Fluorescence is measured using instruments called fluorometers (also known as spectrofluorometers or fluorimeters). These instruments are designed to excite a sample with a specific wavelength of light and then measure the intensity and wavelength distribution of the emitted fluorescence.
Understanding Fluorometry
Fluorometry is a technique used to identify and quantify specific molecules within a sample by analyzing their fluorescent properties. Here's a breakdown of how it works:
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Excitation: A light source (typically a xenon arc lamp or a laser) emits light that passes through an excitation monochromator or filter. This selects a specific wavelength of light, which is directed towards the sample. This selected wavelength corresponds to the excitation wavelength of the fluorophore (the fluorescent molecule) being studied.
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Sample Irradiation: The sample absorbs the excitation light, causing the fluorophore molecules to enter an excited electronic state.
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Emission: The excited fluorophore molecules then rapidly return to their ground state, releasing energy in the form of light (fluorescence). This emitted light has a longer wavelength (lower energy) than the excitation light – a phenomenon known as the Stokes shift.
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Detection: The emitted fluorescent light passes through an emission monochromator or filter, which selects the specific wavelength range to be measured. A detector, typically a photomultiplier tube (PMT) or a photodiode, measures the intensity of the emitted light.
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Analysis: The fluorometer then displays or records the intensity of the fluorescence at different wavelengths. This data can be used to identify the presence of specific fluorophores, determine their concentration, and study their interactions with other molecules.
Components of a Fluorometer
A typical fluorometer includes the following key components:
- Light Source: Provides the excitation light. Common sources include xenon arc lamps (broad spectrum) and lasers (specific wavelengths).
- Excitation Monochromator/Filter: Selects the specific excitation wavelength.
- Sample Holder: Holds the sample being analyzed (often a cuvette).
- Emission Monochromator/Filter: Selects the specific emission wavelength for detection.
- Detector: Measures the intensity of the emitted light. Photomultiplier tubes (PMTs) are common due to their high sensitivity.
- Data Acquisition and Processing System: Controls the instrument, collects data, and displays/analyzes the results.
Types of Measurements
Fluorometers can perform various types of measurements:
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Emission Spectra: Measures the intensity of fluorescence at different emission wavelengths while keeping the excitation wavelength constant. This provides information about the spectral characteristics of the fluorophore.
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Excitation Spectra: Measures the intensity of fluorescence at a fixed emission wavelength while varying the excitation wavelength. This can be used to determine the optimal excitation wavelength for a given fluorophore.
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Time-Resolved Fluorescence: Measures the decay of fluorescence intensity over time after excitation. This provides information about the lifetime of the excited state and can be used to study dynamic processes.
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Fluorescence Polarization/Anisotropy: Measures the polarization of the emitted light. This can be used to study molecular size, shape, and interactions.
Applications of Fluorometry
Fluorometry has numerous applications in various fields:
- Biochemistry and Molecular Biology: Studying protein folding, enzyme kinetics, DNA interactions, and cellular signaling.
- Environmental Monitoring: Detecting pollutants and toxins in water and air.
- Pharmaceuticals: Drug discovery, drug quality control, and drug delivery studies.
- Clinical Diagnostics: Detecting biomarkers for diseases.
In summary, fluorescence is measured using fluorometers, which excite a sample with specific wavelengths of light and then measure the intensity and wavelength distribution of the emitted fluorescence. This technique is widely used in various fields due to its high sensitivity and specificity.
