Nikon Fluophot DIA-EPI Filter Test - Video

[Mitch640]

Testing the effects of the DIA Filters in the FE Arm of the Nikon Fluophot. There is almost no information on these old microscopes anymore, and I have found nothing beyond the PDF owners manual for the scope, given out by Nikon on request. Francisco did post a video of his filtered Fluophot a few weeks ago, but that's about all there is online. My main purpose here, was to find a color that might block out the green fringe I get from my E 40x Nikon lens, cause if I can block that out, between the lens and camera, I can then use the White Balance correcting tool in my video editing software to return the colored version to normal, hopefully, without the green fringe. LOL

The manual does show all the knobs and dials on the scope, and the basics of assembly and how to use the parts, but for the details of what the filters do in flourescent photography, it must take some manual I don't know about. There is almost nothing to be found on the internet about this scope.

Here's the video.

Also, if you can ID the main character in this video, let me know.

Microscope: Nikon Fluophot Flourescence Research Microscope
Camera: Canon T1i w/ EOS Extension Tube adaptor w/Nikon 10x Projector lens
Scope Settings: 15.1 MP Canon 1.6x Camera over 10x and 40x Objective

[NikonUser]

I'm guessing a rotifer.

Those nematodes make my head spin, as do the racing ciliates

A drop or two of Protoslo should work wonders on the racing cars.

[Mitch640]

Thanks NU, I was thinking Rotifer, but I just don't have the experience yet to be sure.

The nematodes are becoming a bane to my existence. A few weeks ago I posted a thread with some pictures of what I thought was an egg. The other day, I updated it with some more links and video, cause I found out it was an egg. Guess what kind? LOL I have nematodes and their eggs in almost all my petri dish cultures now, that began as dry tree moss. You can see what a pest they are.

As for the slowing solution, I have a full bottle here, but for one reason or another, have not used it yet. Now the excuse is, I am waiting for that new 20x CFN Plan Apo to get here. It's coming from Canada, and you know what border crossings can do to even small packages. LOL

[fpelectronica]

Hi Mitch
Interesting test video filters
The "main character" is a rotifer lorica
Greetings
Francisco

[Mitch640]

Thanks for the ID confirmation Francisco.

Do my filters appear to be the same as yours? They do make some interesting colors and contrasts, but I am still deciding if they are useful or not.

[NikonUser]

Mitch:
I'm confused (again).
Your camera's sensor is 22.3mm wide.
At 600x (magn stated for your video), the FOV is only 22.3/600=0.038mm.
This makes the Rotifer and the racing car ciliates about 6µ (0.006mm) wide/long.
I get the feeling that these creatures are much much larger.

[Mitch640]

I add it up this way, 40x objective, times 10x PL lens = 400, times 1.6x for the crop sensor, = 640. Not counting in the extension tubes, cause I don't have any idea how to do that. I'm just doing it like I did with my bird lenses. Probably apples and oranges with microscope lenses, but I haven't figured that out yet.

I'm pretty loose with my math, cause I am simply too old to care anymore. LOL Plus, I was turning 3 or 4 knobs at the same time while trying not to think too hard about killing those nematodes.

[NikonUser]

Sorry Mitch; my apologies.

[Mitch640]

So how should I be adding it up? I think I was good till the 400x part.

Ah, I think you must have looked at the mystery egg video. In there I was way off with my math. I admit that. LOL

This video though was all shot at 4x in the beginning and then 10x.

[rjlittlefield]

I'm pretty loose with my math, cause I am simply too old to care anymore. LOL
...
So how should I be adding it up? I think I was good till the 400x part.

I offer three very different recommendations:

1. Include a scale bar in the image.
Advantage: unambiguous.
Disadvantages: difficult to do at all, easy to get wrong, hard for most viewers to interpret.

2. Specify frame width in text accompanying the image.
Advantage: unambiguous, easy to do, easy to get right if you have a stage micrometer.
Disadvantage: hard for most viewers to interpret.

3. Specify what power microscope you'd have to look through, to see the same thing that is shown in the video.
Advantage: easy for the viewer to interpret, easy to do and to get right.
Disadvantage: not very precise.

Using method #3, your 600X number is fine.

Unless somebody knows exactly how you're shooting the video, magnification on sensor is really irrelevant anyway. Essentially the same video could have been shot with sensor magnifications ranging over a span of almost 10:1, depending on whether you were using a full-frame DSLR or a high quality webcam.

--Rik

[Mitch640]

Thanks Rik. I think #3 would be easiest for me, although it would actually be 640x. This is how I would add it up in my bird shooting days. But even that was entirely arbitrary, using the full frame size of the old 35mm film frame as 1:1, while the new crop sensor would magnify the same scene through the same lens by 1.6x.

Maybe #4 should be no mention of magnification at all, since I use extension tubes with no length markings on them. I don't even know how I would figure that out.

[Charles Krebs]

Mitch,

No need to become a mathematician, but there's actually a very "practical" reason to know, at least roughly, what the actual recorded magnification is... at least at this juncture in your microscope adventure. It can be helpful in insuring that your trinocular tube optics are properly set up, and also be sure that you not losing a large amount of image quality from "over-magnifying".

As I recall you have tried quite a few variations when it comes to camera coupling. (And I really don't have a clue how you are now set up). But the optics and spacing of that "connection" are critical for good photography. (You can almost always get some image in the camera by altering microscope focus and playing around with the camera and trinocular optics, but it might be very far from the best image if the wrong optics are used or their positioning is wrong).

For the camera you are using, with it's 22.3 x 14.9 mm sensor size, you want the magnification provided by the optics in the trinocular tube to be, roughly, in the range of 1.5X to 3.3X. (At the upper end of that range it's best to use a higher NA objective... such as a 10/0.40 instead of a 10/0.25).

(The Microflex HFM that came with your microscope will not be used).

With your "new" microscope, and the Nikon CF (160mm tube length, finite) objectives the best option is a Nikon CF PL 2.5X projection eyepiece. This would be placed in your trinocular tube and "seated" directly against the machined surface that is in the trinocular tube. (i.e. dont raise or alter that location). Then, with the microscope focused on a subject in the viewing eyepieces, the camera is mounted above the CF PL 2.5X so that it too is in focus at the same time. (This may take a little DIY work bit it's generally not difficult. You may be able to make use of the mounting ring from the bottom of the Microflex HFM).

I don't see any way the CF 10X photo-eyepiece you have can be used with your Canon body and get optimal results. You may(?) be able to use the other 2.5X adapter you have mentioned several times, but again, the positioning is important. One way to determine that it is close to the right position is to insure that it is actually providing a 2.5X magnification into the camera. If you use the 4X objective with that 2.5X adapter and photograph a ruler that has mm markings, you should record... left to right... a distance of 2.2mm. If you are recording significantly more or less than 2.2mm on the ruler (with your 2.5X adapter and the 4X objective), then you know your trinocular tube optics are not properly set-up.

Important... always focus on the subject with the viewing eyepieces before fiddling with the trinocular tube arrangement... and don't change that focus! You should not re-focus in order to have the camera in focus. Once set up (if your arrangement does not permit fine camera height adjustment) it might be necessary to do a slight, "fine", touch-up to get perfect focus in the camera. But the eyepieces and camera should be extremely close as far as focus is concerned.

I would strongly recommend that you keep a keen eye out for a Nikon CF PL 2.5X.

Also....

Is your condenser properly centered? And how are you determining the aperture size that you use (in the condenser) when taking pictures? Both of these considerations could be causing you problems.

BTW... knowing, at least approximately, the magnification recorded on sensor is also sometimes very valuable for identifying what you are looking at. Quite a few species may look very similar, but their actual size can be used for differentiation.

[Mitch640]

OK, now we are getting somewhere. It's very frustrating, trying to find any information on the Fluophot. Apparently the only other Fluophot in existance is the one Francisco has in Spain. LOL Google has amost nothing, and I suspect my own webpages will be showing up in Google searches in a few days. Already, Francisco and I are the only two that come up any YouTube search for Fluophot. LOL

With your "new" microscope, and the Nikon CF (160mm tube length, finite) objectives the best option is a Nikon CF PL 2.5X projection eyepiece.

I did bid on that 4x RIK pointed me to a few weeks ago, and lost. The day after he told me about the 4x, I was just puttering around E-bay and found a 2.5x and bid on it and won. It arrived the other day. The bad news is, when I mount it the same way I do the 10x, and take a still image, all I get is about 2/3 of the frame in solid black, and the rest is a round hole with the image of whatever is on the slide. Yes it is in focus, but to use it, I would have to crop the thing to within an inch of it's life, losing any detail I might have gained by using the smaller power lens. The 10x, fills the whole frame on the T1i, just barely.

This I don't understand, cause NU uses the 2.5x and his images are great. Don't look cropped, although they could be. The exact designation of my 2.5, printed on the eye end ring is; CF PL2.5XA Both lenses seat in the trinocular tube as designed.

You may(?) be able to use the other 2.5X adapter you have mentioned several times

While that adapter does work, and the big print on the page I bought it from say's it's a 2.5x, the fine print admits it is closer to 10-15x, which makes sense, cause it and my Nikon 10x PL lens gives about the same size final images, when using the same power objective. Forget that adapter, it has bad green and purple fringe.

I will have to do the measurement test. If I can even find a mm ruler. I think I can get it close to parfocal by messing with my extension tubes, but not perfect. I have gotten quite close with my 10x though. I'll get back to you on the test. I would really like to get this all working before I go totally broke. Your help is very much appreciated.

[Mitch640]

Charles, you are a genius and I am a putz. I am sure you have it figured out already. The little black hole should be the giveaway. All along, since that lens got here, I have been using a full set of Kenko style tubes, what's that, something like 68mm? Well, while setting up for the test I needed, I started thinking about those tubes, and in particular, the 68mm tube length, and then cross referencing that with NU's bellows setup, recorded in my minds eye, and his bellows seems far longer than my piddly 68mm.

Long story short, I slowly added Kenko tubes, from my second set, one ring at a time. Just as I ran out, at 136mm, I hit parfocal on the 4x and got an exact 2.2mm on my frame, side to side.

I should probably run down and buy a Lotto ticket, cause with luck like that, it has to be a winner. All I can say is, thank you for sticking with me on this. I really feel like I made some serious headway today with your expertise. Can't wait till that 20x Plan Apo get's here.

[Mitch640]

Also....

Is your condenser properly centered? And how are you determining the aperture size that you use (in the condenser) when taking pictures? Both of these considerations could be causing you problems.

BTW... knowing, at least approximately, the magnification recorded on sensor is also sometimes very valuable for identifying what you are looking at. Quite a few species may look very similar, but their actual size can be used for differentiation.

The condenser is centered. The Halogen coil is in focus and centered. I use about 70% of the NA on my condenser Iris, then fine tune it by eye. It does make a difference.

I do have a micrometer scale, and I do know how to use it and add it as a layer when I process my images. But, it's a pain to use and means very little to me and I suppose, a lot of other people too. If it was in thousands of an inch, I could get a mental picture of how big something is, but the USA went metric just too late for me to imprint to it.