Fluorescence Technology
What is Fluorescence?
Fluorescence refers to a phenomenon of luminescence where a substance emits light upon excitation. When a substance at room temperature is irradiated with certain wavelengths of light (usually ultraviolet or X-rays), it absorbs light energy, enters an excited state, and immediately relaxes, emitting light with a longer wavelength than the incident light (typically in the visible light range). Once the incident light ceases, the luminescence phenomenon also immediately disappears. The emitted light with this property is called fluorescence.
Principle of Fluorescence Generation
When light irradiates certain atoms, the energy of the light causes some electrons around the atomic nucleus to transition from their original orbit to a higher energy orbit, such as from the ground state to the first excited singlet state or the second excited singlet state. The first or second excited singlet state is unstable, so it returns to the ground state. When the electron returns from the first excited singlet state to the ground state, energy is released in the form of light, resulting in fluorescence.
Additionally, some substances can continue to emit light for a longer period after the removal of incident light, a phenomenon known as phosphorescence. In daily life, people often broadly refer to various faint sources of light as fluorescence without carefully distinguishing and delineating their luminescence principles.
Fluorescence is the emission of light by a substance after it has absorbed light or other electromagnetic radiation. In most cases, the emitted wavelength of light is longer than the absorbed wavelength, and thus lower in energy. However, when the absorption intensity is high, two-photon absorption phenomena may occur, leading to radiation wavelengths shorter than the absorption wavelengths. Resonance fluorescence occurs when the radiation wavelength is equal to the absorption wavelength. A common example is the principle behind fluorescent lamps used in our daily lives, where fluorescent powder coated on the lamp absorbs the ultraviolet light emitted by the mercury vapor in the lamp tube and then emits visible light, making it visible to the human eye.
Parameters Related to Fluorescence
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Excitation Spectrum: The relationship between the intensity or efficiency of a certain emission spectrum or band of the luminescent material and the excitation wavelength of light.
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Emission Spectrum: The variation in the intensity of light at different wavelengths when a luminescent material is excited by a certain light.
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Fluorescence Intensity: The fluorescence intensity is related to the fluorescence quantum yield, extinction coefficient, and content of the substance.
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Fluorescence Quantum Yield (Q): The quantum yield represents the ability of a substance to convert absorbed light energy into fluorescence, which is the ratio of the number of photons emitted by the fluorescent substance to the number of photons absorbed.
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Stokes Shift: The difference between the maximum fluorescence emission wavelength and the maximum absorption wavelength.
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Fluorescence Lifetime: When a beam of light excites a fluorescent substance, after the substance absorbs energy and transitions from the ground state to a certain excited state, then emits fluorescence and returns to the ground state radiatively, the time required for the fluorescence intensity of the molecule to decrease to 1/e of the maximum intensity is the fluorescence lifetime.
Fluorescence Technology
When light shines on a substance, photons strike the molecule and are absorbed within approximately 10^-15 seconds. Electrons originally in the ground state are excited to higher energy levels, leaving the molecule in an excited state. Subsequently, through internal conversion processes, the excited-state molecules transfer some energy to surrounding molecules, causing the electrons in higher excited states to quickly return to the lowest vibrational level of the lowest excited state (also known as the first singlet state). Molecules in the first singlet state have an average lifetime of about 10^-8 seconds. If these molecules emit photons corresponding to various different vibrational energy levels before returning to the ground state, fluorescence occurs, and the wavelength of the fluorescence varies depending on the vibrational energy level it returns to. Therefore, the emission band spectrum of fluorescence is formed. Since some energy is consumed before emitting fluorescence, the energy corresponding to the emitted fluorescence is smaller than the light energy absorbed by the substance, so the emitted fluorescence characteristic wavelength is always longer than the excitation characteristic wavelength.
Whether a substance can produce fluorescence mainly depends on the structure of the substance itself and the surrounding medium environment (such as solvent polarity, pH value, temperature, etc.).
Applications of Fluorescence
- Qualitative and quantitative analysis of substances
- Fluorescent labeling for DNA sequencing
- Physicochemical properties of biomacromolecules and molecular structure and conformation
- Flow cytometry, immunology, clinical testing, cancer cell identification, etc.
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