Diaphot Renewal School Project

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Tommyb
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Diaphot Renewal School Project

Post by Tommyb »

Background
I am trying set up a Diaphot 200 for an Advanced Science Research High School class that I mentor.

The school has 2x AM Fluor Scopes that are quite frankly a joke. I purchased an EBAY TMD and plan to upgrade and donate it for the class use. My mentees research various neurological transgenic C-elegan's typically expressing GFP, but I have chased some filter mods depending on what flour's are being expressed.
I have a background in optics and various engineering trades, but definitely very limited exposure to true optical microscopy. I set up the AM scopes with professional grade B/W USB CCD cameras (Point Grey and Bassler) with Micro-manager + ImageJ for analysis for the students to use.

The condition of the Nikon Scope is pretty good with only the reticle motion frozen. There were three Objective lens and no Epi Light source (U20 0.33, Fluor10 0.5 0.17, E10 0.25 Ph1 DL). There are two Dichroic cubes B2A and G2A.

I have just built a Royal Blue LED and Green + condenser tube for EPI excitation and designed and 3D printed an adapter to fit to the EPI optics train. I would also like to do this for the main illumination.

Now for the bad part, the scope is dusty, the eyepieces were left out in the open and are grimy (more than just dust), the filter cube are dusty as well as the two objects are very dusty. I have not looked at the prism or internal optics. I have cleaned research grade optics in the past of free dust only, but not grime.

I am looking for help/recommendations for:

Cleaning of the external optics? Shall I use reagent grade (what is best)? Should I soak the dirty faces overnight and then blow off? Shall use ultrasonic cleaner on the out glass? I am afraid to wipe! And the objective lens are small and recessed. I am sure I cannot get to the very edges near the bezels.

What is the best approach for the eyepieces. I am sure they are more robust but don’t know if these are coated optics (hard or soft)? Are there any detergents to use or just solvents and gentle wipe.

Filter cube cleaning approach? Are the DM mirrors emulsion type or metal oxide? Should I touch the DM’s at all? What about the filters? Can remove and clean - ultrasonics, water based cleaners, or no? Can I dunk the whole cube in solvent?

Finally, what is the best path/approach for a 1/3 size CCD (or waste of time) imaging solution? I need high sensitivity sensors and can probably purchase a Nikon camera body...but not sure if sensitivity for most SLR cameras are enough. I would love to be able to use the side port and have a lathe and 3D printer.I can make just about any adapter to be a-focal, just need some known optics inputs.
If I use a relay lens on the side port, what factor if I buy a pre-made Tel adapter 0.6x? Can I use a known Thorlabs element? What it is the image size (lens diameter) in the tube and where does it need to be positioned. (mm from ?)? It is amazing that there is very little information on the microscopes!

What modest objectives are recommended? I figure I would add ~b $200 each for 2ea more objectives.

Main illumination conversion to LED suggestions? The halogen condenser is ~50mm, does an LED need to be an area or point source + that condenser good enough? Or use the reflector as is and shine LED to reflector first?

Sorry for such a detailed/long post...just hoping to capture it all in one go and then hope for some inputs.
I would expect to retrieve the unit for home use in the summers for my own photo exploration?

Pau
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Re: Diaphot Renewal School Project

Post by Pau »

Hi Tommyb,

Welcome to the forum!

Having taught elementary Microscopy at High School level before my retirement, I logically sympathize with your project.

I can't answer many of your questions, so just dropping few pointers

Good info on cleaning:
This Zeiss booklet can be useful: https://hcbi.fas.harvard.edu/files/hcbi ... cope_e.pdf
https://microscopy.duke.edu/sites/micro ... scsope.pdf

In most cases I clean the optics very gently with cotton swabs with small quantities of absolute ethanol or isopropanol embedded avoiding the liquid to free flow into the optics casing, but I'm not an expert and this method is not to be recommended in all cases.

Modern fluorescence filters and dichroic beamsplitters are metal sputtered. They are fragile and easy to scratch but they can be home cleaned with care. Sending them to a tech service like Zeiss suggests doesn't seem necessary or economically doable.
Main illumination conversion to LED suggestions? The halogen condenser is ~50mm, does an LED need to be an area or point source + that condenser good enough? Or use the reflector as is and shine LED to reflector first?
Because your microscope illumination train is designed for the halogen lamp small filament, in principle the best option is to have a single die LED placed at the very same location of the lamp filament -and with the same optics after it. (the same with your fluorescence LEDs)

Most single die LEDs have a built in lens. In this case you want it being wide, if too narrow you could experience covering issues.

I've used Cree XML2 LEDs for a few years now, I find them very convenient although now there could be better options.
Pau

PeteM
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Re: Diaphot Renewal School Project

Post by PeteM »

If you're located in the Bay Area, I may be able to help. If so, feel free to message me for contact info.

Scarodactyl
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Re: Diaphot Renewal School Project

Post by Scarodactyl »

Seconding isopropyl and q tips, applied carefully and frequently changed, all after blowing anything loose off. It has worked for me but no guarantees.

No ultrasonic cleaners.

I'd replace the eyepieces with Chinese 10x/22s. They're not too expensove, a bit wider and I believe give a better image--weird but there you have it.

For fluorescence there's the classic 10x/0.50 fluor, not plan but hard to beat the NA.

Tommyb
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Location: California USA

Re: Diaphot Renewal School Project

Post by Tommyb »

Thanks for all the great replies.
@Pau - all great info, thanks for the details and appreciate the empathy and happy retirement!
The LED integration for the main lamp seems straight forward, thank you for confirming. I figure I can go with round planer type SMD 5050.

My filter cubes are the Nikon OEMs and very old. I worry if these are able to be exposed to solvents and cannot find any definitive experience. They seem to be in good shape, just very dirty.

@Petem, thank you for you offer...and yes I am in the South Bay. I will PM you, thank you for the offer.

@Scarodactyl - very useful info...I will look for the objective you mention. I don't really need UV (at this point) glass so this might be perfect.

I have learnt the torn/rolled lens paper edge cleaning technique, seems to still be the best option.

Now for the camera integration in the side port. I recall reading an article about image size and location and somehow with the Diaphot dimensions, a small 1/3 sensor is of little use due to loss of resolution. I cannot find it and just have no idea if that journey is viable. If there are any references (research or otherwise) of such an integration, this would be greatly appreciated.

Scarodactyl
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Re: Diaphot Renewal School Project

Post by Scarodactyl »

Iirc the side port has a 2.5x magnification factor built in. Unless you want to do some serious modification you'd want a larger sensor camera on there. I remember this being a source of frustration for past users but I don't have the links at my fingertips.

Pau
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Re: Diaphot Renewal School Project

Post by Pau »

The LED integration for the main lamp seems straight forward, thank you for confirming. I figure I can go with round planer type SMD 5050.
Could you link the LED you're referring to?
The 5050 models I find are low powered and with 3 actual LEDs under a common phosphor, both are inadequate features. The Cree XML2 is maybe a bit smaller but much more powerful (over 10W) and it's a single chip with uniform light emitting surface. COB LEDs often are inadequate for Khöler illumination
Now for the camera integration in the side port. I recall reading an article about image size and location and somehow with the Diaphot dimensions, a small 1/3 sensor is of little use due to loss of resolution. I cannot find it and just have no idea if that journey is viable.
2.5X is the classic secondary magnification for FF cameras (24x36mm originally). Is there another photo port in the microscope?
1/3 size CCD
Likely there are C mount adapters that reduce the primary image to fill this tiny sensors (1/3x I think) but I don't know how to adapt it to your microscope
https://www.microscopeworld.com/t-micro ... ounts.aspx
http://bestscientificweb.com/wp-content ... re_5MB.pdf
@Scarodactyl - very useful info...I will look for the objective you mention. I don't really need UV (at this point) glass so this might be perfect.
It seems that you already have it :D
There were three Objective lens and no Epi Light source (U20 0.33, Fluor10 0.5 0.17, E10 0.25 Ph1 DL).
Cleaning filters:
https://www.chroma.com/support/technica ... rientation
Pau

Scarodactyl
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Re: Diaphot Renewal School Project

Post by Scarodactyl »

Wow, I definitelty misread that objective list! That'a the main one, but others in the fluor series should be good too. They're basically thr equivalent to today's nikon s fluors.

Tommyb
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Location: California USA

Re: Diaphot Renewal School Project

Post by Tommyb »

The Diaphot has a front camera port with 2.5x..that definitely needs full frame camera.

The side port looks to be direct with no lens in the light path. It was supposed to be for a photo-muliplier from the 80's. The cross sections in the manuals do show some feature (maybe lens?), but I have not found any references on what this might be and not sure if this will be a problem.
There must be a way to relay this image, but I have seen mostly dead end threads like you mention. This should be no different imaging than a CCD eye piece adapter? I think I need to work with a high sensitivity 16 bit B/W) camera sensor and the large formats guys are pretty darn expensive....maybe a dead end for me after all on the side port.

The J5050 LEDS are phosphor based 5w units, but not clear if there are separate discreet emission points. The model I selected is JR5050BWT-K-B50EB0000-N0000001. These units are square, but they do have circular emission zonse which would be even more efficient. Anyway, I may have some XMl2's or the like for some bike lights, I can swap this out and start with those. Thanks for the pointer on COB's. If I can get the camera up, I can confirm with flat frames how bad it is and what needs to be compensated.
Thanks for the filter links. I am still worried about these old filter cubes and looking for a definitive go/no on what is allowed (solvents or not). I guess I could send a note to Nikon?

One last understanding question - inverted microscope will always have the equivalent of a cover slip in the form of the specimen holder (slide or even petri dish). Should all objectives have a fairly large cover slip thickness factor? I understand that this is not as simple focal point adjustment but actually a change in the optics to compensate for the cover slip thickness.

(sorry not sure how to capture quotes yet here)

Alan Wood
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Re: Diaphot Renewal School Project

Post by Alan Wood »

The side port on the Olympus equivalent of the Diaphot was intended to be used with a photo eyepiece, such as 2.5× for 35mm film (same size as full frame).

I wonder if the Diaphot needs a Nikon photo eyepiece in the side port?

Alan Wood

Pau
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Re: Diaphot Renewal School Project

Post by Pau »

Did you see the TMD instruction manual?
You can download it from https://neurophysics.ucsd.edu/Manuals/N ... ot-TMD.pdf

Alan's guess was spot on.
See pags. 21-25, the side port accepts a 23.5mm eyepiece or projective photoeyepiece, so it can accept an eyepiece camera or a C-mount camera with eyepiece tube adapter. In this case the best magnification for 1/3 camera sensor would be 1/3X as formerly said.

The external tube will accept 42 mm smooth tube like the one of the pictured Microflex FX camera or the V-T adapter allowing to make photoeyepiece projection or afocal adaptations.

Are your cameras good enough? Maybe an used outdated FF model like the EOS 5D MkII will still be a better option and not very expensive. For high quality photomicrography it would be preferable, although for classroom projection often small USB cameras are more practical.
One last understanding question - inverted microscope will always have the equivalent of a cover slip in the form of the specimen holder (slide or even petri dish). Should all objectives have a fairly large cover slip thickness factor? I understand that this is not as simple focal point adjustment but actually a change in the optics to compensate for the cover slip thickness.
This is also covered in the manual. The objectives marked 0.17 are not suitable for use with thick glass. In principle inverted microscopes are more limited than upright models for high resolution imaging, although you can use 0.17 normal objectives placing the slide upside down*, use big coverglasses as slides or Petri dishes with a bundled 0.17 bottom window, both three options are pretty usual in some bio labs.
* not very convenient for water mounted fresh samples!
Pau

Pau
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Re: Diaphot Renewal School Project

Post by Pau »

Tommyb wrote:
Thu Dec 09, 2021 6:47 pm
(sorry not sure how to capture quotes yet here)
It's easy, there are two methods, both using the quote icon:
a) on the post you want to quote, click the quote icon in place of the post reply at the end of the thread. This will open a new post window with the quote.
This is useful to have the quoted author at the beginning and for quoting a whole post. Of course once done you can delete the non relevant parts
b) copy the paragraphs that you want to quote and paste them into your post, select them and click on the quote icon over the post reply window on you're writing.

...and you also can use the brackets and quote writing you can see while writing a post
Pau

Tommyb
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Location: California USA

Re: Diaphot Renewal School Project

Post by Tommyb »

Wow, the manual's comments now make total sense with a touch of embarrassment on my part. Thanks for posting link and commentary. The first time I scanned this, the details were missed.

This unit unfortunately did not come with any either of the photo port optics and so now for some trial and error. I do happen to have a .3x Cmount eye piece adapter...will try that after some TLC on the mirrors and optics (prisms and mirrors are fully dusted). I recall being rather disappointed the last time I used this in another application in the eyepiece tube.

I will also look for a "Nikon Photo Eyepiece" as Alan notes. The optical path to photo port has no optics between the objective and the port...this should theoretically be less complex to sort out. But I will say that it is remarkable that there seems to be no successful adaptations noted in the internet gallows for a Cmount in the side port for this imaging approach. Either this is patently obvious or there is a gremlin lurking. I paid $500 for the Scope and hope not to have to pay some multiple to get a reasonable solution.

As far as the camera goes, I have already set up work flow in the school lab with a Point Grey Camera on the AM F scope(s) and use uManager to set up and control the imaging. Both Cannon and Nikon have proprietary interfaces that make this step painful. BUT it is still worth exploring to see if the bit depth and sensitivity is adequate for fluorescence imaging. I have donor cameras in these variants.

Ill report back as we progress.

Scarodactyl
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Re: Diaphot Renewal School Project

Post by Scarodactyl »

It'a the port with the 2.5x built in that gives people trouble. A port with no added optics and a 23mm slot is perfect for a c mount. Nikon optics are non-compensating so you don't need particular corrections done, meaning generic solutions are suitable. Something like one of the older Diagnostic Instruments solutions would probably be the best bang per buck if you can find one with the right magnification.
Nikon photo eyepieces won't be much help here. They only go down to 1x (but cropped down to a 16mm diameter).

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