**2.2 Instrumentation**

220 Macro to Nano Spectroscopy

The fluorescence lifetime, τF, is determined by observing the decay in fluorescence intensity (decay profile) of a fluorophore after excitation. Immediately after a molecule is excited the fluorescence intensity will be at a maximum and then decrease exponentially according to

> − τ= / ( ) *<sup>F</sup> <sup>t</sup>*

Thus after a period of τF the intensity has dropped to 37% of *I0*, that is 63% of the molecules return to the ground state before τF. In many cases the above expression needs to be modified into more complex expressions. First of all it is assumed that the instrument yields an infinite (or very) short light pulse at time zero. In cases where τF is small *I0* must be replaced by a function, which describes the lamp profile of the instrument. Also, more than one lifetime parameter is often needed to describe the decay profile, which is I(t) must be expressed as a sum of exponentials. Finally the concept of anisotropy should be mentioned. Anisotropy is based on selectively exciting molecules with their absorption transition moments aligned parallel to the electric vector of polarized light. By looking at the polarization of the emission the orientation of the fluorophore can be measured. The

anisotropy of the system is defined as (Equation 6) (Rendell, 1987; Lakowicz, 2006):

⊥

*I I*

*r*

= 0 and θ = 0, leading to *r* = 1, the maximum anisotropy.

⊥ <sup>−</sup> <sup>−</sup> = = <sup>+</sup>

2 2

The oval in Figure 10 symbolized the absorption transition moment. Vertical polarized excitation light enters along the x-axis and *I*<sup>⊥</sup> and *I*║ are measured along the y-axis, setting the emission polarizer perpendicular and parallel to the excitation polarizer respectively. Θ is the angle of the emission to the z-axis (see Figure 10), the squared brackets indicates that it is the average value. If all absorption transition moments are aligned along the z-axis then *I*<sup>⊥</sup>

Fig. 10. The absorption dipole is aligned along the z-axis. The excitation light is vertically

aligned and follows the x-axis. Emission is measured along the y-axis.

θ

<sup>2</sup> 3 cos ( ) 1

*<sup>o</sup> It Ie* (5)

*I I* (6)

Equation 5 (Rendell, 1987; Lakowicz, 2006).

The principal sketch of a typical fluorescence spectrophotometer is shown in Figure 11. It consists of a light source, an excitation and emission monochromator (*grooves/mm*), polarizers (*prisms*), sample chamber and a detector (*such as photomultiplier tube*). For steady state measurements the light source usually consists of a 450W xenon arc lamp, and for time resolved measurements it is equipped with nanosecond flash lamp. Most simple spectrometers have a similar geometry, but often extra detectors and/or light sources are fitted resulting in a T- or X-geometry.

Fig. 11. Schematic representation of a fluorescence spectrophotometer.

The light source produces light photons over a broad energy spectrum, typically ranging from 200 to 900 nm. Photons impinge on the excitation monochromator, which selectively transmits light in a narrow range centered about the specified excitation wavelength. The transmitted light passes through adjustable slits that control magnitude and resolution by further limiting the range of transmitted light. The filtered light passes into the sample cell causing fluorescent emission by fluorphors within the sample. Emitted light enters the

Current Achievement and Future Potential of Fluorescence Spectroscopy 223

stray light arising from sources other than the emitting fluorophore—for example, from the excitation source. This stray light must be kept from reaching the light-sensitive detector in order to insure that what the instrument "sees" is due only to the fluorescence of the sample itself. When a single dye is used, a filter that blocks out the excitation light to reduce background noise, but transmits everything else is often a good choice to maximize the signal collected. Such a filter is called a long pass emission filter (Guilbault, 1990; Rendell,

If multiple dyes are used in the sample, a band pass emission filter can be used to isolate the emission from each dye. Careful filter selection helps to ensure that the detector registers

LEDs are relatively new light sources for fluorescence excitation. Single-color LEDs are ideal for low-cost instrumentation, where they can be combined with simple long pass filters that block the LED excitation and allow the transmission of the dye signal. However, the range of wavelengths emitted from each LED is still relatively broad. Currently their application

There are many options for light sources for fluorescence. Selecting the appropriate light source, and filters for both excitation and emission, can increase the sensitivity of signal detection to an astounding degree. Making fluorescence labeling one of the most sensitive

With recent advances in sensitive array detectors, fiber optic wave guides, high speed electronics and powerful software, many new generations of spectrofluorometers have been developed. These new spectrofluorometers use charged couple devices (CCDs) or photodiode arrays to replace the photomultipliers and avalanche photodiodes used in conventional spectrometers. They offer excellent performance for a wide range of spectroscopic applications from the UV to the near IR. Because of their unique combination of outstanding sensitivity, high speed, low noise, compactness, instantaneous capture of full spectra, low cost and robustness, these detectors have revolutionized spectroscopic detection. A quick glance at today's instrumentation market indicates the popularity of the CCD as the detector of choice. The overwhelming benefits of array detectors are simultaneous and multi-wavelength data acquisition. On the other hand, the use of fiber optics as light guidance allows a great modularity and flexibility in setting up an optical measurement system. Recent applications and a critical comparison between simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and chargecoupled device, or photomultiplier for determining proteins in capillary electrophoresis are

In summary, from the preceding discussion, four essential elements of fluorescence detection systems can be identified: (1) an excitation source, (2) a fluorophore, (3) wavelength filters to isolate emission photons from excitation photons and (4) a detector that registers emission photons and produces a recordable output, usually as an electrical signal or a photographic image. Regardless of the application, compatibility of these four

For the sample holders, the majority of fluorescence assays are carried out in solution, the final measurement being made upon the sample contained in a cuvette or in a flowcell.

only the light you are interested in—the fluorescence emitted from the sample.

may also require the use of a filter to narrow the bandwidth.

presented by Casado-Terrones et al. (Casado-Terrones, 2007).

elements is essential for optimizing fluorescence detection.

1987; Lakowicz, 2006).

detection technologies available.

emission monochromator, which is positioned at a 90º angle from the excitation light path to eliminate background signal and minimize noise due to stray light. Again, emitted light is transmitted in a narrow range centered about the specified emission wavelength and exits through adjustable slits, finally entering the photomultiplier tube (PMT). The signal is amplified and creates a voltage that is proportional to the measured emitted intensity. Noise in the counting process arises primarily in the PMT. Therefore, spectral resolution and signal to noise is directly related to the selected slit widths (Guilbault, 1990; Rendell, 1987; Lakowicz, 2006).

Not all fluorimeters are configured as described above. Some instruments employ sets of fixed band pass filters rather than variable monochromators. Each filter can transmit only a select range of wavelengths. Units are usually limited to 5 to 8 filters and are therefore less flexible. Fiber optics are also employed for "surface readers", to transmit light from the excitation monochrometers to the sample surface and then transport emitted light to the emission monochrometers. This setup has the advantage of speed, but has the disadvantages of increased signal to noise, due to the inline geometry, and smaller path length which increase the probability of quenching.

Fluorescence requires a source of excitation energy. There are several main types of light sources that are used to excite fluorescent dyes. This section introduces the types of commonly used excitation sources and presents some of the ways that filters can be used to optimize your experimental result. The most popular sources used for exciting fluorescent dyes are broadband sources such as the mercury-arc and tungsten-halogen lamps. These produce white light that has peaks of varying intensity across the spectrum. In contrast, laser excitation sources, which will be described later, offer one or a few well-defined peaks, allowing more selective illumination of your sample. More recently, high-output light emitting diodes, or LEDs, have gained popularity due to their selective wavelengths, low cost and energy consumption, and long lifetime.

When using broadband white light sources it is necessary to filter the desired wavelengths needed for excitation; this is most often done using optical filters. Optical filters can range from simple colored glass to highly engineered interference filters that selectively allow light of certain wavelengths to pass while blocking out undesirable wavelengths. For selective excitation, a filter that transmits a narrow range of wavelengths is typically used. Such a filter is called a band pass excitation filter.

The high intensities and selective wavelengths of lasers make them convenient excitation sources for many dyes. The best performance is achieved when the dye's peak excitation wavelength is close to the wavelength of the laser. Compact violet 405 nm lasers are replacing expensive UV lasers for most biological work. The most commonly used lasers are the 488 nm blue-green argon laser, 543 nm helium-neon green laser and 633 nm heliumneon red laser. Mixed-gas lasers such as the krypton-argon laser can output multiple laser lines and therefore may still require optical filters to achieve selective excitation. While a given dye's excitation maximum may not exactly match the laser's peak wavelength, the high power of the laser can still produce significant fluorescence from the dye when exciting at a suboptimal wavelength. Filters are important for selecting excitation wavelengths. They are also important for isolating the fluorescence emission emanating from the dye of interest. Detecting the fluorescence emission of a sample is complicated by the presence of

emission monochromator, which is positioned at a 90º angle from the excitation light path to eliminate background signal and minimize noise due to stray light. Again, emitted light is transmitted in a narrow range centered about the specified emission wavelength and exits through adjustable slits, finally entering the photomultiplier tube (PMT). The signal is amplified and creates a voltage that is proportional to the measured emitted intensity. Noise in the counting process arises primarily in the PMT. Therefore, spectral resolution and signal to noise is directly related to the selected slit widths (Guilbault, 1990; Rendell, 1987;

Not all fluorimeters are configured as described above. Some instruments employ sets of fixed band pass filters rather than variable monochromators. Each filter can transmit only a select range of wavelengths. Units are usually limited to 5 to 8 filters and are therefore less flexible. Fiber optics are also employed for "surface readers", to transmit light from the excitation monochrometers to the sample surface and then transport emitted light to the emission monochrometers. This setup has the advantage of speed, but has the disadvantages of increased signal to noise, due to the inline geometry, and smaller path

Fluorescence requires a source of excitation energy. There are several main types of light sources that are used to excite fluorescent dyes. This section introduces the types of commonly used excitation sources and presents some of the ways that filters can be used to optimize your experimental result. The most popular sources used for exciting fluorescent dyes are broadband sources such as the mercury-arc and tungsten-halogen lamps. These produce white light that has peaks of varying intensity across the spectrum. In contrast, laser excitation sources, which will be described later, offer one or a few well-defined peaks, allowing more selective illumination of your sample. More recently, high-output light emitting diodes, or LEDs, have gained popularity due to their selective wavelengths, low

When using broadband white light sources it is necessary to filter the desired wavelengths needed for excitation; this is most often done using optical filters. Optical filters can range from simple colored glass to highly engineered interference filters that selectively allow light of certain wavelengths to pass while blocking out undesirable wavelengths. For selective excitation, a filter that transmits a narrow range of wavelengths is typically used.

The high intensities and selective wavelengths of lasers make them convenient excitation sources for many dyes. The best performance is achieved when the dye's peak excitation wavelength is close to the wavelength of the laser. Compact violet 405 nm lasers are replacing expensive UV lasers for most biological work. The most commonly used lasers are the 488 nm blue-green argon laser, 543 nm helium-neon green laser and 633 nm heliumneon red laser. Mixed-gas lasers such as the krypton-argon laser can output multiple laser lines and therefore may still require optical filters to achieve selective excitation. While a given dye's excitation maximum may not exactly match the laser's peak wavelength, the high power of the laser can still produce significant fluorescence from the dye when exciting at a suboptimal wavelength. Filters are important for selecting excitation wavelengths. They are also important for isolating the fluorescence emission emanating from the dye of interest. Detecting the fluorescence emission of a sample is complicated by the presence of

Lakowicz, 2006).

length which increase the probability of quenching.

cost and energy consumption, and long lifetime.

Such a filter is called a band pass excitation filter.

stray light arising from sources other than the emitting fluorophore—for example, from the excitation source. This stray light must be kept from reaching the light-sensitive detector in order to insure that what the instrument "sees" is due only to the fluorescence of the sample itself. When a single dye is used, a filter that blocks out the excitation light to reduce background noise, but transmits everything else is often a good choice to maximize the signal collected. Such a filter is called a long pass emission filter (Guilbault, 1990; Rendell, 1987; Lakowicz, 2006).

If multiple dyes are used in the sample, a band pass emission filter can be used to isolate the emission from each dye. Careful filter selection helps to ensure that the detector registers only the light you are interested in—the fluorescence emitted from the sample.

LEDs are relatively new light sources for fluorescence excitation. Single-color LEDs are ideal for low-cost instrumentation, where they can be combined with simple long pass filters that block the LED excitation and allow the transmission of the dye signal. However, the range of wavelengths emitted from each LED is still relatively broad. Currently their application may also require the use of a filter to narrow the bandwidth.

There are many options for light sources for fluorescence. Selecting the appropriate light source, and filters for both excitation and emission, can increase the sensitivity of signal detection to an astounding degree. Making fluorescence labeling one of the most sensitive detection technologies available.

With recent advances in sensitive array detectors, fiber optic wave guides, high speed electronics and powerful software, many new generations of spectrofluorometers have been developed. These new spectrofluorometers use charged couple devices (CCDs) or photodiode arrays to replace the photomultipliers and avalanche photodiodes used in conventional spectrometers. They offer excellent performance for a wide range of spectroscopic applications from the UV to the near IR. Because of their unique combination of outstanding sensitivity, high speed, low noise, compactness, instantaneous capture of full spectra, low cost and robustness, these detectors have revolutionized spectroscopic detection. A quick glance at today's instrumentation market indicates the popularity of the CCD as the detector of choice. The overwhelming benefits of array detectors are simultaneous and multi-wavelength data acquisition. On the other hand, the use of fiber optics as light guidance allows a great modularity and flexibility in setting up an optical measurement system. Recent applications and a critical comparison between simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and chargecoupled device, or photomultiplier for determining proteins in capillary electrophoresis are presented by Casado-Terrones et al. (Casado-Terrones, 2007).

In summary, from the preceding discussion, four essential elements of fluorescence detection systems can be identified: (1) an excitation source, (2) a fluorophore, (3) wavelength filters to isolate emission photons from excitation photons and (4) a detector that registers emission photons and produces a recordable output, usually as an electrical signal or a photographic image. Regardless of the application, compatibility of these four elements is essential for optimizing fluorescence detection.

For the sample holders, the majority of fluorescence assays are carried out in solution, the final measurement being made upon the sample contained in a cuvette or in a flowcell.

Current Achievement and Future Potential of Fluorescence Spectroscopy 225

decrease in the fluorescence signal of yellow-green microspheres when exposed to 160°C for

Fluorescence variations due to pH changes are caused by the different ionizable chemical species formed by these changes. The results from these pH variations can be quite drastic since new ionization forms of the compound are produced. Fluorescence spectra may be strongly dependent on solvent. This characteristic is most usually observed with fluorophores that have large excited-state dipole moments, resulting in fluorescence spectral shifts to longer wavelengths in polar solvents. As the polarity of environment decreases, the fluorophore shows a shift to longer wavelength with an increase in fluorescence quantum. Also, in polar environments the fluorescence quantum yield decreases with increasing temperature, while in nonpolar environment very little change in the fluorescence quantum yield was observed. Never the less, the environmental sensitivity of a fluorophore can be transformed by structural modifications to achieve desired probe specificity (Guilbault,

In summary, all fluorescence data required for any research project will fall into one of the following categories: (1) the fluorescence emission spectrum, (2) the excitation spectrum of the fluorescence, (3) the quantum yield, and (4) the fluorescence lifetime. In a typical emission spectrum, the excitation wavelength is fixed and the fluorescence intensity versus wavelength is obtained. Early examination of a large number of emission spectra resulted in the formulation of certain general rules: (1) in a pure substance existing in solution in a unique form, the fluorescence spectrum is invariant, remaining the same independent of the excitation wavelength (known as Kasha's rule), (2) the fluorescence spectrum lies at longer wavelengths than the absorption, (3) the fluorescence spectrum is, to a good approximation, a mirror image of the absorption band of least frequency. These general observations follow from consideration of the Jablonski diagram shown earlier. The fluorescence spectrum gives

information about processes that happens when the molecules are in the excited stat.

spectrophotometry as a powerful spectroscopic tool in several fields of science.

literature. In this section, few recent methods will be summarized.

In this section, the applications of fluorescence spectrophotometry as a powerful tool for quantitative analysis, characterization, and quality control in different fields will be reviewed and discussed in details. This section will include the use of fluorescence

In the period reviewed so many papers using fluorescence spectrophoometry for analysis, characterization, and as a tool for identification of several compounds are appeared in the

For example, the design and development of artificial molecular systems for sensing anions in biologically relevant conditions is a challenging task in supramolecular chemistry. In particular, sensing fluoride anion has attracted increasing interest in the molecular recognition community because of its pivotal importance in many areas of biological and chemical sciences. In recent years high levels of fluoride in drinking water have caused numerous human diseases, creating a crucial need for artificial sensors to detect fluoride anions in an aqueous environment. Recently, highly sensitive fluorescence "Turn-On"

15 minutes (Guilbault, 1990).

1990; Rendell, 1987; Lakowicz, 2006).

**3. Applications** 

**3.1 Organic analysis** 

Cuvettes may be circular, square or rectangular (the latter being uncommon), and must be constructed of a material that will transmit both the incident and emitted light. Square cuvettes or cells will be found to be most precise since the parameters of path length and parallelism are easier to maintain during manufacture. However, round cuvettes are suitable for many more routine applications and have the advantage of being less expensive. The cuvette is placed normal to the incident beam. The resulting fluorescence is given off equally in all directions, and may be collected from either the front surface of the cell, at right angles to the incident beam, or in line with the incident beam. Some instruments will provide the option of choosing which collecting method should be employed, a choice based upon the characteristics of the sample. A very dilute solution will produce fluorescence equally from any point along the path taken by the incident beam through the sample. Under these conditions, the right-angled collection method should be used since it has the benefit of minimizing the effect of light scattering by the solution and cell. This is the usual measuring condition in analytical procedures.

#### **2.3 Sample preparation**

Fluorescence is a very sensitive technique. This is the one criterion that makes it a viable replacement to many radioisotope-labeling procedures. However, it is extremely susceptible to interference by contamination of trace levels of organic chemicals. Potential sources of contamination are ubiquitous since any aromatic organic compound can be a possible source of fluorescence signal. For example, the researcher is a possible source of this type of contamination since oils secreted by the skin are fluorescent. Good laboratory procedure is essential in preventing solvents and chemicals from becoming contaminated with high background fluorescence that could hinder low-level measurements. Solvents should be of the highest level purity obtainable commercially. In addition, care must be taken to eliminate all forms of solid interference (suspended particulates such as dust and fibers). These will float in and out of the sampling area of the cuvette via convection currents, and cause false signals due to light scattering while they remain in the instrument's beam.

Fluorescence spectra and quantum yields are generally more dependent on the environment than absorption spectra and extinction coefficients. For example, coupling a single fluorescein label to a protein reduces fluorescein's quantum yields ~60% but only decreases its molar extinction coefficient by ~10%. Interactions either between two adjacent fluorophores or between a fluorophore and other species in the surrounding environment can produce environment-sensitive fluorescence.

Many environmental factors exert influences on fluorescence properties. All fluorophores are subject to intensity variations as a function of temperature, pH of the aqueous medium, and solvents polarity. In general fluorescence intensity decreases with increasing temperature due to increased molecular collisions that occur more frequently at higher temperatures. These collisions bleed energy from the excited state that produces fluorescence. The degree of response of an individual compound to temperature variations is unique to each compound. While many commercially available dyes are selected for their temperature stability, care should be taken to eliminate exposure of samples to drastic temperature changes during measurement. If possible, the temperature of instrument's sample compartment should be regulated via a circulating water bath. At lower assay temperatures, higher fluorescence signal will be generated. It has been found that a 50% decrease in the fluorescence signal of yellow-green microspheres when exposed to 160°C for 15 minutes (Guilbault, 1990).

Fluorescence variations due to pH changes are caused by the different ionizable chemical species formed by these changes. The results from these pH variations can be quite drastic since new ionization forms of the compound are produced. Fluorescence spectra may be strongly dependent on solvent. This characteristic is most usually observed with fluorophores that have large excited-state dipole moments, resulting in fluorescence spectral shifts to longer wavelengths in polar solvents. As the polarity of environment decreases, the fluorophore shows a shift to longer wavelength with an increase in fluorescence quantum. Also, in polar environments the fluorescence quantum yield decreases with increasing temperature, while in nonpolar environment very little change in the fluorescence quantum yield was observed. Never the less, the environmental sensitivity of a fluorophore can be transformed by structural modifications to achieve desired probe specificity (Guilbault, 1990; Rendell, 1987; Lakowicz, 2006).

In summary, all fluorescence data required for any research project will fall into one of the following categories: (1) the fluorescence emission spectrum, (2) the excitation spectrum of the fluorescence, (3) the quantum yield, and (4) the fluorescence lifetime. In a typical emission spectrum, the excitation wavelength is fixed and the fluorescence intensity versus wavelength is obtained. Early examination of a large number of emission spectra resulted in the formulation of certain general rules: (1) in a pure substance existing in solution in a unique form, the fluorescence spectrum is invariant, remaining the same independent of the excitation wavelength (known as Kasha's rule), (2) the fluorescence spectrum lies at longer wavelengths than the absorption, (3) the fluorescence spectrum is, to a good approximation, a mirror image of the absorption band of least frequency. These general observations follow from consideration of the Jablonski diagram shown earlier. The fluorescence spectrum gives information about processes that happens when the molecules are in the excited stat.
