Hello I was watching an introductory video to two-photon microscopy and came across the concepts of Rayleigh scattering and absorption. As I did not understand these concepts entirely, I would greatly appreciate if you could help me out.
Raileigh scattering
Apparently, it is possible for a photon to get deflected or scattered when it runs into something (i.e. a molecule) that is significantly smaller than its wavelength. Rayleigh scattering in particular indicates that the perceived/relative light intensity after scattering is proportional to 1/lambda^(4). This means that the smaller the wavelength, (or the higher the frequency of light), the more scattering takes place. And conversely, the greater the wavelength, the smaller the scattering, which explains why in two-photon microscopy a laser in the red and near-infrared region is used to image biological samples as opposed to blue/short wavelength light. My question is: why is this? Why can a photon be deflected or scattered by something that is smaller than its wavelength?Does it have to do with the fact that its high frequency makes it more likely to hit molecules?
Absorption
Apparently, light absorption by biological molecules is high in the visible light region, but becomes lower in the region of the red and even lower in the infrared region. So the region in the far red and the near infrared (650-1300 nm) constitutes a good region of wavelengths that can be used for imaging biological samples with a two-photon microscope.
Why do biological (macro) molecules such as hemoglobin and melanin as well as other molecules tend to absorb light in the region of visible light? Why is red and near infrared light not absorbed by biological molecules?
Quick recommendation:
1. Rayleigh scattering is the scattering without changing of the wavelength of the scattered light.
2. Necessary to have in mind that the spectrum of the scattered radiation is determined by the energy of excitation of atoms and/or molecules (and depends on the respective energy levels in atoms and/or molecules).
3. If the molecules are irradiated with monochromatic light with frequency ω0, the intensity of the Rayleigh scattering will occur with the same frequency ω0. When Raman scattering occurs, arise new frequencies in the scattered radiation.
4. For example, the Raman spectra are observed vibrational, rotational, and vibrational-rotational. It is also possible Raman spectrum, wherein the frequency difference represents the energy of electron excitation of molecules. In this case, the combination lines in the spectrum are arranged symmetrically with respect to the unshifted (Rayleigh) line with ω0.
5. Usually power of Rayleigh scattering much more than the power of Raman scattering. Therefore, if the signal of the last few shifted in frequency with respect to the Rayleigh line, it is difficult to register.
6. When considering the various media, such as biological tissues, it is necessary to see their specific scattering and absorption spectra.
References (e.g.)
[1] Craig F. Bohren, Donald R. Huffman, "Absorption and Scattering of Light by Small Particles". - New York: Wiley.
[2] Bruce J. Berne, Robert Pecora, "Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics" (Dover Books on Physics). - New York: Wiley.
Dear Paolo!
Typically Physics prefers to answer on question with word "how " instead "why" ...Neverthless Your last question about biomolecule` love to green light is not true because overall green light of mother-Nature (Flora) provides absorption with injoy red part of Sun power, which corresponds to maximum sun power that reach us after passage of atmosphere. And biomolecule named сhlorofylle has energetic structure that provides the strong dipole singlet-singlet absorption of red light. Gemoglobine absorbs green 530nm because its function differs from chlorofylle: it transfer oxigen in blood of primates while chlorofylle is responsible for photosynthes. However let us return to our oven as people say. Light scattering and absoption are described in the frame of quantum mechanical approach and it isn't nessesary to apply to terms "small" or "big" wavelength in compare to molecules. Pragmatically it is beneficial to undestand what advantages provides two-photon illumination in microscopy. You have connected it with more week light scattering of red and near infrared light and it is true. Two-photon similar one-photon absorption is followed exitation of molecules which can respond on it with luminescence. It is luminescence microscopy.
If my remarks occurs to be usefull for You I would be satisfied.
The charge cloud associated with electrons in atoms and molecules will become distorted in response to incoming electromagnetic waves, oscillating like an electric dipole, and thus absorbing and re-emitting radiation randomly in a dipole pattern (assuming symmetry). The response of the charge cloud to different frequencies of light varies according to the material, and is called the polarizability. The intensity of this response governs the intensity of Rayleigh scattering. We know that accelerated charges emit, and shorter wavelengths cause the dipole moment to oscillate (and thus accelerate) faster. This is why the radiation intensity varies so strongly with wavelength, and also explains why the sky is blue. This also is why red light transmits more cleanly than blue through biological samples with less scattering.
For absorption, each molecule has certain allowed energy levels for its electrons, and when the frequency of incoming light matches an allowed transition, they can be absorbed, causing a change to the internal energy of the molecule. These electronic transitions have typical energies associated with visible and ultraviolet light. The atoms that make up the molecules can also oscillate at (lower) characteristic frequencies, and if their motion results in a varying dipole moment they will absorb near infrared light at those particular resonance frequencies. Often there is an energy gap between the electronic (Vis/UV) resonances and the vibrational (NIR/IR) resonances, and a dielectric material would appear transparent in that region. Some biological molecules with large aromatic structures and loosely bonded electrons do absorb red light, so depends on what you are looking at.
Water the major component in biological tissue have no absorption in NIR range. So the region found to be transparent for study purposes. otherwise all the light energy will be absorbed by the molecules. The absorption and scattering depends upon the molecules present in cells. In blood hemoglobin(coloring pigment) which absorbs light at 413nm. Proteins and DNA absorbs at UV due to carbon and other atoms. The structural composition determines the absorption range.
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