21 Instrumentation for Fluorescence Spectroscopy

Dr. Ajit K. Mahapatro

Learning Objectives

From this module students may get to know about the following

i. The fluorescence spectrometers that provide a tool for analyzing the spectral distribution of the emitted light from the sample

ii. Instrumentation for spectrofluorometry

iii. Applications of spectrofluorometry

1. Introduction

Instrumentation provides tools to measure the realistic data of an experimental process. The successful capturing of the experimental events requires understanding the instrumentation and implementation of techniques for recording the high sensitive signals from a process with high signal to noise ratio. The quantification of actual data from many potential artifacts that can distort the data is the main aim of a perfect instrumentation.

The fluorescence from a material can be measured by fluorescence spectroscopy that detects and records the amount of fluorescence light with high sensitivity. The devices that measure the fluorescence are called fluorometers. Spectrofluorometric methods of analysis are the most commonly analytical techniques. The availability of the instrumentation, the simplicity of procedure, sensitivity, selectivity, precision, accuracy, and speed of the technique still make the spectrofluorometric methods attractive. These features make fluorescence spectroscopy an attractive technique as compared to other forms of optical spectroscopy or other analytical techniques such as chromatography and electrophoresis. Fluorescence spectroscopy has been used widely as a tool for quantitative analysis, characterization, and quality control in the pharmaceutical, environmental, agricultural, nanotechnology and biomedical fields.

The fluorescence signal detected by the detector form the fluorophore of interest may include interfere with the background fluorescence from the solvents, light leaks in the instrumentation, emission from the optical components, stray light passing through the optics, light scattered by turbid solutions, Rayleigh and/or Raman scatter, and efficiency of detectors and other instruments used. Techniques to separate the pure signal of the fluorescence from the observed mixed high amplification data with interference sources is the key to record pure signal for analyzing the fluorescence process of the fluorophore.

The available instruments do not yield true excitation or emission spectra because of the non-uniform spectral output of the light sources and the wavelength-dependent efficiency of the monochromators and detector tubes. The polarization or anisotropy of the emitted light can also affect the measured fluorescence intensities because the efficiency of gratings depends on polarization. It is important to understand and control these numerous factors.

In this lecture we will discuss the properties of the individual components in a spectrofluorometer, and how these properties affect the observed spectral data. These instrumental factors, and the optical density and turbidity can affect the excitation and emission spectra, as well as the measurement of fluorescence lifetimes and anisotropies. Specific examples are given to clarify these effects and rectify the raw data for recording the original fluorescence data by introducing technical filters.

Light of a specific energy is identified in terms of its wavelength (λ), frequency (ν), or wavenumber. The usual units for wavelength are nanometers, and wavenumbers are given in units of cm^–1. Wavelengths and wavenumbers are easily interconverted by taking the reciprocal of each value. Generally, the fluorescence spectra are presented in the wavelength or wavenumber scale.

2. Fluorescence Spectra

When light is incident on a fluorescent material, it excites the electrons to a higher energy level, the fluorophore remains in the lowest vibrational level of the excited electronic state for a period of time (~nanoseconds), generally termed as fluorescence lifetime and come back to its ground state with the emission of a photon of longer wavelength than the incident light. Thus fluorescence occurs at longer wavelength than absorbance. The energies and intensities of fluorescence give information about the structure of and environments of the fluorophores. The fluorescence process is shown in Figure 1. Fluorescence emission occurs as the fluorophore decay from the singlet electronic excited states to an allowable vibrational level in the electronic ground state. This rapid vibrational relaxation process occurs on the time scale of femtoseconds to picoseconds. This relaxation process is responsible for the Stoke shift, which describes the generation of fluorescence photons at longer wavelength than the excitation radiation.

2.1 Excitation and Emission Spectra

An emission spectrum is the wavelength distribution of an emission measured at a single constant excitation wavelength. The recorded emission spectra represent the photon emission rate or power emitted at each wavelength, over a wavelength interval determined by the slit widths and dispersion of the emission monochromator. The emission spectra recorded using different instruments can be different because of the wavelength dependent sensitivities of the instruments.

An excitation spectrum is the dependence of emission intensity, measured at a single emission wavelength by varying the excitation wavelength scanned over a range. This would represent the relative emission of the fluorophore at each excitation wavelength. For most fluorophores the quantum yields and emission spectra are independent of excitation wavelength. As a result, the excitation spectrum of a fluorophore can be superimposable on its absorption spectrum. Such identical absorption and excitation spectra are rarely observed because the excitation intensity is different at each wavelength, and properties of fluorophore a nonlinear response resulting from a high optical density of the sample or the presence of other chromophores in the sample.

3. Fluorometer

The fluorescence spectrometers provide a tool for analyzing the spectral distribution of the emitted light from the sample. The fluorescence emission spectrum includes either a continuously variable interference filter or a monochromator. In more advanced instruments, the monochromators are provided for both the selection of exciting light and to detect the sample emission. Such instruments are also capable of measuring the variation of emission intensity for the fluorescence excitation spectrum. Generally, the fluorescence spectrometers use double-beam optics to compensate the inherent power fluctuations in the source. The technique includes measurement of the fluorescent emission at a right angle to the incident beam. The emitted radiation passes through a second filter or monochromator to isolate the fluorescent peak for measurement.

Figure 2. Block diagram of a fluorometer

The reference beam passes through an attenuator (Figure 2) to reduce its power to that of the fluorescent radiation. The optical paths of the excitation and detection of light are along the orthogonal axis. The orthogonal arrangement ensures minimal leakage of excitation light into the detection side to be captured by the high sensitivity photodetectors such as photomultipliers or charge-coupled device cameras. For spectral measurement, the monochromators or band-pass filters are placed in the excitation and emission light paths to select a specific spectral band. The excitation spectrum is defined as the fluorescent intensity measured as a function of excitation wavelength at a constant emission wavelength, and the emission spectrum is the fluorescent intensity measured as a function of emission wavelength at a constant excitation wavelength.

A fluorometer provides a relative measurement and can be calibrated with a known  concentration standard or correlated to standard laboratory methods to produce quantitative  measurements. Accordingly, based on the technique for measuring the fluorescence, the instrument can be classified into two categories: (i) Filter fluorometer, uses filters to isolate the incident light and fluorescent light, and to restrict excitation and emission beam wavelengths, (ii) Spectrofluorometer, uses monochromator to isolate incident light and fluorescent light. It has two monochromators; one allows choice of excitation wavelength and the other allows fluorescence emission spectra to be scanned.

Both types use the following scheme: The light from an excitation source passes through a filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector.

3.1 Filter Fluorometer: It measures the ability of a sample to absorb light at particular wavelength and emit light at a longer wavelength. It is a good choice when sensitive quantitative measurements are desired for specific compounds. The comparative ease of handling and low cost make filter fluorometers ideal for dedicated and routine measurements. A typical fluorometer includes a light source, a specimen chamber with integrated optical components, and high sensitivity detector (Figure 3)

Figure 3. Schematic diagram of filter fluorometer

3.2 Spectrofluorometer

Most spectrofluorometers record both the excitation and emission spectra. Resolution is obtained with changeable fixed slits. The advantage of spectrofluorometers is that they  provide variations in the wavelength selection and allow the operator to scan over a range of wavelengths. The disadvantage of spectrofluorometer includes, it is several times costly compared to the filter fluorometer and can only provide moderate sensitivity and specificity. For research purposes, the spectrofluorometer with optimal sensitivity and specificity, and monochromators with continuous variable slits and a broad wavelength range (200 – 1000 nm), have been preferred. Such instruments have dual excitation or dual emission monochromators for increasing the sensitivity and reducing stray light. The schematic diagram of the spectrofluorometer is shown in Figure 4.

Figure 4. Schematic diagram of spectrofluorometer

4. Fluorometer Instrument

The typical fluorometer instruments include three basic items: source of light, specimen chamber with integrated optical components and sample holder, and high sensitivity detector.

4.1 Light Source: The first component of the fluorescence spectroscopy is light source. It is used to excite the molecule to higher excited state from which they lose their energy in the form of fluorescence. Generally, the source must be more intense than that required for UV-Visible absorption spectroscopy; magnitude of the emitted radiation is directly proportional to the power of the source.

A number of light sources can be used to excite the molecule:

4.1.1 Incandescent lamp: Incandescent lamps have the advantages that they are low cost, easy to use, long lasting and air cooled. Their disadvantage is that they are not very intense in their light output, not easily focused, and not easily pulsed for short time temporal measurements.

4.1.2 Arc lamps: Arc lamps have much more intense output but are less stable, costly, and cumbersome to use and often require water cooling.

4.1.3 Laser: laser sources are capable of generating monochromatic light with extremely high intensities and are sometimes used as excitation sources. Laser can either be continuous wave type that could emit relatively steady output intensity or pulsed, where the light output is discretely emitted with times. The main disadvantages of laser source are that of their high cost and limited availability of wavelength. Since the radiation produced is monochromatic, there is no need for an excitation monochromator.

Filter fluorometers often employ a low-pressure mercury vapour lamp. This source produces intense lines at certain wavelengths. One of these lines could be suitably utilized for excitation onto a fluorescent sample. The most common light sources for fluorometers are lamp sources, such as xenon arc lamps. These lamps provide a relatively uniform intensity over a broad spectral range from the ultraviolet to the near infrared. Spectrofluorometers need a continuous radiation source, are often equipped with a 75-450 Watt high pressure xenon arc lamp.

4.1.4 Mercury Vapor: Mercury vapor produces ultraviolet radiation when current flows through them. This type of source can be used for the materials that can produces fluorescence through ultraviolet light.

4.1.5 Photodiodes: PIN (p-i-n) photodiodes can also be used as a low-power light source for fluorescence but are having less sensitive.

Different light sources can be used because they provide different irradiation for fluorescence properties because of different wavelength of radiated energies. Such as laser can provide light of wavelength under 0.01 nm that provides an immense amount of energy for fluorescence. There is a big challenge to control the wavelength provided by laser. In the other way, the wavelength provided by the other sources such as lamps, mercury vapor and xenon has a range of 200-800+ nanometer and serves different functions for fluorescence spectroscopy.

4.2 Dispersive Elements: To make fluorescence spectroscopy as a most useful analyzing technique, it is necessary to separate their constituent elements for the excitation energy from the source and emitted fluorescence from the sample. Fluorometers use either interference or absorption filters. Spectrofluorometers are usually fitted with grating monochromators.

There are two types of dispersive elements: Filters and Monochromators

Monochromators are used to disperse white light consisting of various colors or wavelength. This dispersion can be achieved with the help of prism or diffraction gratings. The diffraction grating makes light that enters the grating change the angle depending on the wavelength. This change makes it possible to get the necessary wavelength with the proper adjustment.

Usage of the multiple types of filters or monochromators depends on the type of fluorescence spectroscopy. The primary filters that excite the sample provide the appropriate wavelength necessary, while the secondary filters monochromate the emitted light when sent to the detector.

4.3 Detectors: Fluorescence signals are usually of low intensity, and photomultiplier tubes and diode- array detectors are the commonly used detectors for this purpose. The fluorescence spectroscopy measures optical signals using two types of detectors, single and multiple channel detectors. Single channel detector only detects a single wavelength at a time from the sample and multiple channel detectors can detect multiple wavelengths at a specific time.

4.4 Read-out devices: The output after getting from a detector is amplified or displayed on a read out device. The read out devices may be a meter or digital display.

4.5 Sample holder: To hold the sample of interest the sample cell or cuvette is used with an precaution that the material on the sample cuvette must allow the excitation or the emission light to pass through it.

5. An Ideal Spectrofluorometer

To obtain the correct emission spectra, the individual components must have the following characteristics:

• The light source must give a constant photon output at all wavelengths.
• The monochromators must pass the photons at all wavelengths at equal efficiency.
• The efficiency of monochromators must be independent of polarization.
• The detector must detect photon at all wavelengths with equal efficiency.

The characteristics of ideal spectrofluorometer can illustrate in the following Figure 5. Although it is not possible to obtain such ideal characteristics one is forced to compromise with these components. The above properties are summarized in Figure 5.

6. Phosphorimeters: Instrumentation of Phosphorescence

Instrumentation for molecular phosphorescence should discriminate between fluorescence and phosphorescence because of the difference between the lifetime of fluorescence and phosphorescence. Fluorescence has shorter lifetime than phosphorescence. This discrimination is easily achieved by incorporating a delay between the excitation radiation and measuring the phosphorescence emission. Instruments for measuring the phosphorescence are very similar to those used for fluorescence. The slow process of phosphorescence requires to prevent the excited state from relaxing by external conversion.

Here, two additional components are included in the feature of the instrumentation. They are:

1. A mechanism or electronic circuit is required that allows the sample to be irradiated and allows measurement of phosphorescent intensity after a time delay.

2. Since phosphorescence is aided by low temperature and a viscous medium, a flask for liquid nitrogen with quartz windows is often used.

The Figure 6 shows how two out of phase choppers can be used to block the emission from reaching the detector when the sample is excited or to block the radiation from reaching to the sample from the source while measuring the phosphorescent emission.

Figure 6. Schematic diagram showing the use of choppers to prevent the fluorescent emission from interfering with the phosphorescent emission during (a) excitation and (b) measuring emission from the sample.

7. Advantages of Fluorescence

Comparing the fluorescence with other analytical techniques such as absorbance, it is noticed that the fluorescence has the advantage over the conventional absorbance based technique. Fluorescence is several times more sensitive and selective than absorbance. The enhanced sensitivity arises because of measuring the emitted radiation directly and can be increased by increasing the incident power.

Fluorescence is also referred as zero background technique, and the absorbance spectroscopy measures the difference between the incident and transmitted intensities indicating measurements of small change of signals in a large background.

The fluorescence lifetime can also be measured with high-resolution fluorometers designed in both the time and frequency domains. The accurate lifetime measurement requires photodetectors and signal processing electronics with sub-nanosecond precision.

• In the time domain, femtosecond or picosecond pulsed lasers are often used as excitation light sources. The time delay between the excitation light pulse and resultant fluorescence photon is measured using high-speed electronics, referred as time-correlated single photon counting method.
• In the frequency domain, the specimen is excited by a light source with high frequency light. The resultant fluorescence signal is also modulated at the same frequency but is phase-delayed and amplitude-demodulated. The phase delay and demodulation contain the lifetime information of the fluorophore and can be measured using heterodyning or homodyning detection techniques.

Many fluorophores respond to the environmental changes with lifetime variations. Lifetime measurements are also used to distinguish dynamic and static quenching mechanisms. Lifetime resolved fluorescence resonance energy transfer (FRET) measurement allows determining the distance between two fluorophores by spectrally resolving the relative fluorescence intensities of the donor and the acceptor FRET pairs. An important example is oxygen concentration measurement based on the dynamic quenching of long lifetime fluorophores.

For polarization measurement, polarizers are inserted into the excitation and emission light paths. With the excitation polarizer fixed, the emission polarizater can be rotated to measure the perpendicular (I⊥) and parallel (I||) components of the fluorescence emission. The steady state polarization is defined as:

8. Applications of Fluorescence Spectroscopy

8.1 Laser induced fluorescence spectroscopy for cancer diagnosis of human tissues: Laser spectroscopic techniques have the potential for in-situ diagnosis and the use of non-ionizing radiation ensures that the diagnosis can be made repeatedly without any adverse side effects.

8.2 Study of Marine Petroleum Pollutants: Fluorescence spectroscopy is one of the good techniques for detection of oil slicks on the water surface, determination of petroleum contaminants in seawater, determination of particular petroleum derivative compounds, and identification of pollution sources. Main components of any oil are hydrocarbons. The other components are primarily derivatives of hydrocarbons containing single atoms of sulfur, oxygen or nitrogen. Here, few hydrocarbons show fluorescence and the major of them has no ability to luminescence.

8.3 Accurate determination of glucose: Fluorophotometry was used widely owing to its simplicity for operation and high sensitivity. Recently biomolecule stabilized Au nanoclusters were demonstrated as a novel fluorescence probe for sensitive and selective detection of glucose.

8.4 Study of Biological Structure and Function: The fluorescence spectrum is highly sensitive to the biochemical environment of the fluorophore with their spectra changes as a function of the concentration of metabolites, such as pH and calcium. Protein domain structure and motion on the sub-nanometer scale can be spectrally monitored using fluorescence resonance energy transfer (FRET). FRET is a non- radiative process where the energy is transferred between two fluorophores.

Summary

• The excitation spectrum is defined as the fluorescent intensity recorded as a function of excitation wavelength at a constant emission wavelength and the emission spectrum is the fluorescent intensity measured as a function of emission wavelength at a constant excitation wavelength.
• Filter fluorometer and spectrofluorometer are utilized to record the fluorescence spectra.
• Fluorescence spectophotometry is used in many areas of biology and medicine.
• Usage of filters or monochromators is to remove pollution or stray light from the wavelength that is used to excite the sample.

References

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• Catherine A. Royer, Approaches to Teaching Fluorescence Spectroscopy, Biophys. J, 1995.

• Eftink, M, The use of fluorescence methods to monitor unfolding transitions in proteins, Biophys. J, 66, 1994, 482-501.

• Kim, Intrinsic Tryptophan Fluorescence in Proteins, Biophys. J, 65, 1993, 215-226.

• Leaback DH, Extended theory, and improved practice for the quantitative measurementoffluorescence, J Fluoresc7, 1997, 55S–57S.

• Stelmaszewski A, Application of fluorescence for determination of petroleum content in water, Physicochemical Problems of Natural Waters Ecology, 2007.

• Kommu Naresh, Applications of Fluorescence Spectroscopy, Journal of Chemical and Pharmaceutical Sciences