Nikon Optiphot Fluorescence illumination - BF?

[timothyo]

Hi all - apologies if I word this incorrectly - probably wont use the right nomenclature

I have a Nikon Optiphot with EPI-Fluorescence illumination (has the mercury lamp) I would like to start using it with the M type metallergical objectives, and want to use normal brightfield illumination. Is it possble to modify the existing illumination by adding a Bright field cube? I see them on ebay, but they are normally very expensive. My illuminator comes with slideable ND filters which I assume would allow me to safely use the mercury lamp when not using the Fluorescence cubes? Alternatively I guess I can get a replacement vertical illuminator, and use an LED light source.

[Hobbyst46]

Hi and welcome,
I would strongly suggest that you modify your microscope for brightfield by adding a halogen illuminator (the original illuminator of the Optiphot is a halogen, a 12V 50W I think), or alternatively a LED illuminator, rather than use the mercury lamp. For several reasons, in order of importance (IMHO).
1. The mercury lamp emits UV. For fluorescence, you get reflected light, which is further filtered by the dichroic mirror and the emission filter, so you are not directly exposed to reflected UV. If, on the other hand, you wish brightfield with "white" light, that is, without any color-selective filter, reflected UV goes directly to the eyes. ND filters transmit UV and visible light at about the same efficiency, so they do NOT protect you from UV.
2. Moreover, the light of the mercury lamp is very different from natural day light as well as from the sunlight spectrum etc. It is a sequence of several intense but narrow peaks at discrete wavelengths. No filter can turn it into a continuous spectrum.
Reasons 1 and 2 above mean, that even if you attached a "brightfield cube" that includes a UV filter, it will not be true white light. And not safe enough, IMHO.
3. The life span of mercury lamps is fairly short, and is further shortened by frequent on/off power cycles.
4. The intensity of the mercury lamp is far beyond what is needed for normal brightfield (although it can be attenuated with ND filters).

In case that your plan is to fit the mercury lamp into the bottom port of the microscope, for trans-illumination, then the above drawbacks are intensified. I cannot imagine anyone looking into a mercury-lamp illuminated field of view.

M-type objectives are not ideal for brightfield either, since illumination is directed as a sleeve around the specimen.

[timothyo]

Brilliant - thanks for the information

So what is the most cost effective solution? Modify the existing illuminator, which looks like:


or just get something like:

If I replace the existing FL one with the corrective lenses, I assume I will need 210 objectives - I asume these will these work?

[ChrisR]

Careful , there's more than one epi illuminator. One at least is designed for the BD (Brightfield Darkfield) objectives which have the annular light path. That gives you incident darkfield.
The normal MPlans don't have it. The BD's come as ELWDs as well as normal. They're better for normal front lighting.
Somewhere I have a list...


Found

Mplansnip.jpg (153.83 KiB) Viewed 6890 times

[Hobbyst46]

So the purpose is reflected bright field, not transmitted. Will check some more and be back in a couple of days.

[PeteM]

Greetings.

A bit more information might be useful.

1) There are several types of Nikon metallurgical ("M") objectives, including 160mm tube length, 210mm tube length, and infinity (not likely your Optiphot has those). Which ones do you have or want? 160? 210? Just brightfield. Or with brightfield and darkfield?

2) Not entirely clear if you want only reflected light coming from up top and down through the objectives OR also transmitted light coming from below the stage and through the specimen.

If the latter, you don't need a cube, just the right "air space" (or compensating lens) to match the tube length. That, and an Optiphot with a light source and condenser below -- some don't have that. Recognize that your M type objectives will be limited to lower magnifications if you want to view slides with cover slips.

If the former, reflected light only, you're on the right track. You need a cube for your existing head and must (for safety) replace the mercury illumination source. Or, just buy another vertical illuminator that's either just BF or both BF and DF. Nikon made both types, as well as your fluorescent head and various cube options for the Optiphot.

3) If you have some fluorescence cubes already, could be you can strip the filters and use one to get started for brightfield reflected. What you need is the half-mirror (clear glass, with an anti-reflective coating, set at 45 degrees). This reflects the collimated light source down through the objective and lets the image pass back through the objective to the eyepieces.

For illumination with your existing head you could connect either a proper Nikon halogen head that fits in place of your mercury source or add your own LED source. Try a cheap but bright LED flashlight. Remove any lens in front to expose the LED die. Then get it in the right place (held any way you want so the LED die is centered and the same distance back as the mercury bulb). If you like what you see you can clean up the implementation later. Maybe machine a sleeve or find yet another use for a roll of duct tape and a bit of ingenuity. Again, the position of the LED die (or a halogen filament) should be set about in the same position as the bulb of the mercury lamp now sits.

4) Recognize that metallurgical objectives aren't much good for transmitted light and slides with cover slips above around 200-400x.

I'm assuming you want mainly reflected light -- and it could also be you really want something like reflected BF (brightfield) AND reflected DF (darkfield) illumination. Maybe even reflected polarization. If so, an entire BF/DF head with lamp might not cost all that much more than a proper BF/DF cube to add to your existing illuminator. And, if you don't already have the objectives, might want to search for the more versatile ones (recognizing they need a different turret, to fit the larger diameter objective threads).

What is it that you want to see? Perhaps we can start with that and a photo of your Optiphot and its current set of objectives?

[timothyo]

So here is my scope, including the objectives - the mercury lamp and transformer are not shown here. Perhaps the vertical illumination and transformer are worth selling?

[timothyo]

I guess I should also mention that my primary interest is really pond life, but I am also interested in looking at other biological specimens like pollen, insect parts etc...

This scope has a nice darkfield option in the stage, as well as Phase contrast which is interesting but not visually very appealing. My primary interest in the metallurgical objectives is around trying to get a bit more working distance to the subject.

I would love to get a DIC setup, but I need to learn a whole lot more before I look at that.

[PeteM]

The subjects you're interested in will all benefit from transmitted rather than reflected illumination. They'll sit on a slide, squeezed a bit under a coverslip.

And, once properly set up, I think you'll be more impressed with what phase contrast can do. You have a truly excellent microscope to begin with and a start at a good setup for biological specimens.

So:

1) Forget entirely about reflected illumination for now. I would hold on to what you have for a while, since you might find uses for it as you gain experience. Later you might sell it.

2) Your Optiphot-2 has proper illumination (working?) for light coming up through the base as well as a condenser peeking through the stage. Can I assume (can't see) that it is a Nikon turret type phase condenser under the stage, with all the annuli present?

3) Your present array of objectives is just a bit problematic. The 10x is fine. The non-phase 40x looks to be a Leitz (name not seen) and will be just a bit off in terms of corrections. The Nikon 40x phase contrast oil immersion phase objective is excellent, but not the best to start with. As I believe you are suggesting, it doesn't have very much working distance below the cover slip. And it will want oil immersion between the lens and the coverslip and oil between the condenser and slide.

4) I'd look for Nikon 20x and 40x phase contrast objectives, of the type without a correction collar (not needed for a vertical scope) and not requiring oil. Save the ones you have for when you have greater experience. The "CFN" phase objectives (triple knurled collar) are excellent and the regular "CF" plan phase are very good. Sadlly, anything with phase contrast tends to be a bit expensive used; but a great phase objective should cost less than either the cube or the new reflected head you were considering.

One of the great things about your Optiphot is that you actually stand a chance of finding DIC components in the future -- and for just the arm or leg of the usual "arm and a leg" price. For now, another objective or two might open up new vistas.

[Hobbyst46]

Since the purpose is trans-illumination, a mercury lamp is definitely inappropriate IMO.
Further bits of information:
1. The 40x/1.3 objective is very valuable for fluorescence, since it combines a high light-gathering capacity with resolution.
2. Metallurgical (M) objectives have a different thread and wider than the RMS thread of the non-M objectives, so they can only sit on the same nosepiece if (a) it is a metallurgical nosepiece and (b) thread adapters (M to RMS) are used.

[abednego1995]

Some corrections and follow ups.

Finite CF metallurgical objectives (M Plans) by Nikon are tube length(TL) 210mm. TL160mm pre-CF metallurgical objectives exist, but they are the short barreled variety and will have parfocality problems.

Regarding the mounting thread, M Plans are RMS so they will mount on the same nosepiece as biological objectives. However, BD M Plans have a larger M26 mount due to their having a larger darkfield light path around the lens itself.

BR,
John

[timothyo]

2) Your Optiphot-2 has proper illumination (working?) for light coming up through the base as well as a condenser peeking through the stage. Can I assume (can't see) that it is a Nikon turret type phase condenser under the stage, with all the annuli present?

Yes it is working - the condenser has the following rotatable options - 0, DF, Ph1, Ph2,Ph3,Ph4

The dark field is just the best for viewing pond critters. It is wonderful at 4x, but at 10x, and especially 40x it looses a lot of contrast. Not sure if that is normal - the DF setting doesnt appear to have any adjustments as far as I can see.

[Hobbyst46]

Yes it is working - the condenser has the following rotatable options - 0, DF, Ph1, Ph2,Ph3,Ph4
The dark field is just the best for viewing pond critters. It is wonderful at 4x, but at 10x, and especially 40x it looses a lot of contrast. Not sure if that is normal - the DF setting doesnt appear to have any adjustments as far as I can see.

Likely, the Ph4 condenser position will yield nice darkfield with the 4X and 10X objectives.
Also, try oiling the condenser to the slide when in the DF position. That might create great dark field for NAs as high as ~0.7 .

[timothyo]

You guys are pretty amazing many thanks!

[timothyo]

One other thing - can this scope make use of infinity objectives, or must it use 160 tube length? bit confused about how that works. I was hoping to get a Nikon PLAN APO 20X/.75 1.0 WD (DIC N2) at some point.

[PeteM]

Your scope needs finite (marked 160mm) objectives.

Later in your microscopy life, it's possible to turn it into an infinity microscope. But that requires changing all the optics (head and its tube lens and eyepieces, objectives) and doing a bit of machining work. You'd keep just the mechanical bits and lamp. Cheaper just to buy another scope. And yours can be had with such excellent optics there would be little advantage in doing so, unless you wanted to add (say) laser confocal. Your scope would be a "lifetime" microscope for most advanced hobbyists; just adding optics for advanced techniques like polarization, high mag darfkield, DIC, or the ultimate in objectives.

As noted in another response, getting a new head might just make sense for that 20x plan Apo infinity, best mounted on an interchangeable turret by itself -- but you likely have a bit of a learning curve before venturing off in that direction.

You can adjust darkfield (usable up to 20x, maybe 40x) by offsetting the condenser wheel a bit. As noted above, try some of the phase stops as well.