Fluorescence spectroscopy, an advanced technique within the field of photoluminescence, has established itself as an essential resource not only in areas such as analytical chemistry and molecular biology, but also in food analysis and quality control [1]. This methodology, noted for its exceptional sensitivity and selectivity, is crucial for the accurate identification and quantification of bioactive compounds, contaminants, and adulterants.

 

In this article, we present a compendium of essential strategies and knowledge for the effective application of fluorescence spectroscopy [2]. This includes discussing the fundamentals of the phenomenon, the different types of light sources, the correct choice of cuvettes, and the influence of phenomena such as quenching, inner filter, and background autofluorescence, which are fundamental for the correct interpretation of results.

 

Fundamentals of Fluorescence and its Application in Spectroscopy

Fluorescence is an optical phenomenon in which certain molecules, called fluorophores, absorb photons of light and transition to a higher energy excited state. After this excitation, fluorophores return to their basal state, emitting photons of longer wavelength. The phenomenon has been described in greater depth in the following article.

 

This process, notable for its high specificity and sensitivity, enables the detection of subtle changes in the molecular composition of a sample. Both the intensity and the spectrum of the emitted light provide crucial data about the structure and molecular microenvironment of the fluorophore.

 

Various factors influence the efficacy and properties of fluorescence, including the concentration of the fluorophore, the chemical conditions of the environment, temperature, and pH. Interactions between fluorophores (natural or added) and their environment can induce changes in the intensity and spectrum of emission, which are exploited to investigate the chemical and physical characteristics of the samples.

 

Light Sources in Fluorescence Spectroscopy

The selection of an appropriate light source is crucial in fluorescence spectroscopy. The efficiency in exciting fluorophores significantly depends on the match between the emission spectrum of the light source and the absorption spectrum of the fluorophore.

 

There are various types of light sources based on different technologies:

 

Xenon and Mercury Arc Lamps

Advantages: Provide a continuous light spectrum from ultraviolet to near-infrared, suitable for a wide range of fluorophores. They are versatile and allow the excitation of multiple fluorophores.

Disadvantages: They can cause excessive heating of the sample and have a limited lifespan. Additionally, their continuous spectrum requires additional filters to select the desired wavelength.

 

LEDs (Light Emitting Diodes)

Advantages: Offer light emission with a narrower spectrum for more selective excitation, reducing unwanted light interference. They are energy-efficient, have a long lifespan, and allow for rapid and precise control of intensity. They are less costly compared to other technologies.

Disadvantages: The limited selection of wavelengths may not be suitable for all fluorophores. A single LED does not cover a wide range of excitation and may require multiple LEDs.

 

Lasers

Advantages: Generate coherent and monochromatic light, allowing for highly specific and efficient excitation. Ideal for techniques such as fluorescence microscopy and flow cytometry.

Disadvantages: Lasers tend to be more expensive and can cause photodegradation of the sample due to their high intensity. The wavelength selection is limited and requires different types of lasers for different applications. Additionally, they are sensitive to temperature and require precise temperature control.

 

Pyroistech offers light sources that combine multiple LEDs in a single unit. This setup can be customized with a wide range of LEDs to suit many applications.

 

In addition to the type of light source, the intensity of the excitation light is a critical parameter, as too low intensity may not sufficiently excite the fluorophores, while too high intensity can lead to photodegradation of the sample or saturation of the detector.

 

The choice of the light source and experimental setup must be made carefully to minimize light scattering effects, especially in turbid or heterogeneous samples. Scattering can affect the quality of fluorescence data and lead to misinterpretations.

 

Cuvette Use and Selection Cuvettes

are crucial components in fluorescence spectroscopy, acting as transparent containers for liquid samples [3]. The accuracy of the measurements depends largely on the quality of the cuvettes used, minor defects such as scratches or impurities can disturb light transmission and lead to inaccuracies.

Fluorescence cuvettes have at least three polished sides, with the light source and detector located 90 degrees from each other, minimizing incident light detection and maximizing detection of emitted light. Care should be taken not to confuse them with spectrophotometry cuvettes, as they can lead to problems and inaccuracies because they are designed to measure light absorption with only two polished sides

 

Materials Available in Cuvettes

• Glass Cuvettes: Common and compatible with a wide range of solvents, available in different types of glass like borosilicate and quartz.
• Plastic Cuvettes: Disposable, lightweight, and less prone to breaking. Made from various types of plastic like polystyrene and PMMA.
• Quartz Cuvettes: Used for applications requiring high optical clarity and UV transparency. Resistant to heat and chemical attacks.

 

Parameters of the Cuvette:

• Cuvette Shapes: Rectangular (common for routine assays), square (for applications requiring light from multiple angles), cylindrical (for circular dichroism experiments), and flow (for continuous flow applications).
• Cuvette Volume: Varies to accommodate different sample volumes, from microliters to several milliliters.
• Optical Path Length (Path Length): Crucial, as it affects the amount of light absorbed by the sample. The standard length is 10 mm, but shorter or longer lengths are available for concentrated or diluted samples, respectively. Calibrating and considering the optical path length in calculations is important to ensure accurate measurements when using cuvettes of different lengths.

 

Careful selection of cuvettes is vital for reliability and precision in spectroscopy. Compatibility with equipment, along with consideration of the material, volume, and design of the cuvette, plays a crucial role in the success of spectroscopic analyses.

 

Phenomena Affecting Fluorescence

In fluorescence spectroscopy, accurate interpretation of results depends not only on understanding the characteristics of the fluorophore but also on recognizing and managing various phenomena that can significantly influence the measurement of fluorescence.

 

Fluorescence Quenching

Quenching, or the extinction of fluorescence, is a process that reduces the fluorescence intensity of a fluorophore [4]. This phenomenon can be caused by various factors, including interaction with other molecules, changes in the chemical or physical environment, and energy transfer. This can affect the quantification of fluorescence and should be considered when interpreting fluorescence spectroscopy results. Quenching can be divided into Static and Dynamic Quenching: Static quenching occurs when the fluorophore forms a non-fluorescent complex with another molecule, while dynamic quenching involves energy transfer from the excited fluorophore to another molecule.

 

Inner Filter Effect

The inner filter effect refers to the absorption of excitation or emission light by sample components other than the fluorophore of interest [5]. This can lead to an underestimation of fluorescence intensity, especially in concentrated or complex samples.

Dilution techniques and mathematical algorithms can be employed to correct the inner filter effect. Careful selection of excitation and emission wavelengths can also help minimize this effect.

 

Background Autofluorescence

Background autofluorescence is the light emission by sample components other than the target fluorophore. This phenomenon can interfere with the detection and quantification of the fluorophore of interest, especially in biological or complex samples. It is crucial to distinguish between the specific fluorescence of the fluorophore and the background autofluorescence. The use of filtering techniques and adjustment of experimental conditions, such as excitation wavelength, can help differentiate these signals. In data analysis, it is important to consider the contribution of background autofluorescence and apply corrections if necessary.

 

Conclusion

In summary, fluorescence spectroscopy consolidates itself as an indispensable and versatile technique in the scientific and technological field, valued for its sensitivity and selectivity. This article has covered everything from the fundamental principles of fluorescence phenomenon to practical considerations in its application, such as the choice of suitable light sources and cuvettes, and handling phenomena like quenching and autofluorescence. Fluorescence spectroscopy, with its detailed approach and refined methodology, remains a fundamental tool in research and development across multiple disciplines.

 

 

Bibliography

[1]     Ahmad MH, Sahar A, Hitzmann B. Fluorescence spectroscopy for the monitoring of food processes. Meas Model Autom Adv Food Process 2017:121–51.

[2]     Jameson DM, Croney JC, Moens PDJBT-M in E. Fluorescence: Basic concepts, practical aspects, and some anecdotes. Biophotonics, Part A, vol. 360, Academic Press; 2003, p. 1–43. https://doi.org/https://doi.org/10.1016/S0076-6879(03)60105-9.

[3]     Cuvettes for Spectrophotometer: a Comprehensive Guide n.d. https://qvarz.com/cuvettes-for-spectrophotometer/.

[4]     Lakowicz JR. Quenching of Fluorescence BT  – Principles of Fluorescence Spectroscopy. In: Lakowicz JR, editor., Boston, MA: Springer US; 1983, p. 257–301. https://doi.org/10.1007/978-1-4615-7658-7_9.

[5]     Kumar Panigrahi S, Kumar Mishra A. Inner filter effect in fluorescence spectroscopy: As a problem and as a solution. J Photochem Photobiol C Photochem Rev 2019;41:100318. https://doi.org/https://doi.org/10.1016/j.jphotochemrev.2019.100318.