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.

As described at 3 minutes 20 seconds

Yes, any filter paper will give increased scattering of the excitation light and will need a high quality emission filter to block this. Some filter papers also fluoresce. Other researchers have used polycarbonate track-etch (PCTE) filters to minimize interference with the signal from microplastics. We have only look at microplastics suspended in water in glass dishes or slides. With a stereo microscope it is important to raise the sample dish above the ground glass plate with a spacer to minimize scattering from the plate.

If your water sample has a lot of sediment you can let the sample stand for several minutes - most plastics have as density a bit higher or lower than water and will settle slowly or float to the top. You can also add salt to make the solution denser so practically all plastics will float and most of the sediment will sink.

It should work with the right blue filter. The important thing is that the yellow emission filter blocks most of the blue light i.e. the two filters together should be almost black. Also it is important to focus the LED torch to give a small and bright a spot on the sample as possible using an additional lens.

The width of a standard slide is 1 inch/2.5 cm, so any ring a bit smaller than this should work

​​@@clivebagshaw2805thank you for your response

​@@clivebagshaw2805So how will be the calculation of the density with this dimension?

@@nadjimahmed531 The volume of sample within the o-ring is given by Pi r-squared d, where r is the radius and d the thickness of the ring.

@clivebagshaw2805 great thank you again. It's really useful.

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

@@clivebagshaw2805 I really appreciate for your prompt response.

What do you mean by "it"?

That black circle, what's its name? I tried it today in the laboratory, but I did not find better results. Perhaps I was not able to imitate you well

@@KechidKenzaa-gz9xc They are called O-rings obtainable at hardware stores or on-line (e.g. www.amazon.com/Assortment-Plumbing-Automotive-Mechanic-Connections/dp/B0BJJM91G4/ref=sr_1_19?crid=27NKCBIU57GXU&dib=eyJ2IjoiMSJ9.rMalqnjJK4ptQgIJLXaeol0CN2hMWmSikj0mL1o6aGlH5g0-8UIIJyiXwOtEuSjqxBKzqwW-IMcVi-_6pHMGryygvNELJ9HPp-0vgETRlN_inr_LJkckcwHABGCxr4WD1YOlBKwzdIFHapbaxmIKQghMH8lKh-Z-aK-GKxunq28ntR_F0x6W0id9ajHGwhK3aVH3o7b3rkDwG9otfOWrci5jI2GN2pDwC7mfw3j6JUg.-Gz4sNxGwVz7M4HdpxvjHgJhkWh9_xkTiSzZhAnAiXM&dib_tag=se&keywords=O+rings&qid=1711297114&sprefix=o+rings%2Caps%2C248&sr=8-19 What results were you looking for? The main challenge is making a waterproof seal. O-rings come in different thicknesses - the bigger the depth, the more volume you can examine but the image quality will be reduced.

@@clivebagshaw2805 Thank you very much for the explanation. We will repeat the phytoplancton observation using exactly them

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.

Sorry, I don't have any direct experience with paracetamol. I can only suggest slowing down crystal growth by reducing the evaporation rate (e.g. in a partially closed container)

Thank you, I will certainly try that. The covering glass didn't really worked as the paracetamol cristals started to grow outside of the edge of covering piece. Do you know any other substances that grow interesting cristals? I've tried all typical household accessible chemicals. Take care

I haven't explored crystals much but have seen some interesting patterns when sea water dries out on a slide sfmicrosociety.org/features/living-in-a-polarized-world@@RadBudaspectrum-photography

@@RadBudaspectrum-photography You can try sugar, cafein, vanillin, aspirine, citric acid, salt, borax, boric acid to name common ones.

It might be difficult to use a fluorescence spectrophotometer because Nile Red will emit a low background fluorescence from the solvent that might swamp that from the microplastics. Under a microscope with a thin sample (<2 mm) the stained plastic particles can easily be seen and quantified. Also with our own samples, some plankton are stained with Nile Red (especially zooplankton) and we can visually separate them from plastic, but in a bulk sample in a cuvette this would not be possible

what about the nanoplastics, how can we quantify them?

do you have any suggestions on how to use fluorescence spectrophotometer for quantification of microplastics?

@@PRIYANKASEHRAWAT-d2f No. You would be better off using a cheap microscope with a blue LED light source than using a spectrophotometer that cannot distinguish the fluorescence from particles and solvent.

Polarizing film sheet $9.40 www.amazon.com/gp/product/B004X3XFHU/ref=ppx_od_dt_b_asin_title_s00?ie=UTF8&psc=1 or two circularly polarizing filters at $9.99 each www.amazon.com/Fotasy-Multi-Resistant-Coating-Circular-Filter/dp/B00BAH9AX0/ref=sr_1_11?crid=98NRMY3NYDTZ&keywords=37+mm+polarizing+filter&qid=1684971202&s=electronics&sprefix=37+mm+polarizing+filter%2Celectronics%2C176&sr=1-11

I have only seen them a couple of times (in 2021) - we sample weekly at the Santa Cruz wharf

@@clivebagshaw2805 I wonder if it was correlated with oceanographic metrics, or just abundance of a prey they like, or both?

@@chir0pter The water temperature was 17.1 degrees C, which was near the maximium for that year and 1 degree warmer than the year before

@@clivebagshaw2805 cool, so perhaps a trip down to Sea of Cortez or Baja coast and one could find them?

@@chir0pter No need to travel! An Erythropsidinium showed up in today's sample from the Santa Cruz wharf. Water had warmed up to 14.8 degrees (from 12.3 two weeks ago)

How are you detecting microplastics? How big are they? In favorable cases you can use automatic methods for counting: see www.mdpi.com/2673-4532/4/1/4 For flotation of plastics using NaCl, it is best to use saturated NaCl - it doesn't matter if you have excess salt as it will settle with the soil at the bottom of the tube, while most plastics will float.

@@clivebagshaw2805 appearantly there no Mps on the surfaces but they are detected by microscope adsorbed on my plant sample. For microplastics seperation from sediment sample is challenging for me. Also nile red dye and its protocol related tools are not available for me. For what size of MPs density seperation is effective because they are no larger microplastics that can be seen by naked eye. Also guide me please what to do after adding saturated salt. How to carry those invisible microplastics by density seperation and how to detect them? By simple electric light microscope? Because the only microscope i have is electric light microscope.

@@ham1633 Microplastics usually float in saturated salt solution regardless of their size. After mixing NaCl to soil sample, allow the soil to settle, then suck off the surface layer with a pipette and transfer to a microscope slide. If there are no visible plastics, add some larger pieces of polythene to the sample to check the floatation method is working. Microplastics less than 1 mm in size are difficult to identify just by looking under a microscope. Try using cross-polarizers to examine the sample: kzbin.info/www/bejne/n3-xnJd-rtubmNU Polarizers can be obtained from Amazon: www.amazon.com/Polarizing-Film-Sheet-Gadget-Electronics/dp/B004X3XFHU/ref=sr_1_1_sspa?crid=9X8OOP59XNT1&keywords=polarizing+film+sheet&qid=1681402074&sprefix=polarizing+%2Caps%2C284&sr=8-1-spons&psc=1&spLa=ZW5jcnlwdGVkUXVhbGlmaWVyPUEyM1BIUk04TVpKQUMxJmVuY3J5cHRlZElkPUEwODI3ODE0MzE1Ulo2WlcyV1E2TiZlbmNyeXB0ZWRBZElkPUEwMjQ5ODY0UVg0WDFUVVpDSVQ5JndpZGdldE5hbWU9c3BfYXRmJmFjdGlvbj1jbGlja1JlZGlyZWN0JmRvTm90TG9nQ2xpY2s9dHJ1ZQ== Plastic usually show up as bright or colored objects in polarized light - this method is not infallible, which is why Nile Red staining is a useful additional method. Research labs use infrared or Raman microscopy to be more certain particles are made of plastic.

@@clivebagshaw2805 Do i need to do chemical digestion after density seperation method? I mean Can i use chemical digestion for Microplastics obtaining from both plant and sediment sample.

@@ham1633 You will probably need a digestion step for sediment samples. I have not done this for our plankton samples as we do not have lab facilities. We find Nile Red stains zooplankton, especially their membranes and fat stores, but they do not stain phytoplankton very well because the cell wall slows down Nile Red absorption. Nile Red does not stain fragmented fibers of cellulose, which helps distinguish them from synthetic fibers.

The Nile Red dye was purchased from MP Biochemicals via VWR Ltd. A stock solution of 1 mg/mL in methanol was prepared. Alternatively, Nile Red was dissolved in 91% isopropyl alcohol (rubbing alcohol) available from a local pharmacy. 1 drop (0.02 ml) of Nile Red stock solution was added to 5 ml seawater sample. Formula is at www.mpbio.com/us/nile-red

But if I wanna purchase the nile red then to I have to know whether it same or not ... So please can you tell me the molecular formula of nile red from the bottle which is used??

@@sayrammukri2890 There is no formula on the Nile Red bottle - just a molecular wt = 318.4. The formula of Nile Red is given en.wikipedia.org/wiki/Nile_red

Thank you that uh helped me, but tell yr Nile red dye was already in liquid form pr powder form...?? And then uh made stock solution by adding methanol?

There was 1 mg of dry powder in the bottle as supplied and I added 1 ml of methanol to it, to make the stock solution. There is not enough to weigh out. When dealing with small amounts it is best to dissolve in the estimated volume and then check the concentration from the absorption spectrum if you want a more accurate value.