip-out lens set the other way, they almost trade places.

You might find it instructional to play with a setup like shown at the "Visualizing..." link. That would give you some direct information about what the controls are doing. It will complement what you can see by looking through the eyepieces, and by removing an eyepiece and looking at the rear of the objective as suggested by many sources.

Oh yeah, almost forgot... The Köhler field diaphragm? It's a control for footprint -- the one that has no effect on the image except for increasing contrast by reducing flare. Makes a big difference for some objectives, almost none for others. Lots of scopes don't even have one.

--Rik

Mitch640
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Post by Mitch640 »

Rik, sorry it took so long. I had to get pics, process them and then process screen caps of the Fluophot manual.
Is that control actually labeled "Field Diaphragm"??
Absolutely. Here is a shot of mine;
Image

And the text from the manual;
Image

And here is the condenser;
Image

And the text;
Image

The field diaphragm most definately has a huge bearing on what you see, but especially on how it photographs. If you open it too wide, it shoots light up all over the underside of the nose turret. I generally have been opening it till things look good. What better way to do it? Same with the condenser iris. I think the eye is the best judge. The Fluophot has several more adjustments that really make a difference also, including built in filters to use at the push of a button, which is really handy. I am finding that using a couple filters in combination allows me to crank up the light intensity without burning my eyes or the camera sensor, and yet it shows the internals of thicker subjects, without blowing out the background. something I found by accident, and would make a nice discussion on it's own.

Anyway, I am a self professed noob, and learning more every day. That's the fun of it for me and why I got into it. This forum is the greatest place to learn all that. :)

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