Q-Switched Ruby Laser for Emission Microspectroscopic Elemental Analysis Norman A. Peppers, Earl J. Scribner, Lloyd E. Alterton, and Richard C. Honey Stanford Research Institute, Menlo Park, Calif.
94025
Edwin S . Beatrice, Ingeborg Harding-Barlow, Robert C. Rosan, and David Glick Stanford Unicersity School of Medicine, Stanford,
Gal$ 94304
Instrumental improvements in a laser microprobe system were developed which are significant in the following respects: (1) The long-term stability that is implied by the firing of the laser 20,000 times over a 1year period without repair or significant degradation of performance demonstrates that the laser can be treated as a reliable instrument for micro sampling for emission spectroscopy. (2) The relative standard deviation of the laser energy commonly considered to be the major precision-limiting factor has been reduced to about 4%. (3) The low divergence of the laser beam and the excellent control of its energy, together with the high sensitivity of spectral detection have extended the sample resolution to cellular dimensions. (4) Simultaneous monitoring and control of the laser beam opens the possibility of defining sample size in terms of laser energy delivered.
LASER-INDUCED PLASMAS have been exploited by spectroscopists for analytical purposes since 1962 (1-4). The laser microprobe, because of its potential for sampling to the limit of the light microscope and beyond, offers unique possibilities of elemental analysis of biological and nonbiological materials. Applications to histo- and cytochemistry have been brought up to date recently (5). However, the potential of this analytical technique, particularly in its quantitative aspects, has not been fully realized because of certain problems in the technology. Central to these problems is the nature of the laser unit itself. Pulsed lasers, whether long-pulsed or Q-switched, or whether ruby crystal or neodymium-doped glass is used, have been variable in energy and power output, and the size of sample vaporized is related to laser output. Variations in the output can be measured, but it is not usually done simultaneously with use. This instrumental limitation has greatly hindered the establishment of reliable quantitation. It has been found (6) that the spark excitation usually used to increase spectral emission from the laser-induced plasma must be eliminated for sample sizes less than about 10 p since the spark must be positioned so close to the sample that surrounding material is partially vaporized and included with the sample. However, elimination of spark excitation places more severe demands on the spectrographic and detection systems, and improvements in the latter can lead to greater sampling resolution. Although the limit in spot-size set by diffraction can be achieved with ordinary microscopes and (1) F. Brech and L. Cross, Appl. Spectrosc., 16, 59 (1962). (2) R. C. Rosan, M. K. Healy, and W. F. McNary, Jr., Science, 142,236 (1963). (3) J. A. Maxwell, Can. Mineralogist, 7,727 (1963). (4) E. F. Runge, R. W. Minck, and F. R. Bryan, Spectrochim. Acta, 20, 733 (1964). (5) D. Glick, in “Symposium on Data Extraction and Processing of Optical Images in the Medical and Biological Sciences,” N. Y. Acad. Sci., in press. (6) I. Harding-Barlow, E. S. Beatrice, and D. Glick, Federation Proc., 26,780 (1967).
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lasers (7), a laser of exceptionally fine quality is required to maintain large power densities at these extremely small spot sizes. Thus, improvements in laser quality also can lead to greater sampling resolution. With the aforementioned instrumental limitations in mind, the authors modified an early model Jarrell-Ash microprobe system (8) in several significant respects: a highly stable, Qswitched ruby laser was designed and constructed specifically for analysis of micro samples for emission spectroscopy. The design emphasis was on optical quality as well as on both longterm and short-term stability. A sensitive detector was designed as an integral part of the laser unit to monitor the pulse shape, peak power, and energy per pulse, during actual use of the laser. The laser unit developed was used with a sensitive detection system using multiplier phototubes adapted to an f/6.3 spectrograph. Optical coupling between spectrograph and plasma was established with the use of an f/1.5 lens. The system resulting from these and other modifications has been found to be convenient, versatile, and stable and has permitted the practical sampling resolution to be increased to the point where intracellular analysis appears feasible (6). Significantly, this system provides spectroscopists with a tool that will allow them to develop and evaluate the technique of laser-induced emission spectroscopy in quantitative terms without the encumbrance of major instrumental shortcomings. EXPERIMENTAL
Apparatus. A detailed drawing of the laser, the monitoring equipment, and the microscope is shown in Figure 1. RESONANT CAVITY.To ensure long-term stability, a cavity configuration was selected that did not require optical coatings. Previous experience has demonstrated that optical coatings are more easily damaged in Q-switched lasers than are bulk materials. The resonant cavity is terminated at one end by a fused-silica roof prism (J) and the other end by a Fabry-Perot etalon (L) mounted in a gimbal suspension (M). The etalon is made of high-index glass and has a theoretical maximum reflectance of 27z. The laser rod is a 60°, plane-parallel, in. X 3 in. ruby (E). It is fine optical quality and shows less than three fringes in a Twyman-Green interferometer. All cavity surfaces are flat to within 1/10 with angular tolerances of 2 arc seconds. Q-SWITCH. To minimize interelemental effects and crater depth, a short pulse is desirable. A bleachable dye Q-switch (9), which causes release of energy in a single giant pulse, was chosen because it results in a shorter pulse than does a rotating prism Q-switch and because it is less expensive than either a Pockels cell or Kerr cell Q-switch. The material of the laser rod was chosen to be ruby rather than Nd-doped (7) N. A. Peppers, Appl. Opr., 4,111 (1966). (8) S. D. Rasberry, B. F. Scribner, and M. Margoshes, ibid.,6, 81 (1967). (9) P. P. Sorokin, J. J. Luzzi, J. R. Lankard, and G. D. Pettit, IBM Journal, pp 182-4 (April 1964).
Figure 1. Diagram of laser unit, monitoring equipment, and microscope
I
air pre-filter dehumidifier sub-micron air filter laser pump cavity ruby rod flashlamp air exhaust port flashtube ignitor liquid Q-switch
roof prism transverse mode selector etalon reflector precision gimbal suspension liquid attenuator glass beam splitter dual channel laser detector Q detector power supply R electrometer J K L M N 0 P
glass since the bleachable dye for the former is much more stable than that for the latter. The dye was diluted from a supersaturated solution of purified (10) vanadium phthalocyanine which had been left standing in the dark for at least 48 hours and was sealed in a 5 cm3 silica cuvette (I) with a path length of 1 cm. It is known that exposure both to ultraviolet light and to oxygen over significant periods of time degrades the optical properties of the bleachable dye that determine its ability to Q-switch. Because of this a light shield was used to block from the cuvette ultraviolet light emitted by the flashlamp, and the stopper on the filled cuvette was sealed with vacuum grease. MODESELECTOR.The diameter of the spot to which the laser beam is focused by the microscope objective (V) is (10) F. H.Mosher, “Phthalocyanine Compounds 1963,” American Chemical Society Monograph No. 157, p 128.
adjustable prism adjustable mirror U alignment telescope V microscope objective W spark excitation electrodes X test sample Y safety interlocks S
T
proportional to the divergence angle of the laser beam. A transverse mode selector (K) is placed within the resonant cavity to discriminate against off-axis transverse modes and thus reduce the beam divergence. It consists of an axial circular aperture in an alumina disk. The white ceramic is not damaged by radiation within the cavity. PUMPCAVITY.The pump cavity (D) consists of a helical xenon flash lamp (F) within a 1-in. diameter highly polished aluminum cylinder. The maximum energy stored by the lamp capacitor bank is less than the lamp rating, and the typical operating energy is less than half that of the lamp rating. This was done to prolong the useful life of the lamp and to promote long-term stability. The lamp and ruby are cooled by a current of clean dry air. The air is cleaned by a prefilter (A), dehumidifier (B), and again filtered (C) before flowing past the ruby and flash lamp a t about 4 ft3/min. This causes a slight positive pressure within the laser housing and prevents outside air from entering. VOL. 40, NO. 8, JULY 1968
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IOFT 7CONDUCTOR
VSO90
NE-2
0.1 uf
MODEL 6100
9.IMR
I
WWER SUPPLY
Figure 2.
LASER DETECTOR
Circuit diagram of dual channel detector and power supply
ATIENIJATOR. The energy output of the laser is not easily controlled by varying the energy input to the flash lamp since this implies extremely fine voltage control when the laser is operating near threshold. However, it can he controlled over a wide range by attenuation. Aqueous copper sulfate solution in a 3 cm3 fused-silica cuvette with a path length of 1 cm serves as an attenuator (N). It provides continuous attenuation over several orders of magnitude, is ofexcellent optical quality, and is not damaged by the laser beam. Since the laser beam is monitored after passing through the attenuator, calibration of the attenuator is not required. LASER DETECTORS. Simultaneous use and monitoring of the laser beam is achieved by using a thin uncoated glass beam splitter (0) to deflect a part of the beam into a chamber containing a sensitive dual-channel laser detector (P). Light entering the detector chamber is diffused, attenuated by neutral density filters, transmitted by a narrow hand interference filter centered at 6940 A, and is incident on two solid state ultrafast photodiodes. The current from one diode is integrated to indicate the total energy in the pulse while the current from the other is used to display the power in the pulse as a function of time. The diodes used are low dark current HPA 42011PIN photodiodes (Hewlett-Packard Assoc., Palo Alto, Calif.) with a constant quantum efficiency over 6 decades of light intensity and a time constant of less than 1 nsec. To realize this fast response, it was necessary to restrict the capacitance in parallel with the diode or load to a very small value and to use a coaxial cable that was terminated with a resistance equal to its characteristic impedance. Figure 2 shows the circuit used for the dual-channel laser detector. Power is provided by two 90-V batteries and a small 1.5-V filament transformer. Photo and reverse leakage currents flow in the diodes when external bias is applied in the reverse direction, The leakage current remains constant for fixed bias and temperature while the photo current varies linearly with the intensity of illumination. Diodes D, and DI receive laser light and are used to indicate total energy
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and instantaneous power, respectively. A third diode, Ds is shielded from the laser light but receives light from an incandescent bulb X, that is part of the balance control. Diodes D1 and D, are connected in a bridge circuit. Balancing of the bridge is achieved when the reverse leakage currents at the junctions of D,.and D,areequal. Thisisaccomplished by adjusting the bias on diode Dz or, if the diodesare not well matched, by illuminating diode D2with light from incandes cent bulb Xt.
Figure 3. Laser induced incandescent rapor from 10 g diameter sample in frozen-dried microtome sectinn (5 g thick) of human kidney
wtien diode D, is illuminated by laser light, the current generated is integrated with respect to time, and the total charge is indicated by a Keithley (Model 150B) electrometer. Since the diode current is proportional to laser power, the charge indicated by the electrometer is proportional to laser energy. Thus with the use of a laser calorimeter the dualchanriel laser detector can be calibrated to indicate both laser energ,y and instantaneous power. AND OPTICAL COUPLING. The SPE:CTRAL DETECTORS spectt.a1 detectors are Amperex XP1118 quartz-window multilplier phototubes mounted behind slits (typically 200 p ) locate:d in the film plane of a modified f/6.3 Jarrell-Ash 3/4 m Czerny-Turner spectrograph $grating 1180 linesjmm; recipracal linear dispersion, 11.0 Ajmm). OPltical coupling to the entrance slit (typically 200 j ~ )of the spectrograph is through a 3-in. diameter fj1.5 quartz spher ical lens so that the first mirror in the spectrograph is illuminated to the greatest possible extent. Photographs of the plume, such as shown in Figure 3, are used to determine the approximate magnification from plume to slit that is required, and the final fine adjustments are determined experimmtnllv. Tvnirallv. for crater diameters under 20 u the P
iigurc 4. Electron micrograph of 3 g hole in semiranspared chromium film over 2000 A thick section of at liver
li (.
tl Short-lerm staoi~ity. rne wiarn 01 m e y-swncnea laser pulse is about 15 nsec. The amount of energy in the pulse is determined by the transmittance of the bleachable dye and the size of the transverse mode selector. Typically, for a transmittance of 6 3 Z and a mode selector aperture of 3 mm, the pulse energy is about 0.2 J. The corresponding peak power is thus 13 MW. For these conditions, the full beam divergence angle is nominally 2.4 mradians so that a 1OX objective could focus the radiation to a power density of 9 x 10” W/cm2. However, in normal use the CuSO, attenuator transmits a small fraction of the total laser energy. The laser emits multiple giant pulses when the ruby is overpumped and none when under-pumped. Thus there is a “pumping window” for which only a single pulse is emitted. The corresponding “voltage window” for the voltage applied to the flash lamp power supply is about 400 V, so that the laser emits a single pulse when operated within this range. The emitted energy changes slowly as a function of voltage. The largest variations in laser output occur when a freshly mixed bleachable dye is used. During the first day of use the relative standard deviation of the laser energy may be as high as 2OZ. Similarly, at the end of the useful lifetime of the dye, the relative standard deviation is large. However, when the age of the dye is between 1 or 2 days and 2 or 3 months, the relative standard deviation is usually less than 5%. The relationship of dye age to laser stability is not completely understood. The dye dissolves very slowly and large variations in laser output occur for some time after the solution is prepared. On the other hand, at the end of the useful lifetime of the dye, the transmittance has increased and the “voltage window” for single pulsing has decreased. Long-Term Stability. During the past year the laser was pulsed approximately 20,OM) times. The laser optics were not damaged or replaced and realignment of the laser cavity was necessary only during an experiment in which very small (11) S. D. Rasberry, B. F. Scribner, andM. Margoshes, Appl. Opt.,
6,87 (1967).
mode selectors were used in the cavity. There was a sm decrease in the energy output, probably due to a degradati of the flash lamp although there is no obvious crazing or discoloration of its envelope. The transmittance of the bleachable dye had to be increased gradually in order to maintain a reasonable voltage window. The 2OX microscope objective used with small energies to produce small craters was not damaged. The 1OX objective used with large energies to produce large craters was damaged by action of the beam on the lens cement. The bleachable dye was originally sealed by fusing shut the cuvette with argon over the solution; however, the ground stopper of the cuvette provided a satisfactory seal when lubricated with vacuum grease. The useful lifetime of the dye was relatively independent of the number of times the laser was pulsed. Beam Characteristics. There is some confusion in the general laser field concerning the term, spot siw. It is commonly used to describe the diameter of the laser beam at the target and also the diameter of the crater formed. Clearly these are different concepts. Depending upon the thermal and optical properties of the target, the duration of the pulse, and the absolute power and energy density of the radiation at the target, the crater that i s formed may he either larger or smaller than the laser beam. Here, the term, spot size, refers to the diameter of the laser beam at the target. If good-quality optics are used, spot size is determined by the focal length of the microscope objective and the beam divergence of the laser. To some extent beam divergence can be controlled by changing the aperture of the transverse mode selector. Experiments with the mode selector indicated that a heam-divergence minimum occurred for an aperture of about 1 mm. Aperture sizes either smaller or larger than this led to larger crater sizes. Further experiments with a 1 X 0.5 mm rectangular aperture resulted in an asymmetric crater whose largest dimension corresponded in orientation to the smallest modeselector dimension. This suggested VOL. 40. NO. 8, 1ULY 1968
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aperture. Theoretically, a high-quality 8 mm focal length objective (20X)is capable of focusing a 1 mm diffraction-limited beam to a spot size less than 14 p. The amplitude distribution at the diffracting aperture determines the exact shape of the diffraction image. For example, a uniform amplitude would give rise to an Airy disk distribution with width at half maximum of about 7 p while a Gaussian distribution in amplitude would give rise to a Gaussian image with width at half maximum of about 8 p. In either case the power density in the image would decrease monotonically from its axial peak to less than 2% its peak value at about 14 p. Although the exact shape of the diffraction image was not determined, experiments with a high-quality 8 mm focal length objective (20X)showed that symmetrical craters below 14 p could be
Table I. Precision of Measurements for Magnesium in Human Blood Serum Relative standard deviations Spectral signal/ Number of Laser Spectral measureLaser Sample energy signal ments energy number 4.9 10 3.6 5.9 1 2.4 10 5.2 3.0 2 3.3 11 2.5 4.0 3 3.0 11 4.6 3.9 4 1.9 3.5 5 2.2 4.1 6 4.9 3.5 7 3.0 4.8 8 4.1 4.8 9 3.4 3.1 10 9 3.2 2.3 2.8 11 1.7 11 4.7 5.5 12 3.6 2.3 9 4.9 13 3.3 2.1 10 2.6 14 1.9 4.8 10 4.4 15 1.9 4.3 11 5.0 16 4.4 3.1 11 5.1 17 3.0 4.3 3.9 Mean
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tion, craters less than 1 p diameter could be formed. Figure 4 is an electron micrograph of a 200 A section of rat liver over which a 100 A film of chromium had been deposited. The 20X objective was used to form the 3 p hole in the chromium film. Note that the tissue within the hole is intact. With less attenuation a larger hole in the chromium was made and a small hole was made in the tissue. Because of the beam characteristics demonstrated above and the ability to finely control the energy in the pulse it has been possible to sample single cells, such as the white blood cells in Figure 5, with production of spectral lines of sufficient intensity to be useful for analysis. Laser Monitor. Simultaneous use and monitoring of the laser beam permits the recording of both spectral signal from the sample, and laser energy each time the laser is pulsed. Although the functional relationship between spectral signal and laser energy is unknown, some unique nonlinear relationship exists for a given laser output and sample. Even without a precise knowledge of this relationship, modest improvements in precision of experimental data are achieved by treating the data as though this relationship were linear. Table I indicates the precision of measurements for analysis of magnesium in 17 air-dried 0.1-p1 samples of human blood serum which were totally vaporized. Note that the relative standard d e viation of the ratio of spectral signal to laser energy was usually smaller than that of the spectral signal alone and that the relative standard deviation of the laser energy was less than 6 % for every trial. ACKNOWLEDGMENT
The authors acknowledge the help of B. C. Belt in the construction of the laser unit and of L. Silverman in providing the electron micrograph.
RECEIVED April 5,1968. Accepted May 15,1968. Supported by Grants (to D.G.), GM09227, HE06716 and 5K6AM 18,513, from the National Institutes of Health, U.S.Public Health Service. Work at the Stanford Research Institute supported by Subcontract RA0037 under Grant GM09227.
