Fluorescence microscopy for around $20

[@p16able]

Thank you, Clive! This is fascinating and incredibly helpful work. Have you considered using this to observe nile red stained lipid-rich live organisms, or do you think it might pose a challenge?

[@clivebagshaw2805]

Yes, Nile Red stains membranes and lipid stores of zooplankton very efficiently - see Fig 5d of our paper at drive.google.com/file/d/1w3IJQj8bxUSranxTw7dIPdsFwbv4zM8J/view?usp=sharing . Fortunatley, it does not appear to harm the organisms in the short term and they carry on swimming, so it is easy to distinguish them from microplastics which were the subject of this investigation.

[@alexandrevaliquette3883]

I've look for this video a lot and only KZbin accept to show it to me almost one years after my initial search! Not the best sound quality or video editing and not the few millions views like a cat video.. But very nice content! This is amazing content creator that is taking the time to share his experience with the world. 5 years later, this video is still excellent in my standards Thank you very much for your generosity. Alex, your fellow amateur microscopist from Montréal!

[@sadiebaker5111]

Thank you so much for the detailed explanations! Genius!

[@FulvioGa]

A lot of information in this video!

[@williammauzey8342]

Nice presentation Clive!

[@clivebagshaw2805]

Glad you liked it!

[@rberdan1]

We use something similar in research to record fluorescence in neurons injected with fluorescent dyes - only any 12 V or bright light works by place a BG -12 (Blue Green) filter in front o the light (no yellow filter required). Of course the filters cost more then your set up but can be used with any bright flash light to view fluorescence with a stereoscope and a yellow filter is not needed. Other UV lights also work and is used to view lichen, rocks etc.

[@clivebagshaw2805]

The yellow filter is used to block the scattered (blue) excitation light. You may get away without it for strongly fluorescence samples which have a low scatter. The cheap yellow filters used in this demonstration block most of the scattered light, but a blue background is observed for Nile Red-stained plastic fragments because of Rayleigh scattering. For highest sensitivity, research grade emission filters with an optical density > 4 at the excitation wavelength are required (OD > 6 for single fluorophore detection).

[@RATAMUR1]

Es cierto que añadirle un filtro de color azul QB24 - BG12 se emplea para fluorescencia de bajo coste. Enlaces: canadiannaturephotographer.com/diffential_interference_microscopy.html es.aliexpress.com/item/32889032226.html?spm=a2g0s.9042311.0.0.24ef63c0OB0YTk

[@clivebagshaw2805]

@@RATAMUR1 If you already have a bright white light source, then adding a blue filter in front will also work. But it is more efficient to use a blue LED in the first place because this reduces the problem of other wavelengths breaking through a blue filter. LEDs are cheaper than quality filters. Best results (i.e. selectivity of fluorescence over scattered light) would be obtained using a narrow band LED (or laser) in combination with a sharp bandpass excitation and emission filters.

[@natedoggraymond]

Thank you so much! Brilliant!

[@clivebagshaw2805]

Glad it was helpful!

[@JustDux]

Looks like I can use (or make) dark field condenser, replace microscope LED with UV LED and put yellow film on cover slip

[@clivebagshaw2805]

That might work. I tried putting a blue filter in front of the built-in LED and using a dark field patch stop but it was not as bright as using a separate focused LED flashlight.

[@rajumeganathan3275]

Thank you for your explanation. Excellent work Mam. I have one doubt. Can we adopt this method to quantify Microplastics in soil extract, Leachate (other than freshwater)? We will remove organic content prior to quantification.

[@clivebagshaw2805]

Hi Raju . The microscopy should work, The biggest challenge is to remove all the organic content. There are several papers in the literature which describe the required treatments (e.g. using H2O2 oxidation) but we have not tried them because our seawater samples do not have too much background signal.

[@rajumeganathan3275]

Thank you mam

[@KutaySir]

Hello, do you think yellow filter works with 395 nm flashlight and rhodamine B florescent?

[@clivebagshaw2805]

No. Rhodamine B absorption maximum is around 550 nm so you need a green flashlight and the emission peak is around 580 nm which requires an orange-red filter

[@KutaySir]

@@clivebagshaw2805 I really appreciate for your prompt response.

[@WarMarsM]

Is there any particular yellow filter you recommend?

[@clivebagshaw2805]

The ones I used are no longer available but these look similar: www.amazon.com/LingoFoto-Filter-Orange-Pockets-Cleaning/dp/B08ZK9J737/ref=sr_1_14?crid=1CTRB6MXPCN41&keywords=37mm%2Bacrylic%2Bcolor%2Bfilters&qid=1652151191&s=electronics&sprefix=37%2Bmm%2Bacrylic%2Bcolor%2Bfilters%2Celectronics%2C159&sr=1-14&th=1

[@johndubose6589]

Nice, very simple!

[@legoworks-cg5hk]

Wouldn't a uv light be better?

[@clivebagshaw2805]

That depends on the fluorophore. We used this set up to look at Microplastics stained with Nile Red dye for which blue is best. For autofluoresence would be good.

[@irishguy200007]

Why a yellow filter

[@clivebagshaw2805]

To block the blue light scattered by the sample that would swamp the fluorescence signal (lets through green, yellow and red)

[@xkachlara5616]

hallo i'm chlara from indonesia. i wanna ask to you, how about the wavelength LED? in my country it's difficult to find led blue light in 450 nm wavelength only 395 nm. is it recommendation to use or not?

[@clivebagshaw2805]

395 nm does not work so well with Nile Red, but can be used to excite chlorophyll fluorescence. For 450 nm, we used the WAYLLSHINE Scalable Blue LED 3 Mode Blue Light Flashlight ordered from Amazon

[@xkachlara5616]

@@clivebagshaw2805 could you give me recommendation for amazon's store? because there are many wayllshine options

[@clivebagshaw2805]

Try www.amazon.com/gp/product/B01C2D7SRA/ref=ppx_yo_dt_b_asin_title_o04_s00?ie=UTF8&psc=1

[@albertsiburian6406]

Makasih kak sudah mewakilkan gua ❤️