Paper No. : 06 Atomic Spectroscopy
Module:10 Instrumentation of Fluorescence And Phosphorescence
Principal Investigator: Dr.NutanKaushik, Senior Fellow
The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Dr. Mohammad Amir, Professor of Pharm. Chemistry,
JamiaHamdard University, New Delhi
Paper Coordinator: Dr. MymoonaAkhtar, Associate professor, Dept. of Pharm.
Chemistry, JamiaHamdard, New Delhi.
Content Writer: Dr. MymoonaAkhtar, Associate professor, Dept. of Pharm.
Chemistry, JamiaHamdard, New Delhi.
Content Reviwer: Dr. Mohammad Amir, Professor of Pharm. Chemistry, JamiaHamdard University, New Delhi
Description of Module
Subject Name Analytical Chemistry / Instrumentation Paper Name Atomic Spectroscopy
Module Name/Title Instrumentation of Fluorescence And Phosphorescence
Module Id 10
Pre-requisites
Objectives The student will learn about:
Spectroflourimetry instrument as such
Various types of Light Sources like Arc and Incandescent Xenon, Pulsed Xenon Lamps, LED Light as Sources of light etc
Learn about Distortions in Excitation and Emission Spectra Calibration of the instrument
Keywords spectrofluorometers, Arc and Incandescent Xenon Lamps, LED Light Sources, Thin-Film Filters, Neutral-Density Filters
1. Introduction: The success of fluorescence experiments demands an understanding of the instrumentation and attention to experimental details.
Most of the spectrofluorometers available can be used to record both types of spectra i.e. emission and excitation spectra. An excitation spectrum is the emission intensity, recorded at a single emission wavelength, upon scanning the excitation wavelength. Whereas the wavelength distribution of an emission from a system, measured at a single constant excitation wavelength is taken as an emission spectrum. Such spectra can be presented on either a wavenumber scale or a wavelength scale. The usual units for wavelength are nanometers (nm), and wavenumbers are given in units of cm-1. Conversion of wavelengths to wavenumbers can be done by taking the reciprocal of each value. Since wavelength scale, are easier to interpret visually most commercially available instrumentation produce spectra on the wavelength scale. To record a corrected spectra with any instrument is very difficult and won’t be wise because we don’t need the corrected spectra on a routine basis.
The directly recorded emission spectra represents the power emitted at each wavelength or photon emission rate, over a wavelength interval determined by dispersion of the emission monochromator and the slit widths for an ideal instrument. The excitation spectrum represents the relative emission of the fluorophore at each excitation wavelength. Use of different instruments may lead to different emission spectra recorded because of the wavelength- dependent sensitivities of the instruments. The various parts of the spectroflourimeter are shown in Figure 1.
The excitation and emission spectra recorded normally present the relative intensity of photon per wavelength interval. To obtain such "corrected"
emission spectra is difficult however if such spectra are desirous the individual components of the instrument must have the following characteristics:
1. a constant photon output at all wavelengths from the light source is a must
2. the monochromator having property to pass photons of all wavelengths with equal efficiency;
3. polarization should not affect the monochromator efficiency and
4. the detector efficiency is important as it should detect photons of all wavelengths with equal efficiency.
Figure 1: Parts of a spectrofluorometers
Figure 2: Ideal components of a spectrofluorometer and their properties.
But the instruments components like sources of light, monochromators, and detectors with the kind of ideal characteristics mentioned above are not
available. Due to this compromization on the components selection and response of the instrument obtained.
Figure 3: Corrected and uncorrected excitation spectra of fluorescein.
2. Distortions in Excitation and Emission Spectra
To record an excitation spectrum the emission monochromator is set at a wavelength desired for that particular analysis, generally at the emission maxima and then scanned through the absorption bands of the fluorophore
The signal observed/recorded may be distorted due to number of reasons:
1. The light intensity of the excitation source is dependent on the selected wavelength and affects the response of the reference solution even if it is via the beam splitter and corrected by division
2. The wavelength of the excitation monochromators selected also affects the transmission efficiency.
3. The optical density of the sample may exceed the linear range, which is about 0.1 absorbance units, depending upon sample geometry.
The various components of a spectrofluorometer are shown in figure 1. To compensate the power source fluctuations most of the fluorescence instruments employ double-beam optics. The sample beam first passes through a excitation
filter or monochromator, which transmits radiation that will excite fluorescence but excludes radiation of the wavelength of the fluorescence emission.
The most conveniently method to observe fluorescence is at right angles to the excitation beam because increased scattering from the cell wall and the solution may interfere at all other angles and cause error.
The radiations are isolated by passing the emitted radiation through a monochromator or second filter and then the fluorescence is measured by a phototransducer.
The power of reference beam is reduced by passing through an attenuator to approximately that of the fluorescence radiation. These signals obtained from the sample and the reference photomultiplier tubes are then fed into a difference amplifier and recorded. Fluorescence instruments of null type are also available, this state being achieved by optical or electrical attenuators. Instruments have been designed to use a digital data acquisition or an analog divider circuit followed by data processing to complete the ratio of the fluorescence emission intensity to the excitation source intensity.
There are two types of spectrofluorometers, one that utilizes a suitable filter to limit the excitation radiation and a prism or grating monochromator to produce the fluorescence spectrum. True spectrofluorometers are equipped with two monochromators, one of which permits variation in the excitation wavelength and the other allows production of a fluorescence emission spectrum.
3. LIGHT SOURCES
3.1. Arc and Incandescent Xenon Lamps
The most versatile light source at present for a steady-state spectrofluorometer is a high-pressure xenon (Xe) arc lamp. Xenon (Xe) arc lamps provides a continuous light output from 250 to 700 nm (Figure 4). It provides a number of sharp lines near 450 nm and above 800 nm. The Xe lamps are ozone-free lamps, i.e. their operation does not generate ozone in the surrounding air.
Figure 4: Xenon air lamp and a typical lamp housing.
The xenon lamps are usually placed in specially designed housings because of the danger of explosion due to gas in xenon lamps under high pressure (~10 atm.) (Figure 4).
Pulsed Xenon Lamps
It is somewhat same to that of the continuous arcs except that it is pulsed and the intensity of the peak of the pulses is higher. The pulsed xenon lamps consume less power thereby generates less heat which makes its use advantageous in studying the emission spectra of photolabile compounds. Also the photodamage of the sample is reduced as the excitation is pulsed not continuous.
3.2. High-Pressure Mercury (Hg) Lamps (HPML)
Experiments which requires specific wavelength that are specific to mercury lines, Hg lamps are used. Since the intensity source is concentrated in lines, HPML’s have higher intensities than Xe lamps. Therefore it is good to choose the excitation wavelength that suits the fluorophore, rather than choosing fluorophore according to wavelength. Other lamps that have been used include;
Xenon-Mercury Arc Lamps, Mercury -Argon Lamps, Low-Pressure Mercury and Quartz-Tungsten Halogen Lamps.
3.3. LED Light Sources:
A number of spectrofluorometers use LEDs as light sources as a wide range of wavelengths are available with LEDs. Use of LEDs does not need to use of heat filters as they do not generate significant IR radiations. The application of LEDs is likely to broaden in the near future as an excitation source.
Table 1. Strong Emission Lines from a Mercury–Argon Mercury lines
(nm)
Argon lines (nm 253.7 404.7 696.5 763.5 842.5 296.7 407.8 706.7 772.4 852.1 302.2 435.8 710.7 794.8 866.8 313.2 546.1 727.3 800.6 912.3 334.1 577.0 738.0 811.5 922.6 365.0 579.1 750.4 826.5
“a Data fromOcean Optics product literature, http://www.oceanoptics.com”
Laser: Wavelength ranges from 405 to 1500 nm. Beginning in the 1970s various types of lasers were used as excitation sources as they emits monochromatic radiation. Since the output of Laser diodes is easily focused and manipulated they are convenient and can be pulsed or modulated easily. Of particular interest are tunable dye lasers pumped by a pulsed nitrogen gas laser or Nd:YAG laser.
Figure 5: Spectral output of light-emitting diodes [8]. The black line shows the output of a white LED [Ocean Optics product literature, http://www.oceanoptics.com ].
4. MONOCHROMATORS
Monochromators is a part of instrument that disperses polychromatic or white light into the various wavelengths or colors i.e. isolates the specific wavelength of light from broad-band sources.
The physical size of the monochromator slits in a monochromator is very crucial as the selection of spectral region by a monochromator depends on its design. Most commonly available slits give spectral resolutions from 0.2 nm to 32 nm, range from 0.025 mm to 4 mm. Smaller the base line of the slit, the higher will be the resolution because of different dispersion factors with different monochromators. However this resolution leads to a decrease in the intensity of light, with a two-fold reduction in each slit width (entrance or exit), up to 4-fold decrease in light intensity can be observed.
Dispersion in monochromators can be achieved by using prisms or diffraction gratings, however in commercial spectrofluorometer diffraction gratings are used rather than prisms because a linear scan of the prism assembly will not result in a linear dispersion of wavelengths. The performance specifications of a monochromator include three things i) dispersion, ii) efficiency, iii) stray light levels. Dispersion is usually given in nm/mm. To avoid problems due to scattered or stray light in fluorescence spectroscopy a monochromator should have low stray light levels. Stray light means light transmitted by the monochromator other than the wavelengths and band pass chosen. To increase the efficiency to maximize the ability to detect low light levels monochromators are used.
Figure 6: Figure shows the resolution power of a grating.
The peaks with less than 5 nm line widths are rarely present in emission spectra therefore the resolution of monochromator is usually of secondary. It is the slit width that is generally variable, and a typical monochromator has both the slits, an entrance as well as exit slit. Although bigger slit widths means increased signal levels, it also means increased signal-to-noise ratios whereas if the slit widths is smaller higher resolution can be obtained at that will be at expense of light intensity. It can be generally said that the intensity of light that passes through a monochromator is proportional to the square of the slit width.
The increase in slit width will have no effect on the intensity of light that passes through a monochromator if the entrance slit of the excitation monochromator is wide enough to accept the focused image of the arc. A monochromators can have either a planar or a concave gratings (Figure 7). Concave gratings are produced by photoresist and holographic methods whereas planar gratings are produced mechanically. The imperfections in the gratings give rise to stray light transmission, and ghost images. Ghost images sometimes appears as diffuse spots of white light on the inside surfaces of an open monochromator and light blocks has been used to intercept these ghost images. However efficient monochromators can be developed based on concave gratings as they have fewer reflecting surfaces and lower stray light. In fluorescence spectroscopy use of holographic gratings is usually preferred because these grating can serve both as diffraction and focusing element.
Figure 7: Types of grating available for use in monochromators a) planar and b) concave gratings (Adapted from Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, by Springer.
5. Calibration of Monochromators
All analytical instruments are calibrated periodically for accurate and reliable results. Calibration of wavelength of monochromators is also done for the same reasons. Calibration of monochromators can be done by using a mercury-argon lamp or low pressure mercury as it provide a number of sharp lines. Due to their cylindrical shape and about 5mn diameter, they readily fit into the cuvette holder. A block of size of cuvette is made with a pinhole on the sides is used to hold the lamp stationary. The pinhole in made to allow a small amount of the light to enter the emission monochromator. To reduce the light intensity and improve on precision of wavelength determination, the size of slit width can be manipulated. This also helps in safeguarding the photomultiplier tube and/or amplifiers from getting damaged. Using the emission monochromator the dominant Hg lines are located and the wavelengths measured andcompared with the known values, listed in Table 1. To calibrate the monochromator it is important to observe at least three lines to be certain a line is assigned the correct wavelength. If the values recorded differ from the known values by a definite constant amount, recalibration of the monochromator is required to obtain coincidence.
Analytical Chemistry / Instrumentation
Atomic Spectroscopy
Instrumentation of Fluorescence And Phosphorescence
Figure 8: Spectral output of a low pressure mercury-argon lamp. Courtesy of
Ocean Optics Inc
Calibration of the emission monochromator is followed by calibration of the excitation monochromator is done. The new standard are used i.e. the slits are set as same value on both the monochromators. To do this emission monochromator is set at an arbitrary wavelength and the cuvette is filled with a dilute suspension of Ludox or glycogen to scatter the exciting light. Then the intensity of the scattered light is observed, the intensity of the scattered light will at maximum if the calibration has been done. By setting the emission monochromator at various wavelengths the linearity of the wavelength scale can be determined.
6. OPTICAL FILTERS 6.1. Colored Filters
Inspite of use of monochromators for wavelength selection in the spectrofluorometers, use optical filters have become an essential part of them to compensate for their less-than-ideal behavior. Filters often help to maximum sensitivity when the spectral properties of a fluorophore are known compared to monochromators. Now a days manufactures have bridged the gap for selection of filters by proving a large range of filters alongwith their transmission spectra.
In earlier day’s colored-glass were used as filters as they can transmit a wide range of wavelengths (Figure 9a). Some color filters that transmit above some particular wavelength therefore are called long-pass filters (Figure 9b). The classification of filters has been done according to their colors for example BG, GG, blue glass, green glass, etc.
a
B
Figure 9: Transmission of some typical coloured glass filter (Source Ocean Optics Inc)
A number of filters are available and their use depends on the interest of the experiment. Various types of filters includes; thin-Film Filters, Filter Combinations, Neutral-Density Filters.
6.2. Thin-Film Filters
Despite a wide variety of colored-glass filters are available, thin film filters
have been used to obtain almost any desired transmission curve. With advance
in the technology any filters can be designed according to specific applications
e.g. long-pass filters (Figure 10) designed to reject light from a helium-
cadmium laser at 325nm or an argon ion laser at 488nm. The transmission
above the cut-on wavelength is close to 100% to provide maximum sensitivity.
Figure 10: Long-pass filters designed to reject light from a helium-cadmium laser at 325nm or an argon ion laser at 488nm
6.3 Combination Filters
While it is possible to design almost any desired filter with modern coating
technology, but customizing filters for each and every experiment is just not
practical. To overcome this combination of two or more bandpass filters can be used
to get the spectral properties of choice for any given experiment. Such an example is
shown in Figure 11. To isolate protein fluorescence the combination
of WG-320 and UG-11 filters are often used. A combination of Coming 3-72 and 4-96 filters is used for probes emitting near 450 nm. While selecting a filters, its ability to transmit the desired wavelength is important but it is more important to select the filter on their ability to reject a possible interfering wavelength.
Figure 11: Transmission profile of a combination of Corning and Schott filters
used to isolate protein flourscence and Indo-1 fluorescence
6.3. Neutral-Density Filters:
To attenuate light equally at all wavelengths neutral-density filters are used. The neutral- density filters are composed of sheets of quartz or glass coated with a metal to get the desired optical density.
The name of filter sets for microscopy are now often based on specific fluorophores rather than wavelengths.
Fluorescence microscopy often uses multiple fluorophores to label different regions of the cell. This is done to help in identifying the spectral region of interest by emission wavelength. Similarly Emission filters that allow the emission from multiple fluorophores have been designed.
6.4. Neutral-Density Filters:
To attenuate light equally at all wavelengths neutral-density filters are used.
These filters are composed of quartz or glass sheets coated with a metal to get the desired optical density.
The naming of filter sets for microscopy has now often been done on the basis of specific fluorophores rather than wavelengths.
To label different regions of the cell fluorescence microscopy often uses multiple fluorophores. This is done to help in identifying the spectral region of interest by emission wavelength. Similarly Emission filters that allow the emission from multiple fluorophores have been designed.
7. DETECTORS
Photomultiplier tubes (PMTs) are the first choice for most of the modern fluorometers for detection and quantification of the emitted light. The PMTs work on the principle of the photoelectric effect, i.e., when an incident light hits a metallic surface, an ejection of electrons occurs from it. Albert Einstein was awarded the Nobel Prize for proposing this theory and not his more famous
theory of relativity. The original phototubes were based on a simple arrangement to collect the emitted photoelectrons and produce an electric current, which could then be quantified.
Figure 12: A typical photomultiplier tube
A PMT behaves as a source of current which is directly proportional to the incident light intensity. A photomultiplier tube responds to each photon recieved, and the pulse detected is an average signal of all the signals or counted as individual photons.
The construction of a photomultiplier tube includes a vacuum tube consisting of a photocathode and a series of dynodes for the amplification of signal (Figure 12).
The photocathode is made of thin film of metal placed inside of the window.
The photocathode is held at a high negative potential (-1000 to -2000 V), when incident photons hit photocathode ejection of electrons from its surface takes place. The efficiency of photo electrons generation is depends on the incident wavelength. The dynodes are also held at negative potentials, but the potential of successive dynodes decrease toward zero along the dynode chain. A Zener diode is used to fix the potential difference between the photocathode and the first dynode at values ranging from -50 to -200 volts. This helps an ejected photoelectron to be accelerated towards the first dynode. When the
photoelectron hits the first dynode it causes ejection of 5 to 20 additional electrons, depending on the voltage difference between the dynodes. This process continues down the chain of dynodes till a pulse arrives at the anode.
The size of the current pulse depends on the overall voltage applied to the photomultiplier tube. To increase the ejection of number of electrons from each dynode high voltage is applied, and higher amplification can be achieved.
Photomultiplier tubes are useful for low level light detection because of being low-noise amplifiers i.e. little additional noise is produced as the electrons pass through the PMT. Amplification of signal outside of the PMT results in increase on noise than the signal. To perform the quantitative measurements, the anode current and the light intensity must be proportional to each other. If an excessive current from the photocathode is being drawn a nonlinear response is obtained.
Under high-intensity illumination the electrical potential of the photocathode can be decreased because of its limited current-carrying capacity. This decreases the voltage difference between the photocathode and the first dynode, and also decreases the gain.
Light-sensitive photocathodes are damaged by excessive photocurrents resulting in loss of gain and excessive dark currents. A current in the absence of incident light is the dark current from a PMT. To obtain a linear response the dynode voltages must be constant irrespective of the incident light and anode current.
The dynode chains are designed in such a manner that the total current at the end is 100 fold greater than the maximum anode current. Since a typical PMT causes threefold increase in voltage therefore a small in voltage can cause significant change in the signal. Wide variety photomultiplier tubes are available for use and they have been classified according to the design of the dynode for example size, chain, shape, and spectral response, or temporal response.
CCD Detectors: Charge-coupled devices (CCDs) are very good in sensitivity and have linear dynamic range therefore are very widely used in fluorescence spectroscopy. CCDs typically contain 10
6or more pixels where each pixel acts as an accumulating detector where charge accumulates in proportion to total light exposure. A two-dimensional image can be obtained by reading out the charge at each pixel when desired. Small spectrofluorometers using CCDs are commercially available, have good sensitivity and by using a fiber-optic cable the signal is brought to the device. They are conveniently interfaced via a USB cable and can have no moving parts, when combined with an LED light source it can be turned to a solid-state device.
8. Summary:
The topic discusses about the instrumentation used in fluorescence and phosphorescence. Various parts of the instrument are explained like light sources used; Arc and Incandescent Xenon Lamps, Pulsed Xenon Lamps, High-Pressure Mercury (Hg) Lamps, LED Light Sources. Monochromators there construction and types, calibration of monochromators were also discussed. Different types of optical filters used including colored filters, thin- film filters, filter combinations, neutral-density filters. Discussion about detectors like photomultiplier tubes and charge-coupled devices detectors was also covered in the topic.
