Help needed with new camera and microscope illumination, plz

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twebster
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Help needed with new camera and microscope illumination, plz

Post by twebster »

All right guys, here's my setup :arrow:

First, the microscope lillumination: http://www.oncloserinspection.com/Equip ... ipment.htm. There is a lamp holder at the back of the base. This holds a 6V 30 watt bulb with a tiny, flat, rectangular wound filament. Also in the base right next to the bulb is a series of condensing lenses that focus the light on a 45° angled mirror. The field diaphragm is located right next to these condensers. The light is then bounced by a 45° angled mirror to the bottom of the field collector (?) condensers and up into the substage condenser, etc.

When I achieve true Kohler illumination the windings of the bulb filament are focused at the plane of the subject and the field diaphragm blades are pretty much focused on the diaphragm in the substage condenser (Nikon n/a 1.30). Usually, I will focus the substage condenser a little out of focus, otherwise I get uneven banding from the filament windings. At best, the filament just barely fills the field of view with a 4x objective lens, just barely. To diffuse the light, I've had to place a transparent diffuser on top of the collector condenser. (Don't tell anybody, but I put a thin plastic diffuser between the bulb and the first set of condensing lenses and burned a hole right through the filter :!: I spent an hour cleaning plastic off of the condenser lenses at the back of the base. :oops: )

If I don't diffuse the illumination, then I get "Nikon Rings" as bad as the image on this page, http://groups.yahoo.com/group/Microscope/, actually a bit worse. If I diffuse the light then the lighting is uneven and the shutter speeds are very slow.

The camera setup: I have the Canon G3 attached to macro focusing rails on an enlarger stand. I can run the camera lens down to touch the relay lens if I need to (but I don't). At first, I used the MaxView 40 Plus adapter that MacroMike gave me. I could fill the frame with the image with only a tiny bit of vignetting in the cornes. A little "digital zoom" and the field of view was nicely filled. The only problem is that the MaxView is not corrected at all for my LOMO flatfield objectives and the red wavelengths create an image bigger than the rest of the wavelengths. It's like objects have red shadows and/or looked double imaged in red :!: The higher the magnification, the worse the double imaging. For the photos I posted I used a LOMO flatfield 20x .40 achromatic objective lens. Obviously, this is not going to work.

I next put in a corrected 10x LOMO wide field eyepiece as a relay lens. This corrected the majority of the secondary red image but the camera's lens is so much larger in diameter than the eyepiece that I can't get the whole field of view in the camera filled with an image. I have to jack up the "digital zoom" quite a bit to fill the field of view which is costing me pixels and resolution. I have a spare Nikon HKW10x eyepiece I will try when I get home from work tonight. We'll see if there is any improvement.

Camera settings: I am zooming the lens to the full telephoto setting and using manual focusing. I am running the eyepiece in the trinocular tube up and down to get the camera parfocal with the observing eyepieces.

Questions: Can I get a fiber optic cable larger enough in diameter than the bulb filament and will the light from the cable be more diffuse than the bulb filament. Will this more evenly fill the field of illumination? What can I use as a diffuser and where should I place it? Besides buying new objectives ( :!: ) what can I do to improve my overall setup? I know, questions, questions, questions... :D

Thanks for your help, my friends :!: :D
Last edited by twebster on Sun Dec 19, 2004 8:52 am, edited 2 times in total.
Tom Webster
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Post by S. Alden »

Hi Tom

I cannot help you out, but your posting sure helped me out a lot. One little thing - the link to the yahoo group is broken - a not found error.
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Post by twebster »

The link is fixed now. :D
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Post by Frez »

Hi Tom

Does the secondary red appear on all sides of a specimen, or is it relegated to only one side, for example the north side? Does the G3 lens system have an IR blocker? Does the red shift appear visually with the HKW 10x EP?

I substituted a fiber cable for a 30w 6v square filament lamp in the Nikon. The fiber diameter should be approximately the same as the older filament diameter. I had to settle for one slightly larger that works fine. With the fiber cable the apparent size of the source can be enlarged by moving the cable end farther away from the collector lens which will be the first lens it sees in the scope. If you can move your current filamet away it would also be made larger. If you buy a new fiber cable, try to get a mono-core cable. Mine is a standard multi-core and with Kohler the tiny circle ends of each fiber show up just like a filament does. I put some diffusion material on the cable end in the lamphouse.

Sometimes a different approach can help. Look at it this way. Our eye/brain combo is very different from a camera. When we look at something in the eyepiece, our brain stores information as we move in and out of focus and pastes together a rudimentary 3D mental image. It is also very adaptive and forgiving to contrast issues and more contrast is usually more pleasing. When imaging, the eyepieces are nothing more than tools for obtaining what the camera wants to see. Trying to image a specimen the way our perception tells us that it looks best visually is often not what is best for the digital image. The goal for imaging is to obtain as much information from a specimen as possible without resorting to contrast techniques that can add artifacts that our brains normally compensate for. Start with the condenser diaphragm wide open and adjust it just enough so that contrast just starts to change and image the shot. In the eyepiece it will look washed out, but the scope is operating closer to the full NA of the objective. This provides the best resolution. When adding contrast with the condenser diaphragm, a point will always be reached when artifacts come into play. The first artifact is a thickening of a specimen's borders that obscures fine detail. This is acceptable visually because it is only a single step in assembling a mental image. We don't have that luxury with a camera though. Adding contrast in software, when properly done, will add no artifacts providing the image contains the right amount of information. Now take that image that was done with the diaphragm opened more than what is normally used visually and apply a levels correction to it. If when the levels are applied, the image is grainy, take another image with the diaphragm closed slightly more. The goal here is to use as little contrast diaphragm as possible that will allow a proper levels correction to produce an image that isn't grainy. Less contrast diaphragm will also aid in cleaning up background nuances and make a background selection in software easier.

Software manipulation isn't cheating. Our brains manipulate an image to the same extent that software does. If it was cheating we'd have to remove our brains before using a scope and give them to Steve to stitch together.

Don't get overly concerned about maintaining Kohler. Critical illumination will not sacrifice resolution to any extent that is noticeable, if at all. I compare the two techniques to a car that is either automatic or standard. They both get you to the same store. Hopefully something here will help.

Frez

Charlie K

Post by Charlie K »

Tom... my gears are turning... but it would still be helpful to clarify a couple points...
When I achieve true Kohler illumination the windings of the bulb filament are focused at the plane of the subject
Was this a "misprint". Because in a Koehler set-up the filament should be focused at the plane of the sub-stage (condenser) diaphram.


Are there any optical elements in the lamp holder itself, or when it is removed, it it simply a "naked" bulb, with no other optical elements?

How far into the "base" does the bulb actually go when lamp holder is attached? (Just roughly, as measured from the back surface of the microscope base)

Approximately how far do you think the first fixed optical element in the base is from the bulb when the bulb holder is inserted?

I'm still not entirely clear to me where you have placed the diffuser. From this message it sounds like you are placing it externally between the light "port" coming out of the base and the substage condenser. Am I understanding this correctly?



I don't really understand why you are getting vignetting. How bad is it... very slight, moderate, or circular images?

Lastly... just to be sure... you have done the following and still have a vignetting problem:

1- You have set up the trinocular port to be parfocal with the viewing eyepieces (using the the proper correcting eyepiece for your objectives)

2-You have set the camera zoom to it's maximum optical setting (forget about "digital" zooming), and have set the camera to be permanently focused at "infinity"

3- You then run the camera up and down (centered over the eyepiece) using the focus rail

... and you still can't get a "full frame" image? If this is so... why don't you post an image that shows us the amount of vignetting after doing the above three steps.

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Post by twebster »

Hi guys :!: :D

Yes, Charlie, that was a misprint. I was rushing to get ready for work when I wrote my plea :!: :D I focus it as best I can on the sub-stage condenser diaphragm. I've tried running the bulb in and out and it has very little effect.

Hey, my friends. I've had a very long and tiring day at work, today. I've read everybody's comments and suggestions thoroughly. I couldn't ask for a greate bunch of friends to help me. I am just whipped, tonight. I am going to call it an early night and head for bed. I'll post replies in the morning when I am thinking better. OK :?:
I'll "talk" to you then.

ZZZZZZZZzzzzzzzzzzzzzzzz
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Post by twebster »

Sorry for the late reply, guys. I'm on the downside of the worst cold I've had in years. :shock: :!: No wonder I was so tired the other night. :cry: I'll try to address everyone's questions and suggestions. :D

Microscope Illuminator:

Image

1) The bulb base fits in a socket that seperates from the illuminator housing at arrow 1. I can slide this socket back and forward to focus the filament on the aperture of the sub-stage condenser. The movement is limited and I usually do have the bulb slid all of the way back to make the image of the filament as large as possible.

2) Approximate position of the bulb filament at arrow 2.

3) Illumination condenser lenses at arrow 3. Approximately 1/2" between bulb filament and condenser lenses.

4) Position of the field diaphragm at arrow 4.

You unscrew the silver collar to remove the lamp housing. I think I could make an insert to hold a fiber optic cable in the same position as the bulb filament.

Camera Coverage:

In the following photo, I removed the eye guards from a Nikon HKW10x eyepiece and placed the camera lens as close to the eyepiece as possible. Yes, I have the camera mounted on the focusing rails to run the camera up and down. I have the lens set to max zoom, 28.8 mm (140mm, 35mm equivalent). No digital zoom. I set low sharpening and manual focus. I have the focus set to infinity. AF off. I did create a custom white balance. The diaphragm is set wide open. Aperture priority autoexposure. Here's the result:

Image

I had to back off a bit from the eyepiece. If you look closely you can see a patch of blue haze in the image. The lenses are so close together that there was a bluish reflection between the two lenses. I've since moved the two lenses about 1/4" apart and the blue spot has disappeared. If I add about "5x digital zoom" then I get this:

Image

See the faint "Nikon rings"? I did not add a diffuser to these images. When I did add a diffuser I added it to the filter holder under the sub-stage condenser. I was able to adjust the lighting to get rid of the worst of the rings. I tried the LOMO 10x corrected flatfield eyepiece and the image contrast was a bit better but the HKW10x eyepiece was sharper and had less problems with the red image.

Red Image:

Frez, the "red image" that I referred to is like a copy of the subject but in red and just a bit bigger than the actual image. It appears to radiate from the center outward. The combination of LOMO/Nikon eyepieces with the MaxView 40 Plus adapter caused the red image to record larger than the image formed by the other colors of the light.

Parfocal Issues:

No matter how hard I try I cannot get the camera/eyepiece combo parfocal with the observing eyepieces. I'm not too pleased with the trinoc port. I cannot move the prisms out of the way of the trinoc tube. This makes it impossible to center something in the field of view and have it centered in the trinoc port, too. Also, I don't think my eyesight is helping either. I am diabetic and my left eye is very bad. My right eye has a small cataract that is not big enough to necessitate a lens replacement yet but it might be giving me problems focusing through the microascope. My friend is mailing me the software for the G3. I may just have to wait until the software arrives and I can use the computer monitor to focus.

I am not afraid to use Photoshop. In fact, all of the still photomicrographs on my website have been adjusted in Photoshop to correct color, contrast, and to blur backgrounds. I used Photoshop to sharpen, color correct, and adjust contrast in the amoeba images I posted. They looked much worse before I made the adjustments.

If anybody has any further suggestions, I would appreciate all of the help I can get.

Thanks everybody :D
Tom Webster
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Think about this...maybe Murphy is an optimist!!!

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