Stereo Microscope : Fluorescence Adapter

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MichaelG.
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Stereo Microscope : Fluorescence Adapter

#1 Post by MichaelG. » Thu Nov 01, 2018 5:28 pm

I've just received an eMail which included a link to this:
https://www.emsdiasum.com/microscopy/pr ... htsea.aspx

At a quick glance, it looks
(a) Very promising, and
(b) Simple enough to inspire a DIY project

Any thoughts, fellow tinkerers ?

MichaelG.
.
.
Edit: This colourful brochure is dated 2015, so it's been around for longer than I thought
https://www.emsdiasum.com/microscopy/pr ... e_2015.pdf
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wporter
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Re: Stereo Microscope : Fluorescence Adapter

#2 Post by wporter » Thu Nov 01, 2018 8:59 pm

You're right to call for DIYers, that's a very expensive setup! Nice, though.

If one wants to experiment for VERY cheap, I have used a $5 (but pretty bright) Chinese-made LED UV flashlight. It puts out @ 365nm, fine for say, DAPI, or any of the common fluorescent things around the house: paper, dried urine, dust. Some green algae shows up as red; clear pond water seems like a translucent colloid under UV. One could attach a flashlight like this to a stand to illuminate a dish under a stereoscope.

I always use a pair of amber blue-blocker sunglasses when playing with UV; cheaper than making or buying a UV shield for one's microscope, and it seems to block OK (but use at your own risk, etc).

Here's a small UV flashlight (that looks the same as the one I bought a few years ago):

https://www.ebay.com/itm/UV-WF-501B-LED ... 0005.m1851

Hobbyst46
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Re: Stereo Microscope : Fluorescence Adapter

#3 Post by Hobbyst46 » Thu Nov 01, 2018 9:10 pm

Nice catch MichaelG. Astonishing product, yet is very simple, could be conceived much earlier probably.
I did not look into the details yet, so here is my initial response.
The simplicity of the accessory is achieved by using a LED for excitation light, and this has several advantages over the classic mercury and xenon lamps.
The main advantage is that an excitation filter is not essential.
Further, this arrangement does away with the dichroic mirror. This is possible partially because the excitation beam is not coaxial with the optical axis, but is sideways directed towards the specimen. I am not sure how the observed fluorescence is, how clean from excitation traces, but for the declared purpose and use of the microscope it may suffice.

Is it a possible DIY project? yes, IMHO, provided that you invest in high quality emission interference filters (these can cost 100-200 USD each, depending on size, maker etc). A colored cellophane film will not do. I would also be concerned about eyesight safety when using a UV excitation light. It is important to remember, that the excitation beam must be intense, since fluorescence intensity is very low and is proportional to the excitation intensity. I would start with 10W LEDs at least (my guess). It is also important that the bandwidth of the LED be narrow - otherwise, the addition of an excitation filter will be a serious disadvantage, price-wise (and attenuation of the excitation beam).

And, of course, the chief limitation is that for every type of fluorescence you need a separate LED lamp and emission filter.

A slight elaboration of the above may be in order. On a "biological" epi-fluorescence microscope, the excitation light is reflected off the specimen and back through the objective towards the eyepiece. Unless a dichroic mirror is installed at a 45 angle, the observer (or camera) will see a mixture of the reflected excitation and fluorescence, but the former is more intense, by several orders of magnitude, so the latter is masked. The (long known) solution is a combination of an emission filter and a dichroic mirror at 45 deg above it. Those components are installed within a "cube".
In the present disclaimed accessory for a stereo microscope, if the excitation beam is inclined, so will be the reflected beam (or most of it), which will not enter the objective, so a good emission filter might be adequate.

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Re: Stereo Microscope : Fluorescence Adapter

#4 Post by MicroBob » Thu Nov 01, 2018 9:40 pm

Hi Michael,
I'm sure that it's possible to assemle a fluorescence kit for a halfway acceptable sum. The problem with safety and amount of light can be reduced significantly when a microscope without eyepieces is used, but a camera screen or bigger monitor instead. On ebay I see frequently funny looking filters but the problem is, when they are useful and the seller knows it they aren't cheap. :roll: With the others it will take some frustration tolerance to get a useful set together.

Do you have specific objects that you would like to observe in fluorescence?

Bob

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Re: Stereo Microscope : Fluorescence Adapter

#5 Post by MichaelG. » Thu Nov 01, 2018 10:03 pm

Thanks everyone for the responses so far
MicroBob wrote:Do you have specific objects that you would like to observe in fluorescence?
Not really, Bob ... It's just that this set-up [although still pricey] looks like a big step in the 'affordable' direction, compared with what's available for 'research' instruments: and, of course, it's for the stereo microscope.

... I just 'threw a pebble in the pond' to get us all thinking.

MichaelG.
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Re: Stereo Microscope : Fluorescence Adapter

#6 Post by Hobbyst46 » Thu Nov 01, 2018 10:16 pm

Hi MichaelG,
Bob's question at "what to look at" is highly relevant!
DAPI and other specific fluorescence stains are not abundant outside research/diagnostic labs (I think). Fluorescent proteins are even rarer... on the other hand, when I turn off all my apartment lights, turn on the UV lamp (the home UV tube that I use for curing adhesives) and place a flask of virgin olive oil (NOT any vegetable oil!) near it, it glows in red. To see this red glow of the chlorophyll in a leaf, you need an excitation light of 400-430nm, although 360-380nm UV will also be quite effective, and perhaps a flashlight can work, and indeed, paper glow is revealed under UV. But to get a good clean fluorescence without interference, from tiny microscopical objects, I believe the setup should be better. I hope I am not being didactic!

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Re: Stereo Microscope : Fluorescence Adapter

#7 Post by MichaelG. » Thu Nov 01, 2018 11:11 pm

Hobbyst46 wrote:Hi MichaelG,
Bob's question at "what to look at" is highly relevant!
[ ... ]
But to get a good clean fluorescence without interference, from tiny microscopical objects, I believe the setup should be better. I hope I am not being didactic!
Not at all didactic
Your comments, and Bob's question, are entirely reasonable ... But the fact is that I don't yet know what I might want to view:
I was simply excited to discover that someone has produced a flourescence kit for stereo microscopes. ... That falls into the category of 'a solution seeking a problem'

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Re: Stereo Microscope : Fluorescence Adapter

#8 Post by MicroBob » Fri Nov 02, 2018 9:16 am

Hi Michael,
you might just start with the combinations of light source and filters you are able to aquire. When they are happy to sell you a complete lamp and filter holder with stand, housing, instructions, web page at a profit for 1100$ it can't be too complicated.

With UV light is is also good to be aware that the glue used to cement lens groups might not always withstand it in longer use. So perhaps not use the beloved Wild stereo with planapo objective. :D

Bob

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Re: Stereo Microscope : Fluorescence Adapter

#9 Post by MichaelG. » Fri Nov 02, 2018 9:56 am

MicroBob wrote:Hi Michael,
you might just start with the combinations of light source and filters you are able to aquire. When they are happy to sell you a complete lamp and filter holder with stand, housing, instructions, web page at a profit for 1100$ it can't be too complicated.
Exactly my thoughts, Bob
With UV light is is also good to be aware that the glue used to cement lens groups might not always withstand it in longer use. So perhaps not use the beloved Wild stereo with planapo objective. :D

Bob
:shock: :(

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Re: Stereo Microscope : Fluorescence Adapter

#10 Post by Hobbyst46 » Fri Nov 02, 2018 10:20 am

MicroBob wrote:With UV light is is also good to be aware that the glue used to cement lens groups might not always withstand it in longer use. So perhaps not use the beloved Wild stereo with planapo objective.
Correct me if I am wrong, but with the proposed layout of the fluorescence setup, the objective is not really exposed to UV light. The excitation UV lamp stands at the side of the microscope and illuminates the specimen at an angle of maybe 45-60 degrees. So UV light is reflected from the specimen at the same angle and not upwards towards the objective. The small amount of UV irradiation that, because of refraction, could pass through the objective is absorbed by the emission filter that is located between the objective and the specimen (or better yet, reflected back downwards, if the emission filter is an interference filter, which I would strongly recommend).

The heart of the "invention" is that, in contrast to classical epi-fluorescence, the excitation is not sent to the specimen through the objective.
Last edited by Hobbyst46 on Fri Nov 02, 2018 11:53 am, edited 1 time in total.

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Re: Stereo Microscope : Fluorescence Adapter

#11 Post by MicroBob » Fri Nov 02, 2018 10:57 am

Hi Doron, of cause you are right! So the objective itself is quite safe in this setup.
Bob

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Re: Stereo Microscope : Fluorescence Adapter

#12 Post by Hobbyst46 » Fri Nov 02, 2018 11:48 am

From the literature I learned that, prior to the widespread use of epi-fluorescence microscopy, transmitted light fluorescence was practiced. An example is seen among the specifications of the Reichert Biovert, kindly posted by wporter (Thu Nov 01, 2018 4:14 pm) in a previous post:
viewtopic.php?f=24&t=6606
I do not know whether trans-illumination fluorescence accessories included a dichroic mirror; I guess that a mirror could be installed in the same position as in epi-fluorescence setups, only without the separate excitation source.
Last edited by Hobbyst46 on Fri Nov 02, 2018 3:18 pm, edited 1 time in total.

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Re: Stereo Microscope : Fluorescence Adapter

#13 Post by wporter » Fri Nov 02, 2018 3:14 pm

Older microscopes (Leitz Ortholux, Reichert Univar, etc) that featured transmitted-light fluorescence mostly had the excitation filter at the lamp housing, and no dichroic, just a (yellowish) barrier filter slid into a slot somewhere between the objective and the viewing head.

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Re: Stereo Microscope : Fluorescence Adapter

#14 Post by Hobbyst46 » Fri Nov 02, 2018 10:29 pm

MichaelG. wrote:(b) Simple enough to inspire a DIY project
Any thoughts, fellow tinkerers ?
Hi Michael,
Having looked once again at the brochure and photos of the barrier filters (emission filters), I am now convinced that these are indeed interference filters, and considering their relatively large diameter, they should be expensive.
But, having said my previous recommendation about interference filters, I now think that if one is keen on DIY this fluorescence accessories, it might be a reasonable idea to try and start with simple absorption filters. What one needs is LP filters, that is longpass, that block the wavelengths below the specified level (say below 400nm for the violet). And pass all longer wavelengths. Such filters are available, for example, from Schott (Germany) and should be significantly cheaper than interference filters - and their lifetime is much longer. But, they are not as "absolute" - some light may leak even outside the allowed range.

Hence, if I were keen on fluorescence, and say that my chief interest is fluorescing chlorophyll (say), I would construct a high power (10W LED) violet excitation lamp (400/425nm), and buy a used 530LP or 550LP filter, thickness 3-5mm (the Red fluorescence peaks at 610-650nm). And try it out. So the expense is not excessive if the idea fails.

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Re: Stereo Microscope : Fluorescence Adapter

#15 Post by MichaelG. » Sat Nov 03, 2018 8:55 am

Hobbyst46 wrote:
MichaelG. wrote:(b) Simple enough to inspire a DIY project
Any thoughts, fellow tinkerers ?
Hi Michael,
Having looked once again at the brochure and photos of the barrier filters (emission filters), I am now convinced that these are indeed interference filters, and considering their relatively large diameter, they should be expensive. ... [/i]
Good morning Doron

I've just been looking at the videos, and I am satisfied that the large barrier filters, and the even larger 'sheilds' are made from polycarbonate, and [in each set] the filter material is the same for both items.
... Specifically stated, about 50 seconds into this video:
https://www.emsdiasum.com/microscopy/te ... _vid3.aspx

With patience, we can probably 'reverse engineer' the whole thing. :D

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Re: Stereo Microscope : Fluorescence Adapter

#16 Post by Hobbyst46 » Sat Nov 03, 2018 10:08 am

Hi Michael

I am happy to participate in mind-share, on the theoretical level, since I do not have a stereo microscope.
Some preliminary thoughts though, on key points for this "project":
1. The LED should have a narrow beam. I know that small 5mm dome LEDs of 8, 30, 60 degrees or other angles are available. Here I guess a 10-20degrees is better, for a collimated beam. But I do not know about the options of beam width of higher power LEDs.
2. The barrier filter material can be acrylic, polycarbonate, glass, coated or uncoated, provided that its transmittance spectrum is satisfactory (although I conservatively believe that glass is still the highest quality for optics).
3. The diameter or edge size of the filter should be large enough for the objective. Some mfg. only supply 12.5 or 25mm diameter filters.
4. If the filter diameter is slightly larger than the objective diameter, it may become possible to magnetically attach the filter, by gluing 3 tiny neodymium magnets around the filter rim.
4. In my opinion, the viewing filter - the rectangular one - does not need to be identical to the main barrier filter. It is only there to protect the eyes against the intense excitation (if I am not wrong).
5. The barrier filter carrier ring is really a simple mechanically simple piece that can be fabricated from plastic, and covered with a thin steel/iron flat annulus, as anchor for the filter magnets. Still, this is fabrication on a lathe machine.
:?
BTW the guy in that film says that the two filters are made from the same material, but I did not hear WHAT that material is, or what type of filter it is. Polycarbonate is certainly a possibility; I had the feeling that it is coated.

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Re: Stereo Microscope : Fluorescence Adapter

#17 Post by MichaelG. » Sat Nov 03, 2018 3:00 pm

Hobbyst46 wrote:Hi Michael

I am happy to participate in mind-share, on the theoretical level, since I do not have a stereo microscope.
[ ... ]
BTW the guy in that film says that the two filters are made from the same material, but I did not hear WHAT that material is, or what type of filter it is. Polycarbonate is certainly a possibility; I had the feeling that it is coated.
Many thanks for your interest, Doron ... mind-sharing sounds good !!

Sincere apologies for my referencing error : The explicit disclosure of their use of "optical grade Polycarbonate" is not in the video that I linked, but is in this one:
https://www.emsdiasum.com/microscopy/te ... _vid1.aspx

My best guess is that this will be the C39 / CR-39 material used by Cokin, and others, for camera 'system' filters ... also commonly known as 'optical resin'
https://cokinfilter.com/pages/about

MichaelG.
.
.
PostScript ... https://en.m.wikipedia.org/wiki/CR-39 includes this 'helpful' note:
Although CR-39 is a type of polycarbonate, it should not be confused with the general term polycarbonate, a tough homopolymer usually made from bisphenol A.[3]
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Re: Stereo Microscope : Fluorescence Adapter

#18 Post by Hobbyst46 » Sat Nov 03, 2018 4:38 pm

Hi Michael,
Yes, indeed, the recent link you posted indicates they are polycarbonate. Moreover, from the looks of the filters, I now believe (99% sure) that they are colored plastic filters, that is, absorption filters, rather than interference filters as I previously thought.
However, I think that the material and technology of the filter are not critical for our purpose. What matters in my opinion are the size, transmittance spectrum, and price. High quality filters from the big makers like Newport, Chroma, Omega, Oriel, Edmunds, Schott, should be around 50-100 USD apiece I guess. These are makers of optics for research, like spectroscopy. I am afraid that makers of photographic filters (Hoya? Kenko?) do not supply wavelength edge filters (cutoff like longpass, shortpass) but a more gradual and shallow transmittance slope.
There is a UK supplier called (appropriately for the UK!) :) Knight Optical. Their list shows a nice choice of wavelengths and fairly decent prices, along with relevant spectra, and they look good to me - except that their diameter is 25mm only.

I briefly scanned eBay for long pass filters. Found, for example,

Optical Longpass Filter, 550LP, 10.1mm dia. X 3mm thick (EB00348B) (made by Omega Optical). From the USA. The cost is only 12 USD + H&S expenses. But it is a very small size.

In general, I think that used longpass optical filters are unlikely to appear on the market.

Popular sizes of optical spectroscopy filters (as opposed to camera lens filters) are 25mm/1" diameter circles and 50-51mm squares. Here is a link to a Japanese supplier of good-quality, longpass, 50mm square, glass absorption filters from Schott and Hoya, selling for about 67 and 47 USD, respectively (+taxes and additional costs):

https://www.shibuya-opt.co.jp/eng/Color ... ter01.html

Definitely more expensive than the acrylic filters from Knight; yet less expensive than interference ones.

One more issue of interference filters: the coating deteriorates with time, even when the filter is stored unused.

I forgot to comment about the thickness of the filter. They come in various thicknesses. The thicker the filter, the lower the transmittance at all wavelengths! from my experience, though, 2-3mm is fine. The spectrum given in the mfg. specs usually applies to the shown filter, it is usually NOT normalized to the thickness.

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Re: Stereo Microscope : Fluorescence Adapter

#19 Post by MichaelG. » Sat Nov 03, 2018 8:11 pm

Doron

I think it's time to 'sanity check' my understanding of the NightSea kit:

If you know that I have something wrong, please correct me.
If you think that I have something wrong, please offer an alternative for discussion.

1. Mechanical
1.1 The light sources are interchangeable modules, which attach to a gooseneck stand using BNC connectors. BNCs are convenient, and offer good electrical and mechanical characteristics.
1.2 The 'barrier filter' and 'viewing shield' are both mounted to a simple adapter. We need not discuss the mechanical details.

2. Electrical
2.1 The light heads are LED based, and low voltage DC powered
2.2 The gooseneck supplies power via its BNC connector, and is externally fed with DC

3. Optical
3.1 The light head, although LED based, includes an 'excitation filter' [for some emission wavelengths, I think it may be possible to omit this]
3.2 There is no visible evidence of a lens system in the light heads.
3.3 The 'barrier filters' are mostly 'long-pass' devices, and are made from CR-39 [or similar]. The exception is the 'Green Only' bandpass filter described on p3 of the brochure.

4. Purchasing
4.1 The various components can be supplied separately, but an essential starter kit contains the gooseneck stand, the microscope attachment device, and one matched set of light head + barrier filter + viewing shield.
4.2 Further matched sets, and other accessories are available.

Sorry to labour this, but by presenting it this way it should be easier for you to insert corrections or additional notes.

Thanks
MichaelG.
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Re: Stereo Microscope : Fluorescence Adapter

#20 Post by Hobbyst46 » Sat Nov 03, 2018 9:12 pm

MichaelG. wrote:If you know that I have something wrong, please correct me.
If you think that I have something wrong, please offer an alternative for discussion.

1. Mechanical
1.1 The light sources are interchangeable modules, which attach to a gooseneck stand using BNC connectors. BNCs are convenient, and offer good electrical and mechanical characteristics.
Yes. I have always used BNC connectors to reliably transmit signals, never seen them as high current conductors, so I can only assume they are OK for this as well.
1.2 The 'barrier filter' and 'viewing shield' are both mounted to a simple adapter. We need not discuss the mechanical details.
OK.
2. Electrical
2.1 The light heads are LED based, and low voltage DC powered
2.2 The gooseneck supplies power via its BNC connector, and is externally fed with DC
Yes.
3. Optical
3.1 The light head, although LED based, includes an 'excitation filter' [for some emission wavelengths, I think it may be possible to omit this]
The necessity of an excitation filter depends on the wavelength range supplied by the LED. AFAIK, a 20nm bandpass as shown in the specs of the Nightsea can be met with LEDs without excitation filters, so I am not sure that the Nightsea illumination heads include excitation filters. For our purpose, I would omit all excitation filters. At the worst, it can be added later.
3.2 There is no visible evidence of a lens system in the light heads.
I do not know. But again, there should be narrow angle LEDs, so we can omit the collimating optics.
3.3 The 'barrier filters' are mostly 'long-pass' devices,
Yes.
and are made from CR-39 [or similar polymer]
Yes, but for our purpose, they could be made from any plastic or glass that provides the correct transmittance spectrum.
The exception is the 'Green Only' bandpass filter described on p3 of the brochure.
Not quite. This bandpass filter is suggested to isolate green from red fluorescence. For example, the Nightsea presenter describes GFP; I know vegetable oils, which fluoresce in both regions, but at different intensities, such that mutual masking may result. Hence this green-only filter is supposed to block all light of wavelength shorter than blue-green OR longer than green-orange. It is a bandpass filter. BUT it can be made from the same material as the longpass filters. What I am not sure about is whether it is an absorption type or interference type. It is possible to make absorption bandpass filters, but they do not perform as well as interference bandpass filters. It is perfectly OK to use filters from different makes and of different types, however, within the same set. What I suggest about the filters is to first of all, define what wavelength AND what physical size - and that depends on the microscope and objective diameter.
4. Purchasing
4.1 The various components can be supplied separately, but an essential starter kit contains the gooseneck stand, the microscope attachment device, and one matched set of light head + barrier filter
Yes.
+ viewing shield.
Yes as sold by Nightsea, but not necessarily for us. The viewing shield can be any color filter that blocks the strong sideways excitation light. It is convenient to have it the same as the barrier filter, perhaps, but not an absolute requirement. What counts is what you see through the eyepieces, the shield mainly protects your eyesight.
4.2 Further matched sets, and other accessories are available.
Yes.
Sorry to labour this, but by presenting it this way it should be easier for you to insert corrections or additional notes.
I take pleasure here, it is plenty of fun.

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Re: Stereo Microscope : Fluorescence Adapter

#21 Post by MichaelG. » Sat Nov 03, 2018 10:03 pm

3.3 [ ... ]

The exception is the 'Green Only' bandpass filter described on p3 of the brochure.
Hobbyst46 wrote: Not quite. This bandpass filter is suggested to isolate green from red fluorescence. For example, the Nightsea presenter describes GFP; I know vegetable oils, which fluoresce in both regions, but at different intensities, such that mutual masking may result. Hence this green-only filter is supposed to block all light of wavelength shorter than blue-green OR longer than green-orange. It is a bandpass filter. BUT it can be made from the same material as the longpass filters. What I am not sure about is whether it is an absorption type or interference type. It is possible to make absorption bandpass filters, but they do not perform as well as interference bandpass filters. It is perfectly OK to use filters from different makes and of different types, however, within the same set. What I suggest about the filters is to first of all, define what wavelength AND what physical size - and that depends on the microscope and objective diameter.
Thanks for the very useful responses [I think this process could work nicely!]
I will just mention that, after writing, I discovered that they have another 'wavelength set' which includes a bandpass filter. ... See Table_1 here:
https://www.emsdiasum.com/microscopy/te ... FA-43.aspx

Edit: Confusingly, the list on this page indicates otherwise:
https://www.emsdiasum.com/microscopy/pr ... htsea.aspx
... here, Royal Blue is shown with a long-pass filter


Your point about wavelength, filter technology, and physical filter size is well made ... Most stereo microscopes would probably need a filter of 40mm to 50mm diameter so that rather limits the affordable technologies. It seems to me that NightSea has made expedient, value-for-money, decisions; but that certainly won't prevent me hoping to find something exotic at a bargain price. ;)

Let's keep this going for a while.

Thanks again
MichaelG.
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Re: Stereo Microscope : Fluorescence Adapter

#22 Post by Hobbyst46 » Sat Nov 03, 2018 10:44 pm

The only source of confusion is the name of the set. Nightsea offer two sets with the same excitation. One ("Royal Blue") is excitation 440-460nm, barrier 500nm longpass. The other ("RB-GO", or "GO", or "RB" - they apparently had a dispute over the name:lol:) is THE SAME excitation, 440-460nm, bandpass 500-560nm. There are just two sets along the way and one of them includes a bandpass filter.

I notice, by the way, that the Nightsea inventors checked the usefulness of their device with a huge number of biological (or biophysical) stains: FITC, Texas Red mCherry, Alexa,..... and many many others and fluorescent proteins. I think that the 6 sets they offer as default were tailored to meet popular fluorescent markers. This is interesting, considering that the device is aimed for stereo microscopes rather than biological microscopes, with which live cells are usually monitored. Maybe I should ask colleagues about it.
Last edited by Hobbyst46 on Sat Nov 03, 2018 10:45 pm, edited 1 time in total.

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Re: Stereo Microscope : Fluorescence Adapter

#23 Post by MichaelG. » Sat Nov 03, 2018 10:45 pm

It may be a trick of the light [note there is a green barrier filter fitted to the microscope] but the light unit on the table appears to have a dichroic filter.
Screen-grab from YouTube
Screen-grab from YouTube
CSIMG_6_1.jpg (135.47 KiB) Viewed 15109 times
I will keep looking for something more definitive.

MichaelG.
.
.

https://m.youtube.com/watch?time_contin ... fullscreen
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Re: Stereo Microscope : Fluorescence Adapter

#24 Post by MichaelG. » Sat Nov 03, 2018 10:54 pm

Hobbyst46 wrote:The only source of confusion is the name of the set. Nightsea offer two sets with the same excitation. One ("Royal Blue") is excitation 440-460nm, barrier 500nm longpass. The other ("RB-GO", or "GO", or "RB" - they apparently had a dispute over the name:lol:) is THE SAME excitation, 440-460nm, bandpass 500-560nm. There are just two sets along the way and one of them includes a bandpass filter.

I notice, by the way, that the Nightsea inventors checked the usefulness of their device with a huge number of biological (or biophysical) stains: FITC, Texas Red mCherry, Alexa,..... and many many others and fluorescent proteins. I think that the 6 sets they offer as default were tailored to meet popular fluorescent markers. This is interesting, considering that the device is aimed for stereo microscopes rather than biological microscopes, with which live cells are usually monitored. Maybe I should ask colleagues about it.
Thanks for clarifying that ... and grateful for any additional expert opinion
... It's perhaps worth noting that they suggest the use of the stereo microscope for low-cost 'screening' prior to serious work on more exotic equipment.

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Re: Stereo Microscope : Fluorescence Adapter

#25 Post by Hobbyst46 » Sat Nov 03, 2018 11:20 pm

MichaelG wrote:It may be a trick of the light [note there is a green barrier filter fitted to the microscope] but the light unit on the table appears to have a dichroic filter
Hawk-Eye Michael!!
Indeed both light units on the table give this impression. This is another possibility that I did not consider before: That the illumination head includes an interference excitation filter. IF this is the case - it might mean either: (1) that their "monochromatic" LEDs span a broader wavelength range than the declared 20nm (for example - 440-460nm), and they trim it with interference filters; or (2) alternatively, that the lamp is a WHITE LED, the same one in all the heads, and in each light head they simply isolate the excitation wavelength range by means of the interference filter. Against option 2 is the spectrum of white LEDs as I know it, where the intensity varies a lot along the visible light scale. But these are mere speculations. I guess that their design is dictated by many factors, not only by the physical performance of the components.

So, I cannot decide which is it - option 1 or 2 - or another unknown design.

Actually, the design is affected also by the purpose of the user. For example, if you want to observe only fluorescein fluorescence, you need to excite at about 495nm, but your emission is at 515nm! so you MUST have efficient separation between them! (the difference between exc and em wavelengths is called the Stokes shift). To overcome the Stokes shift, you must cutoff the excitation very efficiently at 500nm, say. An excitation filter is required.
However, if you excite with UV (365nm), and you want to observe all nice fluorescence at 410nm (and above), the Stokes shifts are large, a good monochromatic LED will serve you without excitation filter. To see vitamin E (~550nm) and chlorophylls (~630nm), for example, when you excite with UV, the Stokes shifts are huge, and any reasonable UV LED will do without excitation filter; only that IF the specimen by chance emits in the violet (390-400nm) as well, there will be some interference from the excitation.

BTW, that is how research confocal fluorescence microscopy is set-up: one selects, from within the software, the filters and dichroic mirrors that will provide both intense excitation AND efficient separation between excitation and emission.

So, as long as we limit the purpose to easily separable excitation and emissions, the question of to add or not to add (excitation filter) depends on the availability of powerful, monochromatic LEDs, either of a narrow beam or with an added collimator.

Edit: We did not discuss the necessity of a dimmer. Perhaps the Nightsea goose-neck is equipped with a dimmer. I am sure we can do without it. With fluorescence, usually, the brighter the excitation the better, with two exceptions: (a) a too intense light beam can damage the (live) organism, and (b) a too light beam can cause "photo bleaching" - a chemical change to the fluorescing molecule. These two factors, IMO, are not expected with the stereo microscope, since the illumination is not really focused (since we do not see any lenses between the light head and the specimen).
Cheers.

MichaelG.
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Location: North Wales

Re: Stereo Microscope : Fluorescence Adapter

#26 Post by MichaelG. » Sun Nov 04, 2018 11:58 pm

MicroBob wrote: [ ... ]
Do you have specific objects that you would like to observe in fluorescence?
:idea: This would be an ideal subject to make good use of the relatively low resolution of the stereo microscope:
https://www.researchgate.net/publicatio ... h_Nile_Red

MichaelG.
.


Edit: https://en.m.wikipedia.org/wiki/Nile_red

Reality check ... I have just seen the price of Nile Red stain :o
https://www.sigmaaldrich.com/catalog/se ... us=product
Too many 'projects'

Hobbyst46
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Re: Stereo Microscope : Fluorescence Adapter

#27 Post by Hobbyst46 » Mon Nov 05, 2018 2:30 pm

MichaelG wrote:This would be an ideal subject to make good use of the relatively low resolution of the stereo microscope:
Hi Michael,

I do not know whether you had access to the article, so I did a quick survey of the article main text and supporting information.

Their setup is functionally quite like that of the Nightsea, but it is DIY and is even simpler. They used Crimelite (460-510nm), which to me looks like a LED flashlight without any additional optics, and the "barrier" filter was a simple absorption orange filter. Not any interference filter IMO. They taped the orange filter to the bottom of the objective. The scope itself is "Sanven CNC3020T three-axis micro-milling/engraving machine" which they modified by replacing the milling head (or whatever it was, not my field of knowledge) with a Motic scope, which I guess (from the photos) was a stereo microscope. The LED light was clamped somehow to illuminate the 47mm specimen as uniformly as possible. They used a motorized X-Y stage and software to scan the specimen and take the photos, although the process was mostly manual rather than automated. The camera was Canon EOS 600D.

The Nile Red stain is indeed peculiar in that the fluorescence spectrum strongly depends on the polarity, and when it coats a plastic particle - depends on the type of plastic - PE, PP,Nylon etc. For example, red fluo from Nile Red on Nylon but yellow on HDPE. One of the worst contaminants that I know is polystyrene. Assuming that by the abbreviation PS they mean polystyrene (and not polysulfone for example), Nile REd on it shines yellow, peak at around 570nm. A fairly large Stokes shift.

The price of Nile Red - well, SigmaAldrich prices have always been high... You only need a tiny amount, though, here is a possibly cheaper option:
https://www.scbt.com/scbt/product/nile-red-7385-67-3

Why work with Nile Red? I suspect that these authors hoped that this specific dye, among its other features, will aid in identification of which polluting plastic is the particles they detect. However, this goal was not quite achieved. In addition, they claim that Nile Red is "more efficient" than some other dyes (Eosin B, Rose Bengal and others), they apparently tested; I did not find any data to support this claim in the article text or the SI. Presumably, this claim originates from some other studies on the subject.

However, this does not mean that Nile Red is the only dye that can be used for a similar purpose. Unless one wants to repeat their exact experiments, why not use a different, less expensive, dye that stains plastic?

Another advantage of Nile Red, they claim (and provide substantiation for that) is that it does not stain algae, so it is a selective indicator of plastic and can be used to analyze a raw water sample. This is an important achievement, yet the authors do not say how selective are the other dyes.

Yet another point is that the staining protocol with Nile Red was the result of trial and error. Possibly, such experiments with other dyes will lead to acceptable results.

So far did I get. If you wish to know more details I can try to fetch.

P.S. I just saw that Crimelite offers a multiband flashlight! perhaps the equivalent of Nightsea, except that it is mobile!
their headoffice is in the UK.
Last edited by Hobbyst46 on Mon Nov 05, 2018 3:14 pm, edited 1 time in total.

MichaelG.
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Location: North Wales

Re: Stereo Microscope : Fluorescence Adapter

#28 Post by MichaelG. » Mon Nov 05, 2018 3:14 pm

Hobbyst46 wrote:Hi Michael,

Have you read the article in the link?

Assuming you had access only to the abstract and external references, I made a quick survey of the article as well as the supporting information.
[ ... ]
Thanks for taking the trouble, Doron ... But the article is "open access" so:
Yes, I have read [or at least skimmed] the full paper, and the supporting information; and saved it for more thorough reading.

I much appreciate your well-informed input !!

MichaelG.
.
.
The reference to a "Milling Machine" surprised me at first ... but they mean a little CNC engraver/router, rather than a workshop mill.
From the 'supporting information'
From the 'supporting information'
CSIMG_7_1.jpg (209.01 KiB) Viewed 14909 times
Too many 'projects'

Hobbyst46
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Joined: Mon Aug 21, 2017 9:02 pm

Re: Stereo Microscope : Fluorescence Adapter

#29 Post by Hobbyst46 » Mon Nov 05, 2018 3:43 pm

I could not recognize the Crimelite in the photos of the apparatus. Can you?

MichaelG.
Posts: 3976
Joined: Mon Apr 10, 2017 8:24 am
Location: North Wales

Re: Stereo Microscope : Fluorescence Adapter

#30 Post by MichaelG. » Mon Nov 05, 2018 5:59 pm

Hobbyst46 wrote:I could not recognize the Crimelite in the photos of the apparatus. Can you?
Sorry ... No
The 'Foster and Freeman' website is inaccessible at the moment but I found a brochure elsewhere:
http://www.bvda.com/images/content/down ... (UK)-1.pdf

My guess is that they used one of those ^^^
[see page 3 of the brochure "Blue/Green" version]
and simply substituted it for the little desk-lamp.

MichaelG.
.
Edit: Feast your eyes on the full Crime-lite range:
http://www.bvda.com/en/crime-lite-light-sources

Edit: Perhaps this smaller one would suffice:
http://www.bvda.com/images/content/down ... L2(UK).pdf
... but the paper does mention 'high powered'
Too many 'projects'

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