Instrumentation Principle Laser Induced Fluorescence Biology Essay


Atomic Fluorescence Spectroscopy is a method that has the characteristics of both Atomic Absorption Spectroscopy and Atomic Emission Spectroscopy methods. Laser Induced Fluorescence (LIF) is a type of AFS technique where a laser is used as the excitation source.

Schematic diagram of Laser Induced Fluorescence






The main principle involved in LIF technique is fluorescence emission from gas phase atoms. Fluorescence emission is a two step process which involves excitation of gas phase atoms followed by subsequent relaxation. Excitation of gas phase atoms is caused by the absorption of photon energy from incident laser radiation. The wavelength is selected to be the one at which atoms have their maximum absorption. The excited state atoms then relax by reemitting the absorbed energy in the form of fluorescence emissions. The subsequent fluorescence emission is then detected with a photomultiplier tube. Atomic Fluorescence Spectroscopy is a useful method for quantitative assessments of elements because of its wavelength selectivity, its high signal to noise characteristics and low background noise.

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The LIF instrumentation in this project consists of

Nd:YAG laser (Pulsed laser)

Tunable dye laser

Frequency doubling crystal

Raman shift cell

Optics for focusing and collecting light

H2 Flame

Hydride generation system


Photomultiplier tube

Box-car integrator

Oscilloscope and computer to view and record the spectrum

Argon gas system

Nd:YAG Laser:

An Nd:YAG pulsed laser is used as an excitation source in LIF (Continuum, Surelite SLII-10).

Schematic diagram of Nd:YAG laser


Flash lamp

Laser Rod


MirrorSchematic Nd YAG Laser.png

An Nd:YAG laser consists of four major components including a flash lamp, laser rod, Q-switch and mirrors. The main function of flash lamp is to optically pump Nd:YAG laser. The optical resonator is a closed cavity with a neodymium-doped yttrium aluminum garnet crystal (Nd:Y3Al5O12) as a lasing medium. Nd3+ ions in the gain medium absorb pumping light from flash lamp and they become excited. These excited ions emit photons with the same energy as the laser atomic transition wavelengths. In this process, when a photon passes through the lasing medium and when the frequency of the photon is equal to that of lasing medium, the photon becomes amplified due to the simulation of the decay of the other ions from the upper state to the lower state. The mirrors are arranged in a way to make the amplified light reflect back into the resonant cavity to increase the amplification.

Nd:YAG lasers are one of the most common types of lasers and are used in LIF measurements because of their consistency, efficiency, compactness, and high laser energy production. The fundamental wavelength for an Nd:YAG laser is 1064 nm in the infra red region, which can be transformed to second, third, and fourth harmonic wavelengths.

Table 1 Wavelengths of Nd:YAG laser


Wavelength (nm)

Pulse width (ns)







4 - 6



4 - 6



4 - 6

The fundamental wavelength (1064 nm) is not directly useful for many applications. Different birefringent materials like KDP (Potassium Dihydrogen Phosphate) and KD*P (Potassium Dideuterium Phosphate) can be used to convert the fundamental wavelength to other harmonic wavelengths like 532 nm (second harmonic) and 355 nm (third harmonic) which have two and three times the photon energy of the fundamental wavelength and are useful for many applications, including dye laser pumping.

Table 2 Energy of fundamental and harmonic wavelengths of Nd:YAG laser

Wavelength (nm)

Energy (mJ)




Table 3 Specifications of laboratory Nd:YAG laser (Surelite Laser)

Pulse energy

Pulse duration

Pulse rate

Energy stability

Beam diameter

Beam divergence

Flash lamp lifetime


Tunable dye laser uses an organic dye as the gain or lasing medium and is used to change a laser emission wavelength in a given spectral range. The wavelength of operation of a dye laser can be altered in a controlled manner and hence it can be used over a wide range of wavelengths [Koechner]. Wide bandwidth of Tunable dye lasers makes them particularly suitable for a wide range of wavelengths. Dye molecules absorb pump laser light at one wavelength and reemit at a different wavelength. Organic dyes have broad fluorescence bands. These bands are excited by a pump laser (Nd:YAG laser) and a laser output wavelength is selected using gratings and prisms. The combination of tunable dye lasers and Laser Induced Fluorescence has been shown to provide high sensitivity for many elements [Fassel J D].


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Frequency doubling or second harmonic generation is an optical process, in which two photons passing through a nonlinear crystal are efficiently combined to form new photons with double the frequency of the initial photons [wiki]. This process is used for the conversion of fundamental wavelength (1064 nm) to the second harmonic wavelength (532 nm).

Second Harmonic Wavelength (532 nm)

Fundamental Wavelength (1064 nm)

Nonlinear Crystal

While interacting with the nonlinear crystal, the high intensity fundamental wave generates a nonlinear polarization in the crystal, which provides a new wave at double the fundamental frequency. The generated new photons have half the wavelength and double the energy of the original photons. Frequently used nonlinear crystal materials include lithium niobate (LiNbO3), lithium triborate (LiB3O5), lithium tantalate (LiTaO3), and potassium titanyl phosphate (KTiOPO4) [Rudiger].


Raman cell is designed to convert deep green or visible pulses generated by a dye laser or frequency doubling crystal to the ultra violet range. Wavelength alteration is achieved by the Raman Effect, i.e. Anti-stokes shift. The Raman cell is filled with either nitrogen or hydrogen gas at high pressure. Raman cell produces multiple Stokes and Anti-stokes beams concurrently.

The fundamental beam is focused into the entrance window of the gas cell and an adjustable lens is used after the cell's exit window to collimate the emerging beams. Shifted beams need to be separated from the fundamental beams as they are collinear. Separation of beams is usually achieved by use of Pellin-Broca prisms.


Lenses and mirrors of specific focal length are used for focusing and collecting the laser and fluorescence emissions. Different focal length lens are chosen depending upon the wavelength of the element.


Hydrogen flame uses hydrogen gas and air present in the atmosphere as fuel-oxidant mixture. Hydrogen flame is an invisible flame and as it burns clear it has very less background noise. A H2-Air flame sustained on a quartz tube (1/4th inch o.d.) was used as the atomizer for LIF spectrometry. The laser beam passed through the flame at a height of 2-3 mm above the outlet of the quartz tube. The height of the flame was adjusted with a micrometer stage to optimize the fluorescence signal.


The hydride generation system used in these studies is a continuous flow hydride system. The continuous flow hydride generation system consists of the following components: a four-channel peristaltic pump (Model RP-1, Rainin Instrument Co. Inc., Emeryville, CA, USA) with Tygon or PVC tubing, a U-shaped gas - liquid separator made of pyrex glass, a Nafion tube dryer (MD Series, Permapure Inc., Toms River, NJ, USA) and reaction coils of various lengths (1-2 m, 1mm i.d.) of Teflon tubing.

Two channels of the four channel peristaltic pump were used to pump the sodium tetrahydroborate and acidified samples. The flow rates of sodium tetrahydroborate and acidified sample were controlled by use of tubing with different diameters. These solutions were combined in a mixing chamber, and sent through a reactions coil to a U-shaped gas-liquid separator (GLS). The GLS separates the gas from the liquid waste and purges the volatile hydrides towards the atomizer. An argon gas stream was used to carry the volatile hydrides to the flame. The flow rates of all the gasses and solutions used are shown in table

Table Operating conditions for the Hydride Generation system





Acidified Sample flow rate

NaBH4 flow rate

Argon flow rate (flame)

Hydrogen flow rate (flame)

Air flow rate (Nafion tube)


Monochromator is an optical device that helps in selecting a desired wavelength from a range of wavelengths. In these studies a monochromator (1000 µm vertical slits, Spectra Pro-275, f/3.5, Action Research Corp.) was used to isolate fluorescence emissions. Fluorescence emissions were collected at 90o to the laser beam direction and were transmitted to the entrance slit of monochromator. From the entrance slit the fluorescence emissions were directed towards a diffraction grating. The grating disperses the light by diffracting different wavelengths at different angles. By adjusting the angle of the grating, the desired wavelength can be isolated. The isolated fluorescence emission is directed to a focusing mirror, exit slit and to the Photomultiplier Tube (PMT) detector. The slit widths of entrance and exit slits of monochromator were adjusted to provide the best signal to noise ratio.

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Table Specifications of Laboratory Czerny Turner Monochromator

Focal length

275 mm

Slit width

Upto 3000 µm


1200 grooves/mm


0.05 nm

Focal plane

27 mm wide * 14 mm high


A Photomultiplier tube is an extremely sensitive detector of light that consists of a photocathode, an electron multiplier and a series of dynodes [wiki]. In this study the isolated fluorescence emission from monochromator was detected by a Photomultiplier tube (PMT, R955, Hamamatsu Corp.). A photomultiplier tube amplifies a single incident photon to approximately 106 electrons. A typical PMT consists of the following components: a vacuum tube, several dynodes and an anode. The vacuum tube contains a photosensitive metal called a photocathode. When a photon with enough energy strikes the photocathode, it emits a photoelectron due to photoelectric effect. These photoelectrons are accelerated towards a dynode which generates 2-5 secondary electrons for each incident electron. When these secondary electrons hit another dynode, additional secondary electrons are generated. As there are a series of dynodes many secondary electrons are produced and when all these secondary electrons reach the anode it generates an electrical pulse that can be detected by the Oscilloscope.


A boxcar integrator amplifies and integrates the input signal during a predefined gatewidth, starting at a predefined trigger, ignoring the noise and interference that could be present at other times. Each of these analog input signals can then be averaged by using an analog averager. The boxcar integrator consists of three components: a gate generator, a gated integrator, and an analog averager. The gated generator provides a delay from a few nanoseconds to 100 milliseconds. The delay gate can be adjusted from 2 nanoseconds to 15 microseconds. The gated generator is usually triggered externally. The gated integrator integrates and amplifies the input signal during the delay gate. The output signal from the gated integrator is then normalized to give a voltage. The output voltage is proportional to the average of the input signal during the time the gate is open (Sampling gate). The exponential averaging technique is helpful to recover small signals from noisy backgrounds.


An Oscilloscope is basically a graph displaying device, which converts the waveform of detector signal into an electrical signal and draws a graph of the electrical signal. In most application the graph shows how fluorescence signals (Y - axis) change over time (X - axis). Oscilloscope has a time base control that adjusts the scale of X - axis in seconds and a vertical control that adjusts the Y - axis in volts. By using an oscilloscope the time and voltages values of a signal can be determined and the frequency of an oscillating signal can be calculated.