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Blue Fluorescence and blue color

  1. May 20, 2016 #1
    Hello,

    I have read that blue fluorescent dyes do not exist. Why?

    Optical brighteners do convert UV into blue light but a piece of plastic containing optical brightener dyes appears clear and transparent instead of blue? Why?

    Does the color of the substance not correspond with the fluorescent emission wavelength?

    thanks,
    fog37
     
  2. jcsd
  3. May 21, 2016 #2

    Simon Bridge

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  4. May 21, 2016 #3
    I will provide the citation. But speaking with dye suppliers, they all said that a true substance that is blue in color and fluorescent (in the blue) does not exist. They usually use a regular blue dye mixed with some optical brightener.

    Optical brightners, for example, are colorless but become bluish under strong UV illumination. But even in that case, their color is not a deep blue. My point is that the color of a fluorescent substance is not determined by the color of its emitted fluorescent light. The true color is the combination of the reflectance spectrum and fluorescent emission spectrum...
     
  5. May 21, 2016 #4

    Ygggdrasil

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    There are plenty of substances that fluoresce in basically every region of the visible spectrum: https://www.thermofisher.com/us/en/...uor/alexa-fluor-dyes-across-the-spectrum.html

    Many fluorescent dyes do not appear by eye to be the color that they emit. Rather, the absorption of light dominates the color we see. For example, the fluorescent dye Cy3, which absorbs green light and emits orange light, appears red because it is absorbing green light (and thus appears as red, the complimentary color of green).
     
  6. May 21, 2016 #5

    Simon Bridge

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    I see what you mean - you are asking if a substance can flouresce the same color as it's diffuse color... specifically blue.
     
  7. May 21, 2016 #6
    Hi ygggdrasil,

    Thank you.

    Let me

    Every substance has a reflectance spectrum which tells us how much % of energy at a specific frequency is reflected back into the environment. The rest is absorbed and converted to heat unless the substance is fluorescent. In that case a portion of the absorbed energy is converted into light which adds up to the reflected light.

    If the substance is red and the fluorescent emission is red as well, the substance will have bright red color. But, as you mention, it is possible for the emitted fluorescent light to be different than the reflected light. Your example: the fluorescent dye Cy3 absorbs green light and emits orange light (this is the fluorescent mechanism). You mention the dye powder looks red. The reflectance spectrum is complementary of the absorption spectrum. From the fluorescence standpoint, there is an activation spectrum (equal to a portion of the absorption spectrum) and emission fluorescent spectrum... is that correct?

    I have found several fluorescent powders that absorb in the UV and emit blue light but their appearance is light yellow. There are some yellow powders that in low concentration emit a greenish fluorescent light....

    The moral of the story is that the color of a fluorescent substance does not correspond to the color of the emitted fluorescent light.
     
  8. May 21, 2016 #7

    Ygggdrasil

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    Yes. The absorption will almost always outweigh the fluorescence of a substance in determining the color of the dye as not all photons that get absorbed are re-emitted via fluorescence.

    In case you are interested, here's a photo from my lab work of a plate containing solutions of Cy5 dye (left) and Cy3 dye (right). You can see look up the absorption and emission spectra of the dyes here: http://web.stanford.edu/group/bioconfocal/dyes.html#oragnegreen
    2013-01-19 12.10.26.jpg
    Cy5 absorbs in the orange-red range, fluoresces in the red-near IR range, and appears blue. Cy3 absorbs green, emits orange, and appears red.
     
  9. May 21, 2016 #8
    Thank you!

    Fluorescent dyes used in commercial plastics and textiles have a peak fluorescent emission wavelength that is close enough to the peak wavelength of the reflected light. That is why a yellow fluorescent marker look so bright. But the cyanine dyes you mention, don't seem to be useful to produce bright colors. Cy3 emits a fluorescent orange light which does not really reinforce the reflected light....in fact it looks red....

    How do we find fluorescent dyes whose fluorescent emission light reinforces the reflected light? what criterion (species, etc.) should I follow?
     
  10. May 21, 2016 #9
    One more and last thing if I may:

    When a specific fluorescent dye is chosen for a certain biological application and only its fluorescent emission light is of interested, how do we avoid the fluorescent light to be mixed with the reflected light? How do we keep the fluorescent emitted light separate from the reflected light (light that is not absorbed)?

    The reflected light seems to always be there unless we the illuminating incident light is completely absorbed ( that would be a black substance) and part of it is converted into fluorescent light...
     
  11. May 21, 2016 #10

    Ygggdrasil

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    Right, the application for the cyanine dyes is fluorescence microscopy, in which case it is useful to have a large Stokes shift (difference between the absorbption max and emission max) so that we can separate the light used for excitation from the emitted fluorescence.

    For a blue dye where the emission reinforces the reflected light, you would want something that absorbs in the orange area but emits in the blue region of the spectrum. Obviously, this is impossible as the emitted light must have a lower energy (larger wavelength) than the absorbed light.
     
  12. May 21, 2016 #11

    Ygggdrasil

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    See my reply above. For a more comprehensive explanation see: http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html

    Basically, you use dyes with a large Stokes shift, so that the emitted light has a lower wavelength than the excitation light. You can then use optical filters to block the higher wavelength excitation light from the lower wavelength emitted light.
     
  13. May 21, 2016 #12
    Thank you! I made great progress today in my understanding. In essence:

    Given a uniform white illuminating light, a non-fluorescent substance has a certain absorption spectrum and a reflection spectrum (which is simply the difference between the white light incident spectrum and the absorption spectrum).

    A fluorescent substance, besides the absorption and reflectance spectra, also has a fluorescent excitation spectrum and a fluorescent emission spectrum. If some of the incident light (white light in our case) falls in the excitation spectrum, that light (with a certain % efficiency) will be converted into some new light at longer wavelength (indicated by the fluorescent emission spectrum). The emitted fluorescent light will always superimpose with the reflected light.

    Example:
    1) Non-fluorescent green apple: the absorption spectrum comprises the blue, yellow and red incident wavelengths. The green wavelengths will be reflected. Hence the color green.
    2) Fluorescent green apple: the absorption spectrum comprises the blue, yellow and red incident wavelengths. The green wavelengths will be reflected. Hence the color green. But some of the blue incident light will fall on the fluorescent excitation spectrum and possibly be converted into fluorescent green light which adds to the reflected light. We get a brighter green apple.

    Is there some sort of relationship between the absorption spectrum and the fluorescent excitation spectrum? The fluorescent excitation spectrum does not need to fall inside the absorption spectrum, correct? What is the rule of thumb?

    thanks again. I am very happy and grateful for today....

    fog37
     
  14. May 21, 2016 #13

    Merlin3189

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    I don't know details of any specific flourophores that might be candidates for the OP requirements, but I don't think this is strictly true. With two photon flourescence the excitation can be at longer wavelength than the emitted light. Eg.
     
  15. May 21, 2016 #14

    Ygggdrasil

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    For molecules, the absorption and emission spectra are roughly symmetric, and this has to do with how vibrational states in the ground state overlap with vibrational states in the excited state. The relationships involve some complicated quantum mechanics, which I'm not sure I understand well enough to explain succinctly. However, here's a useful page on Wikipedia explaining some of the principles: https://en.wikipedia.org/wiki/Franck–Condon_principle

    This is true. However, the OP's application is commercial plastics and textiles and these are unlikely to see the light intensities required to see significant multi-photon effects.
     
  16. May 21, 2016 #15
    Thanks to everyone. It was fruitful afternoon.

    -- Ygggdrasil, when purchasing a fluorescent dye both the fluorescent excitation spectrum and fluorescent emission spectrum are always provided with other tech specs (dye strength, solubility, quantum yield, linewidth, etc.) What about the absorption spectrum and related reflectance spectrum of the fluorescent substance? I don't think they are provided. Usually the dye is in powder form and its color is given. Maybe the powder color is all we need to predict the reflectance spectrum. Is that correct? I have searched for fluorescent dyes whose fluorescent light is the same as the light deriving from the reflection process (i.e. the light not absorbed). How would look for those types of dyes?

    -- Fluorescence is not just activated by light having wavelengths within the excitation spectrum. I think the fluorescent mechanism takes place only if there is a certain threshold amount of energy at the wavelengths in the excitation spectrum. Is that correct? Reflection, instead, is independent on the intensity of the incident light.

    -- Optical brighteners are dyes that absorb in the UV and fluoresce in the blue. If we introduce an OB into a clear plastic, the plastic will still look transparent which means the optical brighteners are transparent dyes (i.e. the dye does not absorb in the visible. The reflectance spectrum is zero and all light is transmitted except a portion which converted in the blue fluorescence). There are fluorescent dyes (which are not OBs) that also absorb in the UV and emit in the blue visible region. They appear as light yellow powders.
     
  17. May 21, 2016 #16

    Ygggdrasil

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    Usually the emission and excitation spectrum are provided (at least for the dyes I typically purchase for microscopy applications). The absorption spectrum is essentially the same as the excitation spectrum. The reflectance spectrum is generally not provided, but you would probably be able to get a good idea of it from the absorption spectrum. I'm not sure where would be the best place to look for dyes with the properties you are after.

    At normal light intensities, the intensity of fluorescence should vary linearly with the intensity of excitation light. There is no thresehold intensity of light required to observe fluorescence. Similarly, the amount of reflected light will vary linearly with the intensity of incident light.

    It would make sense if some of the blue emitting dyes appear yellow. These dyes are likely absorbing some light in the violet region of the spectrum in addition to the UV, which would make them appear yellow (the complementary color to violet).
     
  18. May 24, 2016 #17
    Hi Ygggdrasil,

    I was looking at a list of fluorescent dyes. I found some whose excitation spectrum is in the IR and their fluorescent emission in the IR as well. What color should we expect the dyes to be? The absorption is probably in the red-NIR. Under white light illumination, these dyes should look cyan (white - red= cyan if the three color proportions are the same)....
     
  19. May 24, 2016 #18

    Ygggdrasil

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    Yes, that sounds about right. I've worked with the NIR dyes Cy7 and Alexa750 (excitation max ~ 750 nm), and both of these have a pale blue-green color in solution.
     
  20. May 24, 2016 #19
    Thanks!

    I have also been thinking about fluorescent dyes that emit green light and appear green. My little search shows that fluorescent dyes that in powder form are yellow color emit a greenish fluorescent light. I have not found a green fluorescent dye that emits green fluorescent light.

    I think the fact that the powder is yellow and the fluorescence is green is due to self-absorption (re-absorption). Is self-absorption the same as quenching?
    My understanding is that self-absorption is the absorption of emitted fluorescent light by the same species that produced it. This effect happens within the overlap wavelength region of absorbance and fluorescence spectrum. The higher the dye concentration (= higher absorbance), the more re-absorption will occur. Within the overlapping part of absorbance & fluorescence, the emitted fluorescence gets reabsorbed (and re-emitted in consequence). As a result, the short-wavelength part of the emission spectrum gets diminished and the overall result is that the apparent emission maximum is hampered and shifted to greater wavelengths (from green to yellow). The absorbing wavelengths take fluorescence away from the overlapping region, while the re-emitted fluorescence covers the complete fluorescence spectrum.

    Emission spectrum independence of the excitation wavelength: the shape of the fluorescent emission spectrum is independent of the excitation wavelength, correct? If the excitation light was purely monochromatic with wavelength lambda_1 (within the excitation spectrum), the emitted fluorescent light would still contain all the wavelengths of the emission spectrum. What if we illuminated the dye with light having a different wavelength lambda_2 that is still part of the excitation spectrum? Would the fluorescent emission spectrum still be the same? What differences would arise?
     
  21. May 25, 2016 #20

    Ygggdrasil

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    Self-absorption and quenching are different, and I suspect quenching–not self absorption–is affecting the fluorescence of your compounds. quenching is the general name for a number of phenomena that reduce yield of fluorescence emission from a fluorophore. Essentially, an excited molecule wants to get back to the ground state. Fluorescence is one path for the molecule to return to the ground state, but it is not the only path. For example, if the molecule is fairly floppy, it can rotate, twist and otherwise change its conformation, which will re-arrange the energy levels of the molecule to allow the molecule to slowly dissipate the extra energy as heat (this process is called internal conversion). The quantum yield of the dye reflect how many molecules return to the ground state through emission of a photon versus alternative paths to the ground state that do not involve fluorescence.

    Quenching comes about when another substance provides a non-radiative path for the excited molecule to return to the ground state. Sometimes this comes about through electrostatic interactions between molecules (as in the case of Förster resonance energy transfer), but many of these quenching pathways depend on direct physical interactions between fluorescent dye molecules. In particular, fluorescent dyes can engage in intermolecular interactions that change their properties — including their absorption and emission spectra, as well as their quantum yield. Often, these intermolecular interactions between dyes severely reduce the quantum yield of the dyes.

    Thus, many fluorescent dyes will be non-fluorescent in the solid state, where you have many of these dyes packed together and interacting with each other. These powders can also have very different spectra than cases where you have the dye dissolved in solution or dispersed throughout a different solid.

    Yes, the emission spectrum is independent of the excitation wavelength.
     
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