Fluorescence measurement is a powerful analytical technique used across various scientific disciplines. It allows scientists to detect, quantify, and characterize substances by observing the light they emit after absorbing light at a different wavelength. This method is highly sensitive and specific, often proving more sensitive than absorbance measurements, sometimes by as much as 1000-fold. It is used for both quantitative and qualitative analysis in numerous fields, offering a straightforward and cost-effective approach.
The Phenomenon of Fluorescence
Fluorescence is a type of luminescence where certain molecules absorb light energy at one wavelength and then almost immediately re-emit light at a longer wavelength. This process begins when a molecule, known as a fluorophore, absorbs a photon of energy, causing an electron to jump to a higher, excited electronic singlet state. This excited state is unstable, and the electron quickly loses some energy, settling into the lowest vibrational level of the excited state.
From this slightly lower energy excited state, the electron returns to its ground state by emitting a photon of light. Because some energy was lost during the relaxation in the excited state, the emitted photon has lower energy and, consequently, a longer wavelength than the absorbed photon. This difference in wavelength between the absorbed (excitation) and emitted (emission) light peaks is known as the Stokes shift. The efficiency of this light emission is quantified by the fluorescence quantum yield, which is the ratio of emitted photons to absorbed photons.
Basic Principles of Fluorescence Measurement
Measuring fluorescence involves a specialized instrument called a fluorometer or spectrofluorometer. The general setup includes a light source, a sample holder, wavelength selection devices like monochromators or filters, and a detector. The light source provides the energy to excite the sample.
The excitation light then passes through a filter or monochromator, which selects a specific wavelength to illuminate the sample. The sample absorbs this light and re-emits fluorescent light. To prevent the stronger excitation light from interfering with the weaker emitted signal, the detector is positioned at a 90-degree angle relative to the excitation light path.
An emission filter or monochromator is placed in front of the detector to select and transmit only the desired emitted wavelengths, blocking any remaining excitation light. The detector converts the emitted light into an electrical signal. This signal’s intensity is then measured and processed, providing quantitative data about the fluorescent substance in the sample.
Diverse Applications Across Fields
Fluorescence measurement is used across many scientific and industrial disciplines. In biology and medicine, it is used for diagnostic tests, such as detecting specific biomarkers associated with diseases. Cellular imaging uses fluorescence microscopy, where fluorescent probes or naturally fluorescent proteins like Green Fluorescent Protein (GFP) allow real-time visualization and tracking of cellular structures and processes, including protein movement within living cells.
Fluorescence is also used in molecular biology, particularly in protein studies and DNA sequencing. Techniques like Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS) provide insights into protein-protein interactions and molecular distances. Fluorescent DNA probes and labeled nucleotides are used to visualize and sequence DNA, leading to advancements in genomics.
In chemistry, fluorescence is used for environmental monitoring, detecting pollutants in water and soil due to its high sensitivity and selectivity. It helps characterize dissolved organic matter and identify various substances in environmental samples. Material science also uses fluorescence for quality control and to study the properties of new materials.
Forensic science uses fluorescence for detecting latent fingerprints and biological fluids at crime scenes. Fluorescent powders or sprays can bind to residues in fingerprints, making them visible under specific light sources. The inherent fluorescence of biological fluids allows for their identification and even estimation of time since deposition.
In food science, fluorescence measurement is used for authenticity testing and quality assessment. It can detect adulteration, monitor oxidation processes, and identify microbial or mycotoxin contamination in various food products. The technique helps in evaluating freshness, assessing nutritional values, and ensuring the safety of food items throughout their production and storage.