laser raman and fluorescence microprobing techniques

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Analytica Chimicu Acta, 195 (1987) 33-43 Elsevier Science Publishers B.V., Amsterdam -Printed in The Netherlands LASER RAMAN AND FLUORESCENCE MICROPROBING TECHNIQUES P. DHAMELINCOURT Laboratoire de Spectrochimie Infrarouge et Raman, C.N R.S. LP 2641, Universit6 de Lille I, 59655 Wlleneuve D’Ascq Cldex (France) (Received 31st July 1986) SUMMARY The increasing need for non-destructive analytical methods and recent developments in instrumentation have greatly stimulated interest in the use of spontaneous Raman scatter- ing for molecular microanalysis, but only recently has this potential been realized. The fundamentals and the methodology of Laser Raman microspectroscopy are reviewed briefly and some practical uses of the technique in several areas of application are outlined. Modern multichannel detection techniques that offer a significant improvement in perform- ance are emphasized. The very sensitive laser microfluorimeter has great potential for investigations of single living cells in biology. From the time of its discovery in 1928 until ten years ago, Raman scatter- ing has been used for studying bulk samples of macroscopic dimension, furnishing information about fundamental molecular properties and providing an important part of laboratory spectroscopy. Raman spectroscopy is based on the spectral distribution of inelastically scattered light and is a highly- selective technique for investigating molecular species in all phases of matter, as they are fingerprinted by their vibrational spectra. The introduction of lasers as sources has greatly enhanced the possibilities of Raman spectroscopy for instrumental microanalysis; Raman scattering can provide information which previously was nob available from any other widely used technique such as electron microprobes or ion microprobes. The microprobe techniques can readily identify, map out the distribution and determine the quantity of ele- mental constituents present but do not really distinguish the chemical forms of elements present as specific compounds in a microsample. With lasers as sources for excitation of Raman scattering and the ongoing development of instrumentation for optical spectroscopy, Raman microprobing techniques have matured to the point at which non-destructive chemical microanalysis has become routinely practicable for both research and industrial purposes. More recently, laser fluorescence techniques have been developed which offer great possibilities for investigations at the level of single living cells. In this paper, the evolution of the instruments that have been developed is described and then the analytical utility of Raman and fluorescence micro- probing techniques is discussed. 0003-2670/87/$03.50 o 1987 Elsevier Science Publishers B.V.

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Page 1: Laser raman and fluorescence microprobing techniques

Analytica Chimicu Acta, 195 (1987) 33-43 Elsevier Science Publishers B.V., Amsterdam -Printed in The Netherlands

LASER RAMAN AND FLUORESCENCE MICROPROBING TECHNIQUES

P. DHAMELINCOURT

Laboratoire de Spectrochimie Infrarouge et Raman, C.N R.S. LP 2641, Universit6 de Lille I, 59655 Wlleneuve D’Ascq Cldex (France)

(Received 31st July 1986)

SUMMARY

The increasing need for non-destructive analytical methods and recent developments in instrumentation have greatly stimulated interest in the use of spontaneous Raman scatter- ing for molecular microanalysis, but only recently has this potential been realized. The fundamentals and the methodology of Laser Raman microspectroscopy are reviewed briefly and some practical uses of the technique in several areas of application are outlined. Modern multichannel detection techniques that offer a significant improvement in perform- ance are emphasized. The very sensitive laser microfluorimeter has great potential for investigations of single living cells in biology.

From the time of its discovery in 1928 until ten years ago, Raman scatter- ing has been used for studying bulk samples of macroscopic dimension, furnishing information about fundamental molecular properties and providing an important part of laboratory spectroscopy. Raman spectroscopy is based on the spectral distribution of inelastically scattered light and is a highly- selective technique for investigating molecular species in all phases of matter, as they are fingerprinted by their vibrational spectra. The introduction of lasers as sources has greatly enhanced the possibilities of Raman spectroscopy for instrumental microanalysis; Raman scattering can provide information which previously was nob available from any other widely used technique such as electron microprobes or ion microprobes. The microprobe techniques can readily identify, map out the distribution and determine the quantity of ele- mental constituents present but do not really distinguish the chemical forms of elements present as specific compounds in a microsample. With lasers as sources for excitation of Raman scattering and the ongoing development of instrumentation for optical spectroscopy, Raman microprobing techniques have matured to the point at which non-destructive chemical microanalysis has become routinely practicable for both research and industrial purposes. More recently, laser fluorescence techniques have been developed which offer great possibilities for investigations at the level of single living cells.

In this paper, the evolution of the instruments that have been developed is described and then the analytical utility of Raman and fluorescence micro- probing techniques is discussed.

0003-2670/87/$03.50 o 1987 Elsevier Science Publishers B.V.

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Principles of Raman and fluorescence spectroscopy Spectroscopic measurements done with Raman microprobes are based on

the excitation and detection of the normal (spontaneous) Raman effect, which has been fully described [ 11. The effect is an inelastic scattering pro- cess which involves the interaction of a monochromatic beam of radiation, from near-ultraviolet (u.v.) to near-infrared (i.r.), with the molecules of the sample. This interaction produces scattered radiation at different frequencies. These frequency shifts (from the exciting line) are identified with the fre- quencies of the atom oscillations in polyatomic structures contained in the sample.

An energy-level diagram illustrates the vibrational Raman effect (Fig. 1). It shows the changes in vibrational energy, hvi, that occur in collisional pro- cesses involving photons and polyatomic molecular structures. The incident light at frequency v. can be scattered elastically (Rayleigh scattering) or in- elastically (Raman scattering). In this latter process, the incident photons colliding with the molecules can either lose energy to (Stokes Raman scatter- ing, V, = v. - vl), or gain energy from (anti-Stokes Raman scattering, Y, = v. + vl), the target molecules. The molecules raised to electronic states can relax following one or two paths. The frequency differences (v. + vi), called Raman shifts are independent of vo. The overall Raman effect is very weak, typically only 10*-10-9 of th e incident radiation being scattering by Raman process. A schematic representation of a Raman spectrum is presented in Fig. 2. Excitation with the green line (514.5 nm) of an argon ion laser is assumed. The Stokes Raman lines and the corresponding weaker anti-Stokes lines appear symmetrically on both sides of the strong Rayleigh line. The Raman shifts expressed in wave number c1 = vi/C (cm-‘) are read directly on the Raman spectrum recording, which usually consists only of the more intense Stokes part. The intensities of the lines are determined by the Raman cross-section and are directly proportional to the number of polyatomic molecules con- tained in the volume of the sample which is probed.

The kind of information provided by the Raman spectrum is essentially the same as that is obtained from infrared spectra. The vibrational frequencies (Raman shifts) are related to the masses of the vibrating atoms, the bond forces uniting them and their geometrical arrangement. Thus, the Raman spectra can be regarded as unique fingerprints which also contain information on the local molecular environment (e.g., amorphous or crystalline phases).

In the spontaneous Raman effect described above, the incident photon energy is below the energies of any excited electronic levels. But if the excit- ing wavelength is such that electronic excitations occur, then other inelastic processes like fluorescence may be induced. This process is depicted in Fig. 1. In nOrIY&l (nOn-reSOnant) flUOreSCenCe, the light t?IdSiOn at frequencies vi? occurs over a broad range which corresponds to the Stokes region of the Raman spectrum. The fluorescence emission offers less molecular selectivity but has an intensity which is often an order of magnitude or more than that of Raman scattering.

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STOKES ANTI-STOKES

RANAN RAYLEIGH RANAN

3. do 3 3.3 bp&+Y

Fig. 1. Diagram illustrating the vibrational Raman effect and the fluorescence emission process.

ANTI-STOKES RAHAN

HAVEN”

hBSOLUTE) 516A 520OA 5425'4 A WAVELENGTH

0 500 RAPAN SHIFTS

Fig. 2. Schematic representation of a Raman spectrum excited with the green line of an argon ion laser, A, = 514.5 nm [17,, (absolute) = 19436 cm-‘].

LASER RAMAN MICROSPECTROMETRY

The principal advantages of the technique are the molecular specificity, the spatial resolution (the laser focal spot placed on the sample can be as small as 1 pm), the ambient conditions (samples can be examined in air under ambient conditions of temperature and relative humidity), and the range of applications (all phases of matter from inorganic, organic and biological materials can be investigated provided that effective microsampling can be achieved).

The major limitations are the inherent weakness of Raman effects, because the low light level phenomenon places difficult requirements on the perform- ance of micro Raman instruments, the fluorescence interferences, because

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fluorescence emitted by the sample may mask the much weaker Raman signal, and the lack of quantification. The absolute determination of species concen- tration is not feasible as yet principally because of the lack of detailed geo- metrical and optical properties of microsamples.

Instrumentation in Raman microspectrometry The intensity of the signal delivered by the detector of a spectrometer

analyzing a Raman line can be expressed by

where I,, is the laser h-radiance at the sample (W cmb2) defined as the ratio of the effective laser power at the sample over the illuminated area; u is the Raman cross-section, N is the number of molecules in the probed volume, C! is the solid angle under which the instrument collects the Raman scattered light, and T and s are the throughput of the instrument and the sensitivity of the detector, respectively.

When a very small quantity of matter has to be examined, there are only a few parameters which can be modified in order to compensate for the large reduction in the number of molecules N, namely, IO, !C? and s. Extensive work in this laboratory and other laboratories was devoted, several years ago, to exploring techniques for developing micro-Raman instruments [2, 31. From experience gained in this work, it was concluded that the use of microscope objectives was the best way to increase both I0 and a. Microscope objectives, which are high numerical aperture optics, are able to place on the sample a laser focal spot with a size well under 1 pm and to collect a large amount of the scattered light; the angle of collection is 130” for a 0.9 numerical aperture (NA). Thus I0 and R can easily be increased by factors of 1000 and several tens, respectively. That is why all the micro-Raman instruments which are now commercially available possess a good quality light microscope coupled to spectrometers or spectrographs. In these instruments, the same (high NA) objective is used to focus the laser beam on the sample region of interest and to collect the light scattered by the sample (back-scattering geometry). This allows the spectroscopic information to be extracted after or sometimes simultaneously with the visual exploration of the sample.

The first generation of instruments developed in the laboratory on this principle became commercially available (Instrument Jobin Yvon) in 1977 under the name of Mole [4, 51. The instrument comprised a scanning spec- trometer and a monochannel detector photomultiplier tube for the recording of Raman spectra from a microscopic region chosen in the sample. However, this instrument had also an imaging mode of operation which permitted the localization of individual components in a multicomponent heterogeneous sample. Unfortunately, this last mode, though very interesting, has seldom been used because it lacks sufficient spatial resolution and sensitivity to dis- criminate Raman features from fluorescence background, especially for industrial samples. This instrument has now been superseded by a modular

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system, the Ramanor U-1000, in which a conventional micro-Raman scanning spectrometer can be fitted with an optical microscope for micro-Raman measurement.

The main limitation of this first generation of instruments lies in the high laser h-radiance (typically lo’-lo6 W cm -*), which may cause degradation during the long exposure time required to record spectra; several tens of min- utes to several hours may be needed depending on the nature of the sample. This limitation is inherent in the sequential recording of the spectral elements imposed by scanning spectrometers.

For a Raman spectrum composed of N spectral elements, a scanning spec- trometer receives at its detector only one spectral element during the time t = T/N, where T is the total recording time. During this time t, the (N - 1) spectral elements which are available at the exit slit level of the spectrometer are lost to the detector so that the total loss of information is N (N - 1) when the spectrometer has analyzed all the spectral elements during the time T.

In order to overcome this difficulty, the second generation of instruments [6, 71 is based on the use of multichannel detectors which provide simul- taneous recording of all the N spectral elements during the time t with the same sensitivity. Thus, if two identical spectra are recorded (using a mono- channel or multichanneldetector) during the same total time T, the signal-to- noise ratio for the spectrum recorded with the multichannel technique would be superior by a factor N”*. As a result, this signal-to-noise gain can profitably be changed to time in order either to detect very weak Raman signals or to illuminate fragile materials with very low laser power.

Figure 3 presents the optical scheme of a multichannel Raman microprobe (Microdil 28) which has been developed in close collaboration between this laboratory and a French company (DILOR, Lille France). The Microdil28, which has been commercially available since 1982, is equipped with an Olympus microscope. Focusing and coupling optics are specially designed to enable the operatorto explore the whole observed area of the sample without moving the stage of the microscope (optical scanner) [ 81. The dispersive sys- tem uses a zero dispersion double fore-monochromator followed by a spectro- graph. When high-resolution work is needed, an optical device enables the whole system (fore-monochromator plus spectrograph) to be converted to a high-dispersion spectrograph. The multichannel detector comprises a photo- diode array (Reticon) containing a row of 512 diode elements intensified by a miniature distortion-free electrostatic proximity focused microchannel plate intensifier tube (RTC). The detector unit is mounted in a package cooled at -20°C by a Peltier effect, allowing integration times of as long as 100 s without saturation, A computer is used for spectra acquisition, treat- ment, display and data plotting. In addition to this instrument, for which all the optics were conceived and optimized for Raman microanalysis, there are now on the market several multichannel macro-Raman instruments to which a microscope can be attached as a micro-Raman accessory (Omars 89, DILOR; Triple spectrographe Raman, Instruments S. A., France; and Micramate, Spex, U.S.A.).

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SPECTROGRAPH

7s ?A ENTRANCE

SLIT

L

FORE l!WLlCHRWTOR

Fig. 3. Diagram of the multichannel Raman microprobe (Microdil 28) developed at Lille (C.N.R.S.-DILOR company).

APPLICATIONS OF MICRO-RAMAN SPECTROSCOPY

Various kinds of problem are amenable to investigation by micro-Raman spectroscopy.

Geology and gemmology Point and non-destructive analyses of intramineral fluid inclusions have

been achieved [9, lo]. In the course of geological phenomena, whether they be hydrothermal, magmatic or metamorphic, the role played by fluids is very important. Studies of the composition and density of deep fluids, i.e., the intramineral inclusions which may have sizes varying from a few to several tens of micrometers, can therefore provide sound proof of the origin of the minerals. Generally, several generations of inclusions, in terms of age, origin

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and thus composition, coexist in a single mineral. Thus it is very important to examine each inclusion individually and non-destructively. The basic tech- nique currently used to study fluid inclusions is microthermometry; this entails observation of the inclusions under a microscope equipped with a heating/cooling stage in order to establish the homogenization temperature of the fluids and the transition of phases. The results are interpreted in terms of composition, density and pressure by using phase diagrams corresponding to the system trapped in the inclusion. However, whenever the system is not pure, microthermometry permits neither characterization of the fluids pre- sent nor extraction of quantitative data on their molar fractions.

Raman microspectroscopy, through its capacity to examine very small quantities of matter in situ (through the host mineral) and nondestructively is particularly well adapted to the study of fluid inclusions. It provides precise data for identification and determination of molar fractions of the main geological fluids (CH,, COZ, Nz, H& 02, Hz, etc.). The coupling of micro- thermometry with Raman microspectroscopy thus allows a correct definition of the thermobarometric conditions of fluid capture.

In a similar manner, Raman microspectroscopy has proven to be an excel- lent technique for identifying fluid or solid inclusions in gems [ 111. Provided that the host gem is transparent, the laser beam can be focused directly through a crystalline or a cut facet into the inclusion to be characterized. For the gemmologist, this identification is of the utmost importance both for identification of gems and for determination of their geological origin. Moreover, this in-situ non-destructive technique has proven to be very effi- cient in discriminating between natural gems and synthetic minerals made for jewellery (e.g., sapphire, ruby, emerald) [12].

Point and non-destructive analyses of crystallized or vitreous mineral phases in rocks have also provided useful results [ 131. The analysis of polished sections of rock with an electron probe, combined with observation under a photon microscope, is one of the techniques currently used to identify and follow the evolution of mineral phases in studies of metamorphism, magmatic rocks, alteration phenomena, etc. The application of Raman microspectros- copy in the analysis of polished sections of rocks has proven to be an efficient method for rapid identification of minerals during the non-destructive exami- nation of the sample. It is particularly useful for the identification of poly- morphic species indistinguishable with the electron probe and for detecting the presence of vitreous phases and monitoring their evolution, e.g., the phenomena of devitrication, detection of vitreous residues in greatly trans- formed rocks, etc.

Industrial material control Contamination and the formation of defects are problems constantly

encountered in the commercial production of minerals. The chemical identifi- cation of these defects is of particular interest because it can lead to properly selected materials and processes.

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In semi-conductor production, because circuit-board geometries and com- ponents approach micrometer dimensions, contaminants of this size often create reliability problems. The first step towards solving these problems is identification of the contaminant, for which Raman microspectroscopy is often the only method with a chance of success [14]. Inorganic materials (e.g., contaminants from etching and corrosion stains on connector lines) as well as organic materials (e.g., contaminants from packing materials) can be readily identified in situ by Raman microspectroscopy. In addition to the identification of foreign contaminants on devices, Raman microspectroscopy can also provide characterization of the materials used in fabricating the devices [15]. For example, with the development of very large-scale integra- tion (VLSI) technology, considerable emphasis has been placed on devising methods of fabricating high-quality silicon films on a variety of insulating substrates. A wide variety of approaches to the production of such silicon overlayers has been explored, from vapor-phase epitaxy to thermal annealing and lateral epitaxial growth. A good understanding of the crystal growth is essential for further development of correct quality of materials. Raman microspectrometry can be used as a rapid non-destructive method for pro- cessed silicon based on the position and width of the silicon band around 520 cm-’ which is extremely sensitive to local lattice characteristics.

A further example is the examination of contaminants in synthetic textile filaments [16]. Small individual filaments (typically 5-20 ym in diameter) are usually used to make textile yarns. Filament breaks caused by particulate contamination of the polymer pose problems. Inclusions in the filaments arise from particulate contamination of the feed stock polymer. They can be examined directly by recording the Raman spectra through the textile fila- ment. Substances present as part of the polymer recipe or produced during polymerization (internal contaminants such as antiluster, degraded polymer and carbonaceous residues) as well as substances acquired during the material- handling processes (external contaminants such as packing materials, fiber fragments from work clothing and airborne particles) are thus easily identified.

Analysis of airborne particles The analysis of airborne particles in the size range l-10 pm is important

in many fields of human endeavour including pollution monitoring, health hazards, etc. Raman microspectroscopy is eminently suited for characteriza- tion of microparticles of organic and inorganic origin. In particular, organic compounds give informative, diagnostically useful spectra [ 171.

The principal species for which characterization is easy include the com- mon minerals (silicates, oxides, carbonates, sulfates) and some organic com- pounds (pesticides, insecticides, aliphatic acids, polymers, hydrocarbon films, peptides, etc.).

In-situ analysis of ancient works of art Historical materials and works of art are unique and cannot be replaced; no

risk of damage from the analysis can be tolerated. Raman microspectroscopy

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is very suitable in aiding authentication with no major damage to the historic item or objet d’art being examined.

Recent work in this laboratory [18] has demonstrated the ability of this technique to identify pigments on various ancient works of art. Lazurite and mercury(I1) sulphide (cinnabar, natural vermillion) were identified in the blue and red ornamented letters on a page of a twelfth century French missal, and arsenic trisulphide (orpiment) as the yellow paint on a fragment of an Egyptian death mask.

Microanalysis of biological tissues With the advent of Raman microprobe techniques, great possibilities for

unusual investigations appear to be opening up in biology, pathology and tis- sue research to obtain molecular information at the cellular level. Raman microanalysis is generally done on standard histological sections of biological tissues. So far, because of the fragility of biological samples, studies have been restricted to problems where the sample material was sufficiently locally concentrated. Samples examined include. bio-accumulations in cells and tis- sues [ 19, 201, precipitations in vertebrate kidney induced by pathological processes [ 211, hard tissue formation [22] , pellets of Chinese hamster chromosomes [23], and foreign bodies in tissue coming from the degradation of implanted prostheses [ 241.

Improvements in sensitivity are required in order to extend the field of applications to the study of normal tissue in which the biological material of interest is homogeneously distributed. Raman multichannel microprobes will offer unusual opportunities for such studies. For example, in vivo studies of pigments in vegetal and animal single cells are possible by using the reso- nance Raman effect [25] in which the spectral intensity is enhanced when the laser wavelength falls within an absorption band of the pigment.

LASER MICROFLUORIMETRY

The use of laser sources for the excitation of fluorescence spectra offers great advantages, especially in terms of high spatial resolution and high sensi- tivity [26]. However, beside its unquestionable merits, this technique has some limitations connected with the nature of the sample material. The sample material has to absorb light at the excitation wavelength and high u-radiances induce generally rapid modification of the probed area, which is indicated by fast decay of the fluorescence emission. Fragile materials are not able to sustain laser power higher than a few hundred microwatts.

Fluorescence studies usually require fast recordings of the whole fluore- scence spectrum with both good resolution and signal-to-noise ratios [27] . These requirements preclude the use of scanning spectrometers with single- channel detectors. A microfluorimeter which is based on spectrographic dispersion with a sensitive multichannel detection is currently being developed.

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Applications of laser fluorescence microspectrometry Laser sources allow very precise topographic investigations and so are

particularly well adapted to the study of biological mechanisms in single living cells [28]. Studies connected with enzymology, metabolization, drug/intracellular target interactions and intracellular microviscosity measure- ments based on fluorescence polarization have been reported. In recent work, this technique proved to be an efficient approach to examining the mechan- ism of resistance of human tumor cells (K-562 leukemia cells) to an anti- cancer drug (Adriamycin). The fluorescence spectra obtained from the cytoplasm and nucleus of living cells sensitive or made resistant to the drug were compared [ 291.

Pigments can be identified in situ in inks or paints deposited on any support so that no sampling is required; this offers interesting possibilities in forensic science [ 301. Finally, laser-induced photoluminescence studies of semiconductors such as indium phosphide and gallium arsenide, can be con- ducted on a microscopic scale in order to check the uniformity of the elec- tronic properties of the material surface during processing of the devices [ 311.

Conclusion With routine detection limits in the nanogram range and high selectivity,

the Raman microprobing technique has already become a major microanaly- tical technique yielding new or more precise answers to problems left unsolved or incompletely solved by other techniques.

Laser microfluorimetry, with its very high sensitivity and spatial resolution, is a powerful tool for studying intrinsic or extrinsic fluorophores (fluorescent probes) in single cells while preserving their identity so that the future of this technique looks very promising.

REFERENCES

1 D. A. Long, Raman Spectroscopy, McGraw-Hill, New York, 1977. 2 M. Delhaye and P. Dhamelincourt, J. Raman Spectrosc., (1975) 33. 3 G. J. Rosasco, E. S. Etz and W. A. Cassatt, Appl. Spectrosc., 29 (1975) 396. 4P. Dhamelincourt, F. Wallest, M. Leclercq, A. T. N’Guyen and D. 0. Landon, Anal.

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