Chapter 2:
Advantages of Fluorescence

Chapter 3:
Instrumentation

Chapter 4:
Variables of Fluorescence

Chapter 5:
Calibration and Standards

There are two primary kinds of instruments that measure fluorescence: filter fluorometers and spectrofluorometers.

3.11. Filter Fluorometer. A filter fluorometer measures the ability of a sample to absorb light at one wavelength and emit light at a longer wavelength. A filter fluorometer 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 fluorometer provides a relative measurement and can be calibrated with a known concentration standard or correlated to standard laboratory methods to produce quantitative measurements.

3.12. A spectrofluorometer uses an excitation monochromator (device which includes a wavelength-dispersing component as opposed to a filter) and an emission monochromator. Resolution is obtained with changeable fixed slits. The advantage of spectrofluorometers is that they allow for varying wavelength selection; the operator can scan a substance over a range of wavelengths. (For more details regarding spectrofluorometers, see Guilbault[6] and Lakowicz[7].) The disadvantage of spectrofluorometers is that they are often several times as costly as filter fluorometers and can only provide moderate sensitivity and specificity in comparison. If funds are available, optimal sensitivity and specificity can be obtained with a research-grade spectrofluorometer. Such instruments frequently have monochromators with continuous variable slits and a broad wavelength range (200 - 1000 nm). Some have dual excitation or dual emission monochromators for increasing sensitivity and reducing stray light. Data acquisition and manipulation must be mediated by a computer.[8]

3.2 How a Filter Fluorometer Works  

A fixed or filter fluorometer uses optical filters to provide specific excitation and emission wavelengths. To measure different substances, most filter fluorometers allow the user to mechanically change to different optical filter configurations. A filter fluorometer is commonly used for quantitative analysis where sensitivity is a major factor.

A filter fluorometer works as follows: The light source sends out light in the excitation wavelength range of the compound to be measured. The light passes through an excitation filter, which transmits wavelengths specific to the excitation spectrum of the compound and blocks other wavelengths. The light passes through and excites the sample, and the light emitted by the sample passes through the emission filter (which is at a right angle to the exciting light to minimize light scatter). The emission filter further screens the light, the emitted light is measured by the detector, and the fluorescence value is displayed on the instrument.

Figure 2 illustrates the key components of a filter fluorometer: light source/lamp; excitation and emission filters; sample cell/cuvette; and light detector. ®

3.21 Light Source: The lamp or light source provides the energy that excites the compound of interest by emitting light. Light sources include xenon lamps, high pressure mercury vapor lamps, xenon-mercury arc lamps[9], lasers, and LED’s. Lamps emit a broad range of light; more wavelengths than those required to excite the compound. Lasers and LED’s emit more specific wavelengths.

Xenon lamps are very versatile and powerful, providing light output from 190-1200 nm.

Mercury vapor lamps are usually more intense than xenon lamps, but the intensity is concentrated in wavelengths of the Hg spectrum. Various fluorescent phosphors are used to coat the lamps to provide the desired wavelength of exciting light. In general, these lamps are long-lasting.

Lasers. Convenient and inexpensive tunable lasers have long been sought for spectroscopic uses, including absorption, laser-induced fluorescence, Raman-scattering, high-resolution atomic spectroscopy, laser cooling, and environmental monitoring. The wide tuning ranges of external-cavity diode lasers provide a variety of wavelengths and their narrow linewidth with continuous tunability leads to high resolution scanning capability.

LED’s. The latest revolutions in LED technology have just begun with the introduction of LED’s based on AlInGaP and InGaN. The AlInGaP LED’s offer better efficiencies than filtered incandescent light bulbs in the yellow through red portions of the spectrum. The InGaN LED’s offer higher efficiencies than filtered incandescent light bulbs in the blue to green portions of the spectrum. Continuous advances in the performance of LED’s have opened up a host of new applications for these solid-state light sources.

 3.22 Excitation Filter: The excitation filter is used to screen out the wavelengths of light not absorbed by the compound being measured. This filter allows a selected band of light energy to pass through and excite the sample; it blocks other wavelengths, especially those in the emission spectrum. (Refer to Section 3.3 on Optical Filters.) 

3.23 Sample Cell/Cuvette: The sample cell or cuvette holds the sample of interest. The cuvette material must allow the compound's absorption and emission light energy to pass through. Also, the size of the sample cell affects the measurement. The greater the pathlength (or diameter) of the cell, the lower the concentration that can be read. Fluorometers commonly hold 10 mm square cuvettes, and/or 13 mm or 25 mm test tubes. Adaptors are available for 9m l capillary tubes and 100 m l minicells for small volumes. Cuvettes are made from borosilicate or quartz glass as well as various plastics that can pass the selected wavelengths of light.

Fluorometers are also available for flow-through studies, where samples are pumped through a flow cell in the instrument’s sample chamber. This allows for continuous, on-line monitoring of samples. Flow cells, too, are available in various diameters, and made of borosilicate or quartz glass. The TD-4100 On-Line Monitor does not have a glass flow cell. Aromatic hydrocarbons are detected in a stream of water which falls through an open chamber. This flow cell is referred to as a falling stream flow cell.

3.24 Emission Filter: Stray light such as Rayleigh and Raman scatter is also emitted from the sample. In addition, stray background light may be present that has not passed through the sample. The emission filter screens out these components, allowing primarily wavelengths of light specific to the compound to pass through. (Refer to Section 3.3 on Optical Filters.) 

3.25 Light Detector. The light detector is most often a photomultiplier tube, though photodiodes are increasingly being used. The light passing through the emission filter is detected by the photomultiplier or photodiode. The light intensity, which is directly proportional (linear) to the compound's concentration, is registered as a digital readout.

A photomultiplier tube (PMT) contains a material which creates an anode current proportional to light intensity. Typically, a chain of 6-12 dynodes, which amplify (multiply) the current, are present. High voltage is supplied to the PMT, which determines the intensity of the signal and also affects the noise. The higher the high voltage, the more sensitive the instrument and the greater the noise. Adjusting the operating level or sensitivity of a fluorometer involves finding the right balance between sensitivity and noise. Most PMT’s used in fluorometers are sensitive to light in the 300 - 600 nm range; special red-sensitive PMT’s are available, which provide sensitivity above 600 nm (necessary for applications such as chlorophyll measurement).[10] PMT’s also give off some dark current (current present even when no light is on the PMT). In addition, temperature affects a PMT, as does aging over a period of time. Thus, for stability of readings, it is important when purchasing a fluorometer, to find out if the fluorometer is closed-loop (with stabilizing reference circuitry built in) or open-loop. Refer to Section 3.5 for details.

"A silicon photodiode (PD) converts incident light into an electric current. Two electrically dissimilar semiconductor layers create a potential barrier, usually a PN or NP junction. Incident photons with energy greater than or equal to the bandgap of approximately 1.12 eV create electron-hole pairs. Pairs produced within a diffusion length of the depletion region will eventually be separated by the electric field, producing a current in the external circuit as carriers drift across the depletion layer."[11] A photodiode is better suited to applications with low to moderate light levels in the ultraviolet and near-infrared range. Since it is small and rugged, with low power consumption, it is useful for compact and field instrumentation.

Optical filters are chosen to be optimal for each application, cost effective, and durable. Filters are used to selectively pass a portion of the ultraviolet or visible spectrum.

In combination with a light source, the excitation filter allows only light which excites the molecule of interest to strike the sample. The emission filter allows the fluorescence from the sample to pass to the detector and blocks stray light from the light source or interfering components in the sample. The reference filter is used in the reference path of the Turner Designs 10-AU series fluorometers and is a factor in determining the basic operating level of the instrument.

Filters can be used alone or in combination to select the desired spectral band. Optical filters obey the Bouguer-Lambert Law, which states that the spectral transmittance of two or more optical filters used simultaneously is equal to the product of the spectral transmittance of each filter.[12]

Filters with four types of spectral characteristics are used in Turner Designs fluorometers: broad or narrow bandpass (Figure 3), sharpcut (Figure 4), and neutral density.

Figure 3. Bandpass Filter.

Figure 4. Sharpcut Filter

• Broadband filters pass a broad band of light. For instance, a broadband filter may transmit light from 300 - 400 nm, but block light with wavelengths shorter than 300 and longer than 400.
• Narrowband filters pass a narrow band of light (as little as 1 nm). For example, a 436 nm filter with a bandpass of 10 nm, will pass light from 431 - 441 nm (5 nm on either side of 436 nm).
• Sharpcut or edge filters are used to block light that is longer or shorter than a nominal wavelength. A 450 nm long-wave filter will allow transmission of light that is longer than 450 nm, but it will block light that is shorter than 450 nm. A 450 nm short-wave filter will transmit light that is shorter than 450 nm and block light that is longer than 450 nm.
• Neutral density filters, primarily used as reference filters, can be used to decrease the transmitted light across a very broad spectrum. For instance, a neutral density filter can be used to decrease the total light transmission by a factor of 10 or 100.

Three types of optical filters are used in Turner Designs Fluorometers: Optical Glass, Interference, and Gel Wratten.

• Thermal shock caused by a rapid temperature change.
• Solarization caused by prolonged exposure to ultraviolet light (can cause an increase in absorption, decrease in transmission).
• Exposure to high humidity or corrosive environments (can cause 'spotting' or 'staining', which changes the surface, resulting in increased light scattering off the surface and decreased transmission through the glass.)[13]

When selecting a fluorometer, there are several factors to consider:

• Will it be used in the field in harsh conditions or in the lab or other protected environment?
• Is long-term stability necessary? For example, is frequent calibration practical? And is the instrument going to be used for on-line monitoring?
• Will the instrument be used for discrete sampling or flow-though studies or both?
• Is battery operation required?
• Is portability required?
• What are the data collection requirements?
• Is temperature compensation for changes in sample temperature required?
• Is 4-20 mA signal output required?

Since fluorescence is a flexible technology, consider future applications to be undertaken as well as current needs when selecting a fluorometer.

When selecting a filter fluorometer for your study, sensitivity, dynamic range, and stability are important instrument factors to consider.

3.51 Sensitivity: Signal-to-Noise and Signal-to-Blank. Sensitivity of a fluorometer refers to the minimum detectable quantity of a compound of interest under specified instrument conditions. It is related to two factors: signal-to-noise and signal-to-blank. Practically, sensitivity means the minimum concentration that can be measured above background fluorescence of the interferences. Note that when comparing two instruments for sensitivity, absolute numbers are meaningless. One cannot simply read a sample and blank in two instruments and say the instrument with the "highest" numbers is more sensitive.

Signal-to-noise. Signal refers to the reading of light passed through a sample. Noise refers to the output from the instrument’s electronics, which is present whether or not sample is being read. Instrument noise can be seen by placing a black/opaque sample, which allows no light (signal) into the instrument and looking at the readout. The signal-to-noise ratio can be improved either by increasing the signal or reducing instrument noise. The user can increase signal by using a larger diameter cuvette or increasing slit/window width. The manufacturer can reduce noise by installing a higher-quality, "less noisy" PMT and electronics. Note that merely increasing the instrument’s operating level (sensitivity) will provide a larger signal, but will probably also increase instrument noise.

Signal-to-blank. This is related to signal-to-noise but not the same. Signal refers to the reading of a sample. Blank refers to the matrix liquid containing none of the compound to be measured and scattered light. Signal-to-blank ratio can be determined by measuring blank against sample concentration and determining the ratio. Signal-to-blank ratio can be improved by employing better optics for the specific chemistry. For example, reduce the amount of light scatter (light that hits the detector that has NOT passed through the sample or blank) with a narrower slit (light attenuator, especially on the emission side); or use optical filters with spectra and bandwidths more specific to the compound to be measured. Square vs. round cuvettes may even improve signal-to-blank. Note that merely increasing the instrument’s operating level (amount of light to the sample and sensitivity of the detector and the electronics) will NOT improve signal-to-blank (and it may increase instrument noise). It will simply give larger numbers for both signal and blank with little change in the ratio between them. To improve signal-to-blank, the amount of stray light (light that has not passed through the sample; light not due to fluorescence of the compound) must be reduced.

A comparison of minimum detection limits among fluorometers is often made by using quinine sulfate or some other stable compound as a reference standard. This can work well in many cases, provided the instruments are properly and "equivalently" set up and operated. The standard must be pure and properly diluted and stable. Cuvettes must be clean and properly handled. (Note that at very low levels, quinine is not linear.[16] Currently, there are better compounds to use.[17])

3.52 Dynamic Range. Dynamic range refers to the range of concentrations an instrument can read, from the minimum to the maximum detectable. The minimum detectable concentration is determined by signal-to-noise and signal-to-blank ratios. The maximum detectable concentration is determined by the compound’s chemistry and by factors such as instrument sensitivity ranges, optical pathlength, specificity of optical filters, etc. Some instruments with a wide dynamic range, like the Turner Designs 10-AU series, can read compounds over a range of 5,000,000 to 1 when optimally set.

3.53 Stability: Closed-Loop vs. Open-Loop Fluorometer. Creating an electronically stable fluorometer is especially important for insuring that it produces consistent analytical results over long periods of time.

An open-loop fluorometer is one in which there is no correction for electronic sources of instability. This means that changes in components like the PMT and lamp (due to aging, temperature changes, etc.) are not compensated for by the instrument. These changes can cause errors in readings which necessitate frequent recalibration.

A closed-loop fluorometer compensates for electronic drift in the instrument. This means that if the lamp or PMT changes its performance, the signal output will remain stable. Accordingly, readings change only with changes in sample concentration. The fluorometer does this by correcting for any drift resulting from variations in lamp intensity and/or PMT sensitivity (see Figure 5). Readings are compared and realigned with very stable reference circuitry resulting in excellent stability of readings over time. Closed-loop instruments, like the TD-4100 and 10-AU-005-CE allow for long-term studies, less frequent calibration, and accurate on-line monitoring.

Continue... to Chapter 4