Fluorescence on Optiphot

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JohnyM
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Post by JohnyM »

By the way, if you find useful references such as lists of old cubes/filters/handy fluorophores or equipment documents, I'll put a link in the original post. Send a pm if you prefer.
This link that i already posted:
https://www.microscopyu.com/techniques/ ... ilter-sets

Includes all legacy Nikon cubes, as they havent changed much/ at all (except multibands).


@Pau quite a task you gave me. Allow me to answer in parts.
Pau wrote: Do you know the emitting power of Colibri LEDs?
... I have the impression that they are low powered
I do not have any resource, althrough i do have acces to Colibri (atm it's 300km away, so cant do a test) but they are very weak indeed. Weaker than mine (referenced further in this post). Althrough Zeiss setup provide EXCELLENT pure black background.
Pau wrote: I've seen lots of filters heavily damaged, mainly excitation ones, although not knowing their history, likely they came from labs and had been in not careful continuous daily use for many years, a very different situation of amateur use.
I've seen quite a few damaged too. From my experience it's due to cleaning... with shirt etc. Scope have 4 holes for cubes (some newer scopes accept 6, im working on mod for 8 atm). We got like 20 cubes. In storage, they got dusty / fingerpints when swapping them. Since there are many users with many needs, and none of them ever read the manual... they often attempt to quickly "fix" their error of touching the filter and ruining it in the process.

Pau wrote: At least for UV I don't think that halogen could compete at all with modern LEDs (if so it would be extremely dangerous!)...
-Power, it's no competition. Imagine using F5.6 zoom in a church. Then switch it to F1.4 prime. It's something like that.
Again, could you kindly provide exposure data of an example?
My led setup:
Image
All leds are 3W "high power" 365, 405, 455 nm
They were mounted in place of mirror in HBO lamphouse.

Nikon CFN PlanFluor 10x/0.3 through 1,5x teleconverter on A6300. ISO set on 100, camera in A-mode will chose shutter according to its whim.

Target is low photobleaching, high autofluorescence... office paper:
Image


Now the comparision:
-First row is LED
-Second row is 12V100W halogen
-3rd row is 100W HBO mercury burner

1st column is UV 365nm (UV-2A)
2nd column is V 405nm (V-1A)
3rd column is B 470nm (B-2A)

Image
Full res screen capture montage:
https://drive.google.com/open?id=19N5dW ... Rm1526DOyD


Ok i admit, i was wrong. Difference i've mentioned is between Halogen and LED. HBO is like power of sun at your fingertips...

Halogen have:
-2,66 stops advantage over 3W led in UV
-2,33 stops advantage over 3W led in V
-2,33 stops advantage over 3W led in B

HBO have
-7,66 stops advantage over 100w halogen in UV
-6 stops advantage over 100w halogen in V
-1 stop advantage over 100w halogen in B
(in green light, halogen takes advantage over HBO btw)

HBO have
-11 stops advantage over 3W led in UV
-8,33 stops advantage over 3W led in V
-3,66 stops advantage over 3W led in B

Differences might be slightly larger/smaller as i didnt payed much attention to focusing / centering.
Last edited by JohnyM on Wed Jan 16, 2019 6:00 am, edited 1 time in total.

Pau
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Post by Pau »

ChrisR wrote:Prompt for query:
Image

Query: how important is Köhler illumination ?
I understand that not using it wastes light and may affect contrast but is there a problem other than that as long as there's no image of the led/filament appearing anywhere?
You need the "1-Up" model for sure, the 2-Up will provide 3X light but only a tiny fraction of it will enter in the light path.
Köhler is not only much superior, it's easy if you use the original optics of the lamphouse and illuminators because they have been designed for it.
Critical works well enough in low end microscopes for transmitted illumination because they use strong diffusers that act as the actual light source, for epifluorescence you are not going to find this approach because it wastes a lot of light.

Peter, nice paper you linked.
---
To all:
By the way, if you find useful references such as lists of old cubes/filters/handy fluorophores or equipment documents, I'll put a link in the original post. Send a pm if you prefer.
Take a look at the documents I formerly sent to you, there are lots of relevant readings on this subjects
Pau

JohnyM
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Post by JohnyM »

This is flash test in UV.
Image

1st row: Full power and 1/2 power
2nd row: 1/4 power and Full power WITH FRESNEL LENS AND FILTER NOT REMOVED


I dont have osciloscope to test flash duration. I would say that it matches HBO output (1/1600 sec) on 1/2 power. Things get better when we move to right side of the spectrum.

So stopping power might not be impressive, althrough big flash advantage is greatly reduced photobleaching in fluorescence stacking.

ChrisR
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Post by ChrisR »

That's a lot of work Johnny, a useful reference. Don't sell tickets, do you? :)

I'm surprised at:
Halogen have:
-2,66 stops advantage over 3W led in UV

So there's 2^2.66 = 6.3x as much UV coming from a 100W halogen as a 3W UV led? Wow!
Chris R

JohnyM
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Post by JohnyM »

I was suprized myself. On the other hand it's mirrored 100W vs 3W. Anyway, those LED's are fishy anyway. They look all good on paper, but 365 is leaking violet and supposed to be blue is shining through GIF.

Pau
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Post by Pau »

Johny, thank you very much for the tests, most interesting!

The differences are really high. Specially surprising -and scary- is the amount of UV emitted by the 100W halogen.

I think that at least some of my LEDs are more powerful than yours although will not approach the other sources. Will test the exposure times on office paper when I mount again my microscope for fluorescence, all I can say at this moment is that the light emitted by a white paper 365 UV excited dazzles the eyes with the 80/20 trinocular enabled and my exposure times with a 10/0.30 (I think) and 1.6X relay magnification were around 1/30s at ISO 100 in a test I did time ago for other purposes.

I also need to try electronic flash, would be great for quick photobleaching subjects as you say.
Pau

pbraub
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Post by pbraub »

As Pau said, thank you very much Johny.

Do you have optics beside a mirror in your 3-way Cube? I am thinking about building a similar thing to mount an HBO and a LED at the same time. It would be nice to switch them without much mechanical work. Any would be appreciated.

Kind regards
Peter

ChrisR
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Post by ChrisR »

Funny stuff this UV. I could try this highly technical experiment with a tungsten lamp. Second picture:
http://www.photomacrography.net/forum/v ... 296#207296

FIlter is ZWB2
Chris R

ChrisR
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Post by ChrisR »

In the current edition of the Journal of the Quekett Microscopical Society there's an interesting article by H.J. Swatland; The Colourimetry of Chromatic Aberration.

In it is this graph, where Xenon is flash. That makes me wonder if there's a Xenon bulb we could use, perhaps from a vehicle :
Image
Chris R

Greenfields
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Location: Nottinghamshire, England

Post by Greenfields »

What a fascinating discussion.

ChrisR asked on Jan 16:
Query: how important is Köhler illumination ?
I understand that not using it wastes light and may affect contrast but is there a problem other than that as long as there's no image of the led/filament appearing anywhere?
I believe that unlike dia-illumination, epi-Kohler illumination has less and maybe no effect on resolution because the specimen is likely to reflect or (in fluorescence) radiate light in all the directions needed to fill the objective pupil.

However, just as in dia-illumination the aim is to completely de-focus the light source so that nothing of its structure is superimposed on the image, and it is designed to deliver as much light as possible from the source.

To do these things the optics project an image of the source in to the rear pupil of the objective - just as in conventional Kohler illuminagtion - but in epi-illumination this is done from behind the objective rather than through the objective as in conventional Kohler illumination.

I suggest that an important feature is that the epi-illumination optics are designed to magnify the source to fill the rear pupil of the objective.

A mercury HBO lamp's arc is conventionally taken to be about 0.25mm in diameter, so the combination of the collecting system in a HBO lamphouse and the optics in the vertical illuminator have to magnify it by more than 10x to fill the rear pupil of an objective.

The (Genuine) Nikon 20x 0.75 Plan APO VC which recently became available has a rear pupil diameter of something like 14mm but I assume that the Optiphot objectives were smaller.

The first step is a collector lens in the lamphouse which has as short a focal length as the thickness of the HBO bulb will allow

A halogen lamp lamp filament is larger, so its collector lens can have a longer focal length.

I guess that a single LED die without a lens will be of the order of a millimetre in size so would ideally need a collector lens focal length somewhere between that of an HBO lamp and a halogen lamp to use it efficiently.

I think that the three-die LED illustrated by Chris would be too big, but say a 3x3 die array from a 10W array may be OK and with a heat sink still run cool enough not to need forced cooling [= vibration risk]

That does not mean it would not work. Just like JohnnyM's flash unit, if enough power it produced it will overcone any inefficiency in the optics.

Henry
Feel free to edit my images.

Pau
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Post by Pau »

Greenfields wrote:I suggest that an important feature is that the epi-illumination optics are designed to magnify the source to fill the rear pupil of the objective.
...

The first step is a collector lens in the lamphouse which has as short a focal length as the thickness of the HBO bulb will allow

A halogen lamp lamp filament is larger, so its collector lens can have a longer focal length.
Henry, you're right. I have both HBO and 100W halogen lamphouses and the collector lens of the HBO has shorter focal length. I don't use either lamps but only the lenses with LEDs, did you see my setup?
http://www.photomacrography.net/forum/v ... hp?t=32736 (now improved with better UV and blue LEDs)
I guess that a single LED die without a lens will be of the order of a millimetre in size so would ideally need a collector lens focal length somewhere between that of an HBO lamp and a halogen lamp to use it efficiently.
With mine (all the LEDs have built in lenses) the HBO collector lens is adequate, always better than the halogen lamp lens even with a bigger 10W LED (although as I formerly said the Nichia has a too powerful lens and has some issues with certain objectives)
I think that the three-die LED illustrated by Chris would be too big, but say a 3x3 die array from a 10W array may be OK and with a heat sink still run cool enough not to need forced cooling [= vibration risk]

That does not mean it would not work. Just like JohnnyM's flash unit, if enough power it produced it will overcone any inefficiency in the optics.
I think that placing a single chip LED at the position of the original lamp source works better, at least it does with a 10W single die LED I use for transmitted illumination with the 100W halogen collector lens.
Pau

Greenfields
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Post by Greenfields »

Pau,

You have the most elegantly implemented aftermarket solution I have seen. It shows that testing and adjusting something until it works is worth any amount of theory,

Henry
Feel free to edit my images.

ChrisR
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Post by ChrisR »

Temporarily I can't find the first Epi-illumunator with its two cubes.
Regarding "no filter", here are the second two, which I think are common types,, "B" and UV-2A.
As you can see the latter appears to have removable filters, the B ones are more fixed.

I imagine that's normal? I doubt I'd want to change them unless some odd filters came along.

Is that correct?
Image

Image


Two other questions:

What is the 4mm (or so) filter slot in the upper part of the housing normally used for? Pol or, other colour filters?

Image

AND
What are the electrical requirements of a power supply for an HBO burner?
Do they need a slow voltage or current start?
(I know they usually have hour counters.)
Thanks
Chris R

JohnyM
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Post by JohnyM »

All my cubes are Nikon original and look like the right one. I know nothing about the left one.

I had 4 different versions of that "4mm slider". One was empty, one had polarizer, one had auxiliary barier filter and one was blocking the light.
Auxilary barier filters are actually usefull to tune the emmision colors to your needs / taste.

Electrical requirements... i dunno. I just plug the power supply in and it works... im from EU tho.

Pau
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Post by Pau »

Time ago I got a 3 cube set for an old Nikon *..phot model with 18mm Chroma filters (I think yours are 25mm) that came glued exactly like in your left cube. I contacted Chroma tech service and they explained me how to dismount them, (very kind people!), because I wanted the filters alone for my Zeiss epi illuminator. They were glued with a very soft rubber-like glue, easy to dismount without damaging the filters
Pau

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