I have a proposal to prsent tomorrow on the quantification of Microplastic in the Philippines and one of the methods is using Nile Red thank you for this video. Very informative! Love from the Philippines!
Very interesting, congratulations! One question, is it useful to observe microplastics inside the insects after having depigmented the cuticle? Thank you!
Hi, thanks for the video. The sample needs to be in the dark after adding the nile red, until further analysis? How do you distinguish plastics from chitin, for example, or other organic material present in the sample?
The sample is best stored in the dark, or at least away from direct sunlight. Chitin is a potential problem that gives false negatives. Maes et al 2017 (Scientific Reports | 7:44501 | DOI: 10.1038/srep44501) consider that solvent density separation helps as chitin is denser than most plastic. By "organic", I assume you mean derived from living material and not carbon-based chemistry. In our samples most other stained objects are living plankton that are motile, or at least show intracellular structure, and are easily distinguished from microplastic. The main problem we had was from plant-derived cellulose fibers that did not show clear cellular structure. Nile Red did not stain these fibers.
Great video! Would you get a better signal-to-noise ratio if you used a glass filter over the objective lenses instead of plastic? I imagine some of the blue light bouncing off the dish will cause autofluorescence in the plastic filter itself, reducing contrast in the images?
The plastic filter may show some autofluorescence, but the main problem is that these cheap filters do not block all the blue light, so there is a high background of blue scattered light. Actually, this can be an advantage in this application because all objects can be seen either from their intrinsic scattering or from the yellow, orange or red fluorescence from bound Nile Red. It does however limit the detection of small weakly fluorescent objects.
Hello thank you for the video! I am currently conducting a similar experiment in which I am looking for microplastics in a water sample and I wanted to know if there are any specific colors that the microplastics might give off when observing using the nile red? Could you provide the link to purchase the yellow filter and a source that furhter explains why the yellow filter is best for identifying microplastics? Sorry for the long request!
The color of Nile Red fluorescence depends on the kind of plastic e.g. yellow for polyethylene and red for polyamide. A broad cut-off yellow filter only blocks the blue excitation light, allowing green, yellow and red light to be detected. See drive.google.com/drive/u/0/folders/18Qaq6wmz5V5iuGKYw6a1IEt33-dGnXrQ or email me for more details crb5@att.net
Is it possible to highly intensify the brightness of fluorescence of a given chemical?? I know this may sound a bit strange but I’m a big Star Wars fan and there is a huge market for custom made light sabers, these have polycarbonate blades with either a tri Cree LED in the hilt or a neo pixel strip running inside the blade (Please look up Neo Pixel light saver to see what I mean) with capabilities of colour changing … Now my theory for getting the blades to look a bit more ‘realistic’ (For the want of another term) than just an illuminated tube of plastic would be to add high intensity fluorescent chemicals and add an extra UV LED to the neo pixel strip, Now these blades would be colour locked to a chosen colour depending on the chemical added to the polycarbonate tube during manufacture unlike the standard neo pixel. So for example let’s say a red blade, the neo pixel strip would contain 3 red, 1 white & 1 high intensity UV (of a safe wavelength), orientated facing outwards from the epicentre of the polycarbonate tube with a defusing material of some kind The chemical would have to be translucent enough to allow the visible red light through the tube BUT the UV would make the chemical fluoresce (Highly, intensely hopefully IF the defusing material doesn’t hinder it) creating a red ‘hue’ around the blade … The same could be done with other chemicals to get other colours …. Does this sound feasible??
It should work if you can find the right fluorescent dye. This is the same principle used in washing powders that claim to be "whiter than white". The added brighteners absorb in the uv and fluoresce in the blue. However fluorescent light is relatively weak - it may only be effective in the dark and will not be as bright as colored LEDS.
Thank you for making this video and especially the protocol. This is going to help me in my research. I am from India and I am having problems with acquiring blue light source. I will really appreciate it if you share the link of website or details of the light source. Thanks!!
You need to use a saturated yellow filter, not a pale yellow correction filter. See www.amazon.com/gp/product/B0924F3B51/ref=ox_sc_act_title_1?smid=AEJSLTSVSZ3PF&psc=1
WAYLLSHINE Scalable Blue LED 3 Mode Long Range Blue Beam Blue Light www.amazon.com/gp/product/B01C2D7SRA/ref=ppx_yo_dt_b_asin_title_o04_s00?ie=UTF8&psc=1
In the video, we dissolved the Nile Red in 100% methanol to make the stock solution. We have also used 91% isopropyl alcohol (rubbing alcohol). We add around 20 ul of stock Nile Red to 5 ml seawater sample, so the final conc of alcohol is around 0.4%
Lots of table top tripods on Amazon (best to avoid those with flexible legs) e.g. www.amazon.com/Vanguard-VS-82-Table-Top-Tripod/dp/B0002J2TLC/ref=sr_1_21?crid=1C6GCMQIT35PU&dchild=1&keywords=table+top+tripod+for+camera&qid=1615682740&s=electronics&sprefix=table+top+tripod%2Celectronics%2C233&sr=1-21
@@muradurrashedin5409 Hi Muradur, I used a JVC camcorder attached to a wide field eyepiece as shown on my webpage: sites.google.com/ucsc.edu/cbagshaw/outreach/mbnms-plankton?authuser=0 . Unfortunately, my USB eyepiece camera that I use for bright field was not very sensitive in the red region and did not image weakly stained microplastics in plankton samples - it just about worked with strongly stained pieces of test plastic. A dSLR also worked as I could use long exposures (1/2 to 2 seconds) for weakly stained samples.
Unfortunately Nile Red is difficult to buy as an individual, but can be purchased by schools and colleges that have a business account with chemical companies. For small quantities (10 mg) MP biochemicals is a good source www.mpbio.com/0215174410-nile-red-cf but it can be obtained from other suppliers such a VWR. 10 mg is plenty as it needs to be between 1 and 10 microgram per ml in the sample .
I have a proposal to prsent tomorrow on the quantification of Microplastic in the Philippines and one of the methods is using Nile Red thank you for this video. Very informative! Love from the Philippines!
Best of luck!
This video is very helpful. Thanks for posting!
Very interesting, congratulations! One question, is it useful to observe microplastics inside the insects after having depigmented the cuticle? Thank you!
Hi, thanks for the video.
The sample needs to be in the dark after adding the nile red, until further analysis?
How do you distinguish plastics from chitin, for example, or other organic material present in the sample?
The sample is best stored in the dark, or at least away from direct sunlight. Chitin is a potential problem that gives false negatives. Maes et al 2017 (Scientific Reports | 7:44501 | DOI: 10.1038/srep44501) consider that solvent density separation helps as chitin is denser than most plastic. By "organic", I assume you mean derived from living material and not carbon-based chemistry. In our samples most other stained objects are living plankton that are motile, or at least show intracellular structure, and are easily distinguished from microplastic. The main problem we had was from plant-derived cellulose fibers that did not show clear cellular structure. Nile Red did not stain these fibers.
I keep getting recommended this because I watch nilered the UA-camr..
Sorry, but our video is about Nile Red, the dye which I think came before the UA-camr - but we are glad he came up with these T-shirts.
Great video! Would you get a better signal-to-noise ratio if you used a glass filter over the objective lenses instead of plastic? I imagine some of the blue light bouncing off the dish will cause autofluorescence in the plastic filter itself, reducing contrast in the images?
The plastic filter may show some autofluorescence, but the main problem is that these cheap filters do not block all the blue light, so there is a high background of blue scattered light. Actually, this can be an advantage in this application because all objects can be seen either from their intrinsic scattering or from the yellow, orange or red fluorescence from bound Nile Red. It does however limit the detection of small weakly fluorescent objects.
Hello thank you for the video! I am currently conducting a similar experiment in which I am looking for microplastics in a water sample and I wanted to know if there are any specific colors that the microplastics might give off when observing using the nile red? Could you provide the link to purchase the yellow filter and a source that furhter explains why the yellow filter is best for identifying microplastics? Sorry for the long request!
The color of Nile Red fluorescence depends on the kind of plastic e.g. yellow for polyethylene and red for polyamide. A broad cut-off yellow filter only blocks the blue excitation light, allowing green, yellow and red light to be detected. See drive.google.com/drive/u/0/folders/18Qaq6wmz5V5iuGKYw6a1IEt33-dGnXrQ or email me for more details crb5@att.net
Is it possible to highly intensify the brightness of fluorescence of a given chemical??
I know this may sound a bit strange but I’m a big Star Wars fan and there is a huge market for custom made light sabers, these have polycarbonate blades with either a tri Cree LED in the hilt or a neo pixel strip running inside the blade (Please look up Neo Pixel light saver to see what I mean) with capabilities of colour changing … Now my theory for getting the blades to look a bit more ‘realistic’ (For the want of another term) than just an illuminated tube of plastic would be to add high intensity fluorescent chemicals and add an extra UV LED to the neo pixel strip, Now these blades would be colour locked to a chosen colour depending on the chemical added to the polycarbonate tube during manufacture unlike the standard neo pixel. So for example let’s say a red blade, the neo pixel strip would contain 3 red, 1 white & 1 high intensity UV (of a safe wavelength), orientated facing outwards from the epicentre of the polycarbonate tube with a defusing material of some kind The chemical would have to be translucent enough to allow the visible red light through the tube BUT the UV would make the chemical fluoresce (Highly, intensely hopefully IF the defusing material doesn’t hinder it) creating a red ‘hue’ around the blade … The same could be done with other chemicals to get other colours …. Does this sound feasible??
It should work if you can find the right fluorescent dye. This is the same principle used in washing powders that claim to be "whiter than white". The added brighteners absorb in the uv and fluoresce in the blue. However fluorescent light is relatively weak - it may only be effective in the dark and will not be as bright as colored LEDS.
Thank you for making this video and especially the protocol. This is going to help me in my research. I am from India and I am having problems with acquiring blue light source. I will really appreciate it if you share the link of website or details of the light source. Thanks!!
Hi Kshitij - send me an email to crb5@att.net and I will send you details
hi.. i wanna ask. what the specific yellow film that covering on microscope lens? can you suggest me to find it on marketplace?
You need to use a saturated yellow filter, not a pale yellow correction filter. See www.amazon.com/gp/product/B0924F3B51/ref=ox_sc_act_title_1?smid=AEJSLTSVSZ3PF&psc=1
Hi great video! Can you post a link to the LED torch. Thanks
WAYLLSHINE Scalable Blue LED 3 Mode Long Range Blue Beam Blue Light
www.amazon.com/gp/product/B01C2D7SRA/ref=ppx_yo_dt_b_asin_title_o04_s00?ie=UTF8&psc=1
How many ml of methanol is needed to dissolve the nile red dye?
We dissolved 1 milligram Nile Red in 1 ml of methanol (isopropyl alcohol can also be used)
What percentage of ethanol was used
In the video, we dissolved the Nile Red in 100% methanol to make the stock solution. We have also used 91% isopropyl alcohol (rubbing alcohol). We add around 20 ul of stock Nile Red to 5 ml seawater sample, so the final conc of alcohol is around 0.4%
- #POW #OOTUS #king
hi, can i get the links to buy tripod equipments?
Lots of table top tripods on Amazon (best to avoid those with flexible legs) e.g. www.amazon.com/Vanguard-VS-82-Table-Top-Tripod/dp/B0002J2TLC/ref=sr_1_21?crid=1C6GCMQIT35PU&dchild=1&keywords=table+top+tripod+for+camera&qid=1615682740&s=electronics&sprefix=table+top+tripod%2Celectronics%2C233&sr=1-21
@@clivebagshaw2805 Thanks for your reply. I will use that. Just want to know how you took the pictures? Which camera setup you used?
@@muradurrashedin5409 Hi Muradur, I used a JVC camcorder attached to a wide field eyepiece as shown on my webpage: sites.google.com/ucsc.edu/cbagshaw/outreach/mbnms-plankton?authuser=0 . Unfortunately, my USB eyepiece camera that I use for bright field was not very sensitive in the red region and did not image weakly stained microplastics in plankton samples - it just about worked with strongly stained pieces of test plastic. A dSLR also worked as I could use long exposures (1/2 to 2 seconds) for weakly stained samples.
Where can I buy nile red?
Unfortunately Nile Red is difficult to buy as an individual, but can be purchased by schools and colleges that have a business account with chemical companies. For small quantities (10 mg) MP biochemicals is a good source www.mpbio.com/0215174410-nile-red-cf but it can be obtained from other suppliers such a VWR. 10 mg is plenty as it needs to be between 1 and 10 microgram per ml in the sample .
😮