Anal. Chem. 1993, 85, 2938-2940
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CORRESPONDENCE
Photothermal Microscopy with Excitation and Probe Beams Coaxial under the Microscope and Its Application to Microparticle Analysis Masaaki Harada, Kouji Iwamoto, Takehiko Kitamori, and Tsuguo Sawada' Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
INTRODUCTION
beam offset? and alignment of the pump and probe beams and the sample becomes more critical when the sample particles are smaller. Moreover, microparticles having a diameter less than 100 bm are hard to handle and it is impracticable to selectivelymeasure a particular microparticle in the matrix. Now, we have conceived a plan to incorporate a microscope in the photothermal beam deflection measurement system. However, the distance between the objective lens and the sample is extremely short and it is impossible to pass the probe beam just above the sample perpendicular to the vertical excitation beam, as in the transverse photothermal deflection experiments. The situation becomes worse as the objective lens with higher magnifying power is used. We irradiated the sample by the probe beam, which was pumped under the microscope objective coaxiallythrough the microscope's body tube. With such geometry for the excitation and probe beams, troublesome optical alignment among the excitation and probe beams and the sample could be reduced greatly. When a sample is transparent to the probe beam, it is expected that the spatial intensity distribution of the probe beam transmitted from the sample is photothermally altered. In this paper the modified measurement system was applied to quantitation of substances distributed inside the microparticles.
Many polymer and semiconductor materials and biological samples have microscopically heterogeneous structures, which frequently contribute to their physical and chemical functions. Examination of these samples necessitates microanalysis techniques with high spatial resolution. With respect to elemental composition, methods using electrons or ions as a probe are powerful tools and they include electron probe microanalysis, Auger electron spectroscopy, and secondary ion mass spectroscopy. These methods have a high spatial resolution down to several nanometers, but they require measurements be made in a high vacuum and sometimes the samples are damaged. On the other hand, optical microspectroscopic methods, which are free of the above shortcomings, have attracted much attention in recent works. These methods, though their spatial resolution reaches no more than the submicron level, give some information about molecular structures, which becomes of great significance in characterizing the samples. Among them, fluorometry is highly sensitive and widely used, but it is only applicable to fluorescent or easily derivatized compounds. So highly sensitive absorption methods are desirable, but conventional absorption microspectrophotometric methods offer insufficient sensitivity. Confocal laser scanning microscopy has recently been applied to fluorescent and Raman spectroscopy. This method is quite ingenious and promising; however, spectroscopic applications are still limited. Photothermal spectroscopy has proved to be a highly sensitive technique,l4 and it has been applied to microscopic measurements. Photoacoustic microscopy (PAM) measurements have been popular, but recently photothermal beam deflection microscopy is preferred to PAM as no sample cell is needed and there is no contact with the detector. We have theoretically and experimentally shown that the photothermal beam deflection method has higher sensitivity for a single microparticle than the photoacoustic method.6 With the photothermal deflection method, 28 fg of Fe-oxine chelate complex adsorbed on a 50-pm-diameter microparticle was detectable and the adsorption spectrum of a single human white blood corpuscle (5-15 pm size) was measurable.' The signal amplitude, however, strongly depends on the probe
EXPERIMENTAL SECTION The experimental setup is schematically shown in Figure 1. The excitation beam was the 488.0-nm emission line of an Ar ion laser, and ita intensity was modulated mechanically or acoustooptically. The modulated light was passed through the body tube of the microscope and then focused on the sample, which was placed on a piece of slide glass in the air. The 633-nm beam of a 1-mW H e N e laser, the probe beam, was reflected off a dichroic mirror and directed coaxially, with the heating beam, onto the sample. Transmitted heating and probe beams were collected with another objective lens and reflected out by amirror. After the excitation beam was cut off by a filter, only the probe beam intensity was monitored by a single small-area photodiode. The signal from the photodiode was preamplified by a gain of 100 and synchronously detected and amplified by a lock-in amplifier. No pinhole was used in this experiments, which would usually be required in thermal lensing experiments. The probe beam, tightly focused by the first microscope objective lens, diverged to a larger spot than the second objective. In addition, a photodiode with a small area (2 mm X 2 mm) was used in this experiment. So it is quite possible that the second microscope objective and/or the photodiode substituted for a pinhole and the thermal lens measurement could be done. Two kinds of samples that differed in the chromophore distribution inside the microparticles were prepared. The first
* To whom all correspondence should be addressed.
(1) Morris, M. D.; Peck, K. Anal. Chem. 1986,58, 811A-822A. (2) Murphy, J. C.; Aamodt, L.C . J. Appl. Phys. 1980,51,4580-4588. (3) Jackson, W. B.; Amer, N. M.; Boccara, A. C.; Fournier, D. Appl. Opt. 1981,20, 1333-1344. (4) Mandelis, A. J. Appl. Phys. 1983, 54, 3404-3409. (5) Chen, T. I.; Morris, M. D. Anal. Chem. 1984, 56, 19-21. (6) Wu, J.; Kitamori, T.; Sawada, T. Appl. Phys. Lett. 1990,57,22-24. (7) Wu, J.; Kitamori, T.; Sawada, T. Anal. Chem. 1991,63, 217-219. 0003-2700/93/0365-2938804.0010
(8)Harada, M.; Kitamori, T.; Sawada, T. J. Appl. Phys. 1993, 73, 2264-2271.
D 1993 Amerlcan Chemical Society
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Excitation beam power (mW) Flgurs 2. Signal dependence on the excitation beam power. was polystyrene-divinylbenzene copolymer (XAD-2) resin microparticles,on which an Fe-oxine chelate complex was adsorbed, so only a surface layer, from surface to several microns deep into particle, was an optically absorbing layer.9 Detailed descriptions of this sample preparation have been reported elsewhere.lO The second was polystyrene microparticles, which were polymerized from styrene monomer containing red dye. Since the original monomer held the dye, the excitation light was assumed to be absorbed homogeneously in the particles.
RESULTS AND DISCUSSION Signal Dependence on the Excitation Beam Power. To c o n f i i that the detected signal is photothermallyinduced, the dependence of the signal amplitude on the excitation beam power was measured. The Fe-oxine adsorbed XAD particle with a diameter of about 250 pm was used. The output power of the Ar ion laser was set at 100 mW, and the excitation beam power was reduced by a neutral-density filter. The results are shown in Figure 2. The signal intensity increased linearly with increased excitation beam power in the range of less than 100 mW. Moreover, no signal was observed when a sampleparticle was not under the microscope or when a sample particle contained no absorbing species, as shown in Figure 4. So it could be concludedthat the detected signal results from the photothermal conversion of the light energy absorbed by the sample particle itself. Figure 3 shows several expected photothermal phenomena, which include a thermal lens in the sample, a thermal wave in the air over the sample, and thermal expansion of the sample. At modulation frequencies higher than 1kHz or on a time scale shorter than thermal conduction, acoustic radiation resulting from periodical particle expansion must be taken into account.ll At the modulation frequency of 319 Hz,however, the photoacoustic contribution is considered to ~~
(9)Heueh,Y.-M.;Harata,A.;Kitamori,T.;Sawada,T. Anal. Sci. 1990, 6,71-76. (10)Yoshinaga, A.; Hsueh, Y.-M.; Sawada, T.; Gohshi, Y. Anal. Sci. 1989,5,147-149. (11)Diebold, G. J.; Khan, M.I.; Park, S.M. Science 1990,250,101104.
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be negligible. The thermal diffusion length in the air and in the particle were respectively about 137 and 9 pm in this measurement. So only the part of the particle along the excitation beam path was thermalized and contributed to the signal. The detected signal results from several photothermal effects, but judgingfrom the temperature distribution generated outside and inside the particle, the thermal lens effect of the sample seems to be dominant. The gradient of the temperature field outside the particle was generated parallel to the probe beam, while that inside the particle was generated normal to the probe beam. Which mechanism makes the main contribution to signal generation would probably depend on experimental conditions and sample kinds, for example, distance between the focus of the excitation and probe beams and the sample particle, which will be also mentioned later, analyte distribution in the sample particle, and the particle size. To understand signal generation in detail, further experimental examination such as measurement of modulation frequency dependence and theoretical analysis must be made. In any case, it has been shown that the signal is photothermally generated. QuantitativeMeasurements. The proposed microscopic method was applied to quantitation of the substances distributed in the microparticle. First, the homogeneously absorbingparticles (diameter, 40 pm) were quantitated. Three particles were measured for samples which differed in their pigment content (0,0.1,0.3, 0.5 wt %). The result is shown in Figure 4. The output power of the Ar ion laser was 10 mW. A linear relationship was obtained between the pigment content and the signal intensity. Taking the particle mass density as 1.05, the amount of the pigment contained in one particle of the 0.1 wt % samples was estimated as 33 pg. The signal-to-noise ratio was fairly good (about 30 for the 0.1 wt
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power of 10 was used. The focal point of the excitation and probe beams was changed vertically near the particle surface. The focus height dependence of the signal intensity is shown in Figure 6. The signal intensity was considered to be approximately proportional to the probe beam intensity incident on the detector, which changed slightlywith changes of the focus height due to the change in refraction and reflection by the microparticle. So without the excitation beam the intensity of the probe beam was measured at each focus height position, and using the results, signal intensity was compensated by the probe beam intensity. Though the present reproducibility of the result was not so good, a signal peak was observed when the focal point was in the vicinity of the particle surface. The fact that the signal maximum was observed inside the particle surface showed that the signal was mainly generated inside thasample. The full width at half-maximum of the peak was about 20 pm, which was considered to correspond to a kind of convolution of the absorbing layer thickness (6 pm) with the probe beam spatial profile. To extract a true concentration depth profiie from these data, further considerationsabout the signalgeneration, beam spatial profie, and optical path change are needed. The detailed examination will be reported in a subsequent paper. The lateral resolution was decided by the spot size while the vertical one yas decided by the depth of focus of the excitation and probe beams. The lateral resolution was nearly 10 pm in this experiment, but by tight focusing of the probe beam it is ultimately expected to reach the micron to submicron levels. The proposed microscopic technique has proved to be a highly sensitive and selective microspectroscopic method for quantitation, and it is expected to be applicable to in vivo three-dimensional distribution analysis of biological samples in various matrices, which is under investigation. ACKNOWLEDGMENT
7% particles). The detection limit calculated from double signal-to-noiseratio was 8 pg. The spot size of the excitation and probe beams (several microns) was somewhat smaller than the particle size, and the virtually measured absolute amount would reach the subpicogram level. Then the samples which had adsorbed 1.9,3.0, and 4.7 pg of Fe-oxine per one 50-pm-diameter XAD particle (density of 1.08) were quantitated. The output power of the Ar ion laser was 130 mW. Similar to the measurements of the pigment-containing particles, three particles were measured for each sample. The results are shown in Figure 5. The horizontal axis presents the adsorbed amount of the Fe-oxine complex per the excited and probed surface area of the XAD particle. The signal intensity linearly depended on the adsorption amount in the range of several tens of femtograms. The regression curve was represented by the formula y = 0.175~- 0.717, and the signal-to-noise ratio for the 19-fg samples was 18. So the detection limit was calculated as 6 fg. This value was comparable to that of the usual transverse photothermal beam deflection method.' The detection limit of optical absorbance was le,which was about 1 order of magnitude superior to that of the conventional UV-visible absorption microspectrophotometricmethod and equivalent to that of the photoacoustic method for similar experimental and sample conditions.12 Regardless of the internal distribution, ultratrace substances in the microparticle could be determined by the proposed microscopic method. The microparticles,the diameters of which were less than 100pm, were detected with much difficulty by the conventional photothermal beam deflection method, and it is impossible to selectively measure one from among a group of particles. The proposed microscopic method, however, can measure a particular particle easily. Spatial Distribution Analysis. For plane samples, lateral distribution of the analyte is easily obtained by scanning the sample. This is not the case with microparticulatesamples, but a depth distribution of the d y t e should be obtainable by moving the focus up and down in the microparticle. The test sample was the 260-pm-diameter XAD particle, which had adsorbed Fe-oxine chelate complex in a surfacelayer of about 6-pm thickness. The sampleparticle was fixed on the slide glass, and an objectivewith a magnifying
We thank Prof. S. Matsuzawa and Dr. H. Kimura of the Department of Forensic Medicine, School of Medicine, Juntendo University, Japan, and Mr. H. Nishi of the Department of Research & Development, Ganz Chemical Co., LM.,Japan, for preparation of the polystyrene microparticle containing a homogeneous distribution of red dye.
T.;Sawada, T.A m l . Sci.
RECEIVEDfor review May 13, 1993. Accepted July 28,
(12) Hsueh, Y.-M.; Hnrata, A.; Kitamori,
1990,6,67-70.
1993.
