What is Laser-Induced Fluorescence (LIF)?

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- GoPhotonics

Jun 12, 2023

Laser-Induced Fluorescence (LIF) is an optical spectroscopic method in which a laser is employed to excite a sample, and a photodetector is used to capture the resulting fluorescence emitted by the sample. LIF can be categorized as a type of fluorescence spectroscopy that replaces conventional lamp excitation with a laser source. Although lasers are now commonly employed as excitation sources in photoluminescence spectrometers, laser-induced fluorescence was initially developed as an independent laser spectroscopy technique, rather than for commercial instrument purposes.

The intensity of the excitation spectrum can be used to identify gas species like acetone, OH, CH, NO, etc, as well as determine the concentrations of atoms and molecules present. Also, the spectral distribution enables temperature measurement. This technique finds applications in diverse fields, including Planar Laser-Induced Fluorescence (PLIF), which allows simultaneous measurement of multidimensional species, temperature, speed, and more. It is also used for liquid film thickness measurement.

Working Principle of Laser-Induced Fluorescence

A laser beam, typically a high-energy and monochromatic light source, is directed towards the sample containing the target molecules. The laser light must have a wavelength that matches the absorption characteristics of the fluorophores present in the sample. The fluorescent molecules in the sample absorb photons from the laser beam. This absorption causes the electrons within the molecules to transition from their ground state to a higher energy state, known as the excited state. Once in the excited state, the fluorescent molecules possess excess energy. They can transfer this energy to nearby molecules through processes such as collisional energy transfer or Förster resonance energy transfer (FRET)

Then the excited electrons eventually return to their ground state, releasing the excess energy in the form of fluorescence. This emitted fluorescence typically has a longer wavelength than the absorbed laser light, allowing it to be easily distinguished and detected. The emitted fluorescence is collected using appropriate optical components, such as lenses or fiber optics, and directed towards a detector. The detector measures the intensity, spectrum, and lifetime of the fluorescence. This data provides valuable information about the properties of the fluorescent molecules, including their concentration, location, and interactions within the sample.

Types of Laser-Induced Fluorescence

Laser-Induced Fluorescence spectroscopy is of various types, depending on the specific laser and detection system utilized. Typically, the technique is categorized as either excitation or emission LIF spectroscopy. A laser is employed to stimulate molecules from their ground state to an electronically excited state. Subsequently, as the molecules return to the ground state, the emitted fluorescence is detected using a photomultiplier tube (PMT).

In excitation LIF, a tunable laser is used to vary the excitation wavelength, enabling the resolution of the vibrational structure of the excited state as in the above figure. In a liquid sample, molecules emit light from their excited state and gradually return to a series of vibrational levels in the ground state. However, the detection system cannot separate the different wavelengths of this emitted light. For this, a special filter is placed between the sample and the detection system. This filter allows the detection of all emitted light from the sample while blocking any unwanted scattered laser light.

In emission LIF, a specific pump wavelength is used to excite the sample, and the emitted light from the sample is examined by analyzing its spectrum. This analysis is done by utilizing a monochromator, which helps select the desired detection wavelength for measurement. The figure above illustrates the use of a photomultiplier tube (PMT) for single-point detection. It is also possible to utilize an array detector (such as CCD or CMOS) to capture the complete spectrum in a single measurement.

Laser-Induced Fluorescence can also be classified into two types: continuous wave (CW) LIF and time-resolved LIF. This technique provides valuable information about the lifetimes of chemical intermediates and their corresponding spectral changes over time.

Continuous wave LIF employs a continuous laser for excitation and is suitable when only spectral information is needed. The significance of CW LIF lies in its ability to provide steady-state fluorescence measurements. It allows for the detection and quantification of fluorescence intensity, which can be correlated with the concentration of fluorescent molecules in the sample. CW LIF is commonly used in various fields such as biochemistry, molecular biology, environmental analysis, and pharmaceutical research. It is particularly useful for rapid and real-time monitoring of fluorescence signals, enabling the study of dynamic processes and interactions.

On the other hand, time-resolved LIF involves using a pulsed laser to excite the sample, and the emitted light (either a single wavelength or the entire spectrum) is detected over a specific time period. Time-Resolved LIF has the ability to provide additional information about the fluorescence lifetime of the sample. The fluorescence lifetime is the average time a fluorophore remains in the excited state before returning to the ground state by emitting fluorescence. By analyzing the fluorescence decay kinetics, valuable information about the molecular environment, molecular interactions, and photophysical properties of the sample can be obtained. Time-Resolved LIF is commonly used in fields such as molecular spectroscopy, biophysics, pharmaceutical research, and materials science.

Applications of Laser-Induced Fluorescence

Laser-Induced Fluorescence plays an important role in environmental studies. It enables the detection and monitoring of pollutants in air, water, and soil. For example, LIF techniques have been used to identify and quantify harmful compounds like polycyclic aromatic hydrocarbons (PAHs) in contaminated sites. The ability to selectively target specific molecules enhances our understanding of environmental processes and aids in the development of effective mitigation strategies.

It has applications in biomedical and pharmaceutical research also by assisting in drug discovery, allowing researchers to monitor drug interactions, metabolism, and distribution within living organisms. Also, LIF techniques aid in cancer diagnostics by detecting fluorescent biomarkers associated with specific types of cancer cells. This non-invasive approach holds promise for early detection and personalized treatment.

LIF has been used in the field of combustion and plasma diagnostics. By introducing a tracer molecule into a flame or plasma, researchers can study the fundamental processes occurring within these environments. It facilitates the measurement of temperature, species concentration, and velocity fields, providing crucial insights for optimizing combustion efficiency, reducing emissions, and developing cleaner energy sources.

It has also found application in the field of art conservation. Researchers can gain insights into the age, authenticity, and degradation processes affecting artworks by analyzing the fluorescence emitted from pigments. This non-destructive technique aids in the identification of materials and assists conservators in making informed decisions about restoration and preservation.