Epi illumination without Dichroic mirror maybe?

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Gasman
Posts: 153
Joined: Sat Jan 09, 2016 1:36 pm
Location: North East UK

Epi illumination without Dichroic mirror maybe?

#1 Post by Gasman » Sat Jan 15, 2022 12:12 pm

Hi guys
Been trying to have a go at epi illumination but I don`t have a Dichroic mirror nor can I find one the size I want so had the mad idea of trying to illuminate the sample with a 2mm ring of light using an elliptical hole at 45 degrees and a small piece of foil (the pics might describe it better than my feeble attempt ;-)) 3d printed adaptation. Sorry no idea why all these images are shown on a side as they are all the right way up in other apps??.


open box
open box
imgonline-com-ua-resize-8ADBqpVcVhSFNLTp.jpg (116.82 KiB) Viewed 4291 times

on microscope
on microscope
imgonline-com-ua-resize-XNUmwwppwYLd.jpg (120.29 KiB) Viewed 4291 times


View through bottom hole before microscope mount was attached
2mm ring
2mm ring
imgonline-com-ua-resize-a9LjiXSq59vgjM.jpg (142.26 KiB) Viewed 4291 times


Erythritol using normal illumination from below, no filters
Erythritol lit from below
Erythritol lit from below
imgonline-com-ua-resize-nVXIKzifFmKpdu.jpg (152.46 KiB) Viewed 4291 times


Illumination via small 5mm bright led on 45degree plate
via led
via led
imgonline-com-ua-resize-K7vCPmCxh50Q6f.jpg (170.35 KiB) Viewed 4291 times

I intend to fit excitation and emission filters to see if any fluorescence is visible

Steve

Gasman
Posts: 153
Joined: Sat Jan 09, 2016 1:36 pm
Location: North East UK

Re: Epi illumination without Dichroic mirror maybe?

#2 Post by Gasman » Sat Jan 15, 2022 12:13 pm

Forgot to attach pic of parts before assembly!


imgonline-com-ua-resize-0p8FC5mo22e.jpg
imgonline-com-ua-resize-0p8FC5mo22e.jpg (140.53 KiB) Viewed 4290 times
Regards
Steve

Greg Howald
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Re: Epi illumination without Dichroic mirror maybe?

#3 Post by Greg Howald » Sat Jan 15, 2022 12:58 pm

Been there. Done that. But my stuff wasn't as nice as yours. I don't have sophisticated equipment to make it pretty. My stuff comes out looking pretty crude.

I bought the dichroic mirror on ebay. It was a circular mirror one inch in diameter. I couldn't be absolutely certain that my contraption was made at 45 degrees so I mounted the mirror with a circular frame having straight rods on the side of the frame at 180 degrees so I could rotate the mirror and get as much light as possible through the objective.

I did achieve fluorescence but my rig was rather rickety and somewhat scary. I didn't feel good about playing with a $1000.00 microscope that way so I stopped after proving to myself that I could do it. It made we want an older and less expensive scope to play with, but if you find one the optics are usually finite rather than infinite.

Your equipment looks like it is well designed and solid enough to safely continue.
One other thing I found interesting with the dichroic mirror with the ability to rotate the mirror was that if the mirror is mounted between the illuminator and the condenser, as you rotate the mirror you can get the full spectrum of color filtration depending upon the angle of the mirror and the colors are very bright and vivid.

In finding such versatility I highly recommend experimenting with a dichroic mirror, no matter the shape or size.

You have taken the time to design a good adaptation and you are off to a good start. I encourage you to continue.

I have a metallurgical scope with epi lighting using a 30 watt halogen bulb and it is very tempting to mount a dichroic mirror in that apparatus. It and a fluorescence objective or two is all I would need to create a fluorescent scope, but once again, who does that to a $2000.00 microscope? I just can't convince myself to make modifications to the scope.
Enjoy the challenge. I certainly did.

Greg

Gasman
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Re: Epi illumination without Dichroic mirror maybe?

#4 Post by Gasman » Sat Jan 15, 2022 1:38 pm

Many thanks for the tips Greg!. I don't pretend for one minute that this setup will replace a Dichroic mirror but it does give some useable illumination and fortunately I'm not playing with a $1000 scope 😁.
Steve

Hobbyst46
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Re: Epi illumination without Dichroic mirror maybe?

#5 Post by Hobbyst46 » Sat Jan 15, 2022 2:05 pm

It would be difficult to imagine an alternative to dichroic mirror - at least for epi-fluorescence. A good-quality DM can even fulfill the whole fluorescence function without additional filters, or with just an added cheap long-pass emission filter.
About the required size: while not an elegant and perfect solution, a large and thin (say, ~1mm thick) dichroic mirror can be cut to smaller pieces.

MicroBob
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Location: Northern Germany

Re: Epi illumination without Dichroic mirror maybe?

#6 Post by MicroBob » Sat Jan 15, 2022 6:50 pm

How about this half silvered mirror: https://de.aliexpress.com/item/40011567 ... 4c4dQBVd0T

Chas
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Re: Epi illumination without Dichroic mirror maybe?

#7 Post by Chas » Sat Jan 15, 2022 7:57 pm

I know it sounds daft...but have you tried a coverslip ?

microb
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Re: Epi illumination without Dichroic mirror maybe?

#8 Post by microb » Sat Jan 15, 2022 8:23 pm

Chas wrote:
Sat Jan 15, 2022 7:57 pm
I know it sounds daft...but have you tried a coverslip ?
Camera set-ups use a sheet of glass, so a microscope slide or, as suggested, a cover slip would project the light and yet give a good image. The light source might have to be brighter is all. But in the camera rigs, ghosting doesn't seem to be an issue.

Also in your design, you want a black light-sink opposite the light source, something fuzzy or dark matte will do. It's probably not noticeable, but when a beam splitter is installed, the light hitting that blue plastic wall will bounce back and then 50% goes back into the light source, and 50% goes up to the camera adding possibly with your 3D print chassis blue giving a blue tint.

You can also get 50/50 Beamsplitter, 3.00mm BK7, 38mm x 25mm on ebay. Or as someone else suggested a large piece for a projection system on aliexpress. There is a person in California selling lots of 3 so that's $8 each. You'll probably not notice the QA flaws mentioned in that listing. Aliexpress also sells BK7 beam splitter prisms. Prisms are best if used with collimated light, not focusing light. Don't know if the conjugate planes for an epi design going with the collimated light planes count in that case.

Hobbyst46
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Re: Epi illumination without Dichroic mirror maybe?

#9 Post by Hobbyst46 » Sat Jan 15, 2022 8:24 pm

Chas wrote:
Sat Jan 15, 2022 7:57 pm
I know it sounds daft...but have you tried a coverslip ?
Slides and coverslips can serve as (less than optimal) beam splitters, but they do not perform the separation in wavelength between reflected and transmitted light, that is the essence of a dichroic mirror.

microb
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Re: Epi illumination without Dichroic mirror maybe?

#10 Post by microb » Sat Jan 15, 2022 9:12 pm

Hobbyst46 wrote:
Sat Jan 15, 2022 8:24 pm
Chas wrote:
Sat Jan 15, 2022 7:57 pm
I know it sounds daft...but have you tried a coverslip ?
Slides and coverslips can serve as (less than optimal) beam splitters, but they do not perform the separation in wavelength between reflected and transmitted light, that is the essence of a dichroic mirror.
Seemed like the original poster was just asking about basic lighting, and was implementing a vertical BF epi set-up with a ring mirror made with aluminum foil to get it.

But with two filter slots (one towards camera, one from the light source) a beam splitter that isn't just a visible spectrum 50/50 could be used to filter wavelengths in combo with the two filters. I don't know a good source for super cheap filters and dichroic mirrors made for specific wavelengths. They start getting expensive then. Although maybe with LED lighting, you could cut down the light source to what is needed (skipping one filter), and then maybe with some cheap high band and low band filters, you could build a stack seen from the camera. There are some notch filters on ebay that center around common laser colors. There are also some projector RGB filter cubes and laser combiner mirrors that a survey of common projector parts for epi could be an interesting article for someone to write based on testing cheap commercial parts with a spectrometer and maybe Thorlabs epi-fluorescence test slides. That'd be cool to know about.

Gasman
Posts: 153
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Location: North East UK

Re: Epi illumination without Dichroic mirror maybe?

#11 Post by Gasman » Sat Jan 15, 2022 10:24 pm

Interesting idea the cover slip will give that some thought although the light source would have to be much brighter of course thanks. Yes I have coated all the inside Matt black to remove reflections.
Actually thinking about it the upstreaming image would have to compete with the bright led light source coming down whereas the setup I have the light coming back up from the sample through the hole has an unrestricted path to the detector.
I am wondering what the specific purpose of the Dichroic mirror is as I gather it can reflect light of a certain wavelength but if excitation and emission filters are used anyway??.
Steve

Gasman
Posts: 153
Joined: Sat Jan 09, 2016 1:36 pm
Location: North East UK

Re: Epi illumination without Dichroic mirror maybe?

#12 Post by Gasman » Sat Jan 15, 2022 10:28 pm

Gasman wrote:
Sat Jan 15, 2022 10:24 pm
Interesting idea the cover slip will give that some thought although the light source would have to be much brighter of course thanks. Yes I have coated all the inside Matt black to remove reflections.
Actually thinking about it the upstreaming image would have to compete with the bright led light source coming down whereas the setup I have the light coming back up from the sample through the hole has an unrestricted path to the detector.
I am wondering what the specific purpose of the Dichroic mirror is as I gather it can reflect light of a certain wavelength but if excitation and emission filters are used anyway??.
Steve
Ps sorry just reread Hobbyst46’s excellent explanation!

MicroBob
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Location: Northern Germany

Re: Epi illumination without Dichroic mirror maybe?

#13 Post by MicroBob » Sun Jan 16, 2022 10:03 am

Hi Steve,
a dichroic mirror is used for fluorescence work, a half silvered mirror for normal epi immumination. A cover slip or foil works as a half silvered mirror, but only a little part is reflected down, the rest ends possibly as stray light. So a real half silvered mirror improves image quality here. I built a mirror insert for my Zeiss epi illuminator and didn't get a satisfying result with a cover slip, much better with the half silvered mirror.

Bob

Chas
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Joined: Tue Jan 19, 2021 3:11 pm

Re: Epi illumination without Dichroic mirror maybe?

#14 Post by Chas » Sun Jan 16, 2022 11:54 am

To be honest, I think that your foil-with-a-hole works pretty well.
It seems that the illuminators with the half-silvered mirrors are the ones where the light comes straight through the axis of the microscope and the user is viewing at right angles to this axis ..e.g. the Baker inverted metallic:
viewtopic.php?t=5700

The others,the majority, where the light comes in at right-angles and and the viewer is looking down the axis, seem to have had unsilvered reflector plates.
-Something to do with the transmission being greater than the reflectance, for a 45 degree glass plate and the need to maximise the light reaching the eyepiece.

I had a book that claimed that a thin film of nitrocellulose (liquid plaster?) was much better than a glass reflector ..I think it was painted onto a glass slide and floated off.

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