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    Lecture_07

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    Nguyen Tri Tuan

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    Lecture_07

    Uploaded by

    Nguyen Tri Tuan
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    1 views14 pages

    Lecture_07

    Uploaded by

    Nguyen Tri Tuan

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    © All Rights Reserved
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    Biophotonics

    Lecture 07

    Fall 2024
    Energy-level diagram of molecules
    • Singlet-to-Singlet and Triplet-to-Triplet transitions
    are allowed, while Triplet-to-Singlet and Singlet-to-
    Triplet transitions are not allowed

    • Vibrational modes
    (형광)
    (인광)
    Energy diagram for a diatomic molecule and molecular orbitals

    homonuclear molecules
    (H2)
    Symbols:

    𝜎𝑢∗ 1𝑠 𝜎𝑔 1𝑠 𝜋

    • 𝜎: molecular orbitals with cylindrical symmetry


    about their inter-nuclei axis
    • u: odd indicating antisymmetric molecular orbital
    • g: even symmetry
    • asterisk (*): antibonding (unmarked: bonding)

    Anti-bondingMolecular Orbital: Energy is greater than that of


    constituent atomic orbitals. less stable than the parent atomic
    orbitals.

    BondingMolecular Orbital: Energy is less than that of constituent


    atomic orbitals. More stable than the atomic orbitals.
    The ground state configurations simple homogeneous and heteronuclear molecules

    • For the heteronuclear molecules, the g or u symmetry is lost.


    • The energies of the combining molecule are now different due to the difference in nuclear charges.
    Energy diagram of CO molecule

    • Ground state: 1𝛴, 𝜎2𝑠 2


    • Partially occupied molecular orbitals in the excited state
    LUMO ∗ 1
    configuration: 𝜎2𝑠 1 𝜋2𝑝

    • The two unpaired electron give rise to singlet and triplet


    states: 1Π and 3Π respectively.

    HOMO

    2𝑠 2 2𝑝2 C CO O 2𝑠 2 2𝑝4
    Anthracene molecule (C14H10)

    • The absorption and emission spectra of Anthracene molecule involving different vibrational levels:
    Absorption and Emission spectra of Alexa series of fluorescent markers

    These markers can be conjugated with the protein/lipid


    of interest for biological imaging.
    Absorption and Emission spectra of membrane binding fluorescent markers

    • These fluorescent markers are tagged with specific


    membrane protein for understanding and quantizing the
    specific biochemistry of the membrane protein. This gives
    the estimate of the concentration of these lipids in
    different intracellular compartments.
    Absorption and Emission spectra of DNA binding fluorescent markers
    Kasha’s Law, Stokes Shift and Frank-Condon Principle

    • Kasha’s law: Irrespective to the nature of electronic excitation, the emission occurs from the
    lowest vibrational state of the excited electronic state.

    • Stokes Shift: The emission always occurs at higher wavelengths or equivalently at lower energy
    Stoke shift for GFP and its variants
    Quenching and Photobleaching

    • Quenching: reduces the ability of a molecule to fluoresce


    • In a solution, the fluorophores undergo random collisions with other species of molecules
    called quenchers. Upon contact with the quencher, the excited fluorophores gets deactivated
    by various means including, energy transfer to other molecules and complex formation.

    Radiative decay:
    Non-radiative decay:

    • Photobleaching: The phenomenon that the light emitted by the fluorescent markers continuously
    fades with time. These molecules get switched off for small time, and can be switched on again.
    • Photobleaching can happen due to a lot of reasons including, molecular collisions, energy
    transfer and presence of metastable triplet state.
    Controlled Light Exposure Microscopy (CLEM) Microscopy
    Two-photon absorption and fluorescence spectra

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