Histochemistry 63, 323 328 (1979)
Histochemistry 9 by Springer-Verlag 1979
Staining and Relative Determination of Elemental Contents by the Laser Microprobe H. G o t o h 1 ,, M. Murota 2, and A. Kamiyama 3 Department of Physiology,Yokohama City University School of Medicine, Yokohama 232, Japan, 2 Research Fellow of Japan Electron Optics Laboratory, Akishima City, Tokyo, Japan, and 3 Department of Physiology, Teikyo University School of Medicine, Itabashiku, Tokyo, Japan
Summary. An improved laser microprobe procedure is developed and applied to the measurement of calcium content in microareas of right and left subepicardial muscles. The staining of canine cardiac muscles by Methylene Blue solution (1% w/v) was found to improve sampling efficiency. Elemental content is proportional to T - 1/,~, where T is the transmittance of the characteristic emission line of the element of the photographic plate and 7 is its contrast. In the present system, the calcium content is analyzed using T -32. We find that the staining of samples and the determination of T 1/~ are useful procedures in the application of laser microprobe to the study of elemental content in biologic microareas.
Introduction The laser microprobe is a modern apparatus which is useful in determining the elemental contents of many kinds of materials (Rosan et al., 1963; Sherman etal., 1965; Glick, 1966). With this instrument, a microsite of usually 10~300 gm in diameter or a microgram in dry weight is vaporized by a laser shot. The laser beam can deal with single cells or even subcellular bodies. Thus, the area of destruction is so minute that the preparation may be retained for other types of investigation. Also from the viewpoint of preparation of samples, the laser microprobe has some advantages compared with other instruments; i.e., living or wet samples can be analyzed without fixation, dehydration or ashing. However, the laser microprobe has two problems. The first is its low efficiency in vaporizing biological tissues. Recently, the laser microprobe has been found to be very useful when combined with a mass analyzer (Hillenkamp * Please send offprint request to . Dr, H. Gotoh. Departments of Physiology,College of Physicians and Surgeons of Columbia University, 630 West 168th Street, NewYork, N.Y., 10032, USA
0301-5564/79/0063/0323/$01.40
324
H. Gotoh et al.
eta!., 1974; Wechsung et al., 1978). This approach combines the advantages of high lateral resolution (< 1 gm) and high detection probability (detection limit, 10 - i s to 10 . 2 0 nag). However, it requires tedious handling of the mass analyzer, and this limits its applicability. Moreover, it cannot analyze wet biologic tissues because of its vacuum chamber for samples. Marich et al. (1970) stained samples with Methyl Blue to intensify the laser energy absorption by the samples, but this resulted in little improvement. We report here the successful use of Methylene Blue for staining of biologic tissues. The second problem with the laser microprobe is the difficulty of obtaining suitable reference standards. Nonetheless, the relationship between the emission intensity and the transmittance of the recorded characteristic emission line of an element permits an estimation of the relative content of the element. One of us has reported differences in electrophysiological response due to differences in the calcium environment or as the result of blockage of the calcium current in the canine right and left subepicardial muscles (Kamiyama and Saeki, 1974). In the present study, laser microprobe is used to compare the calcium content in local regions of canine subepicardial muscles so as to clarify physiocochemical characteristics of these muscles.
Materials and Methods Mongrel dogs were anethetized by an intravenous injection of pentobarbital sodium, 30 mg/Kg body weight, and the hearts were immediately removed from thoracic cavities. The free walls of the ventricles were frozen at - 2 0 ~ and excised perpendicularly to the wails using a freezing microtome (Yamoto, EFM-A). Sample slices of 100 gm thickness were obtained. The slices were placed on a plate of vicor glass and stained by immersing them in Methylene Blue solution (1% w/v) for 40 s. A microarea at 0.2 mm depth from the ventricular surface was shot with the laser beam. The laser microprobe of Japan Electron Optics Laboratory, JLL-200 was used. The wavelength of the laser beam was 1.06 gm from neodymium-glass. The fluctuation of the laser output energy was less than 5%. The sample vapor was cross-excited by a spark. The light emission was recorded on a Kodak SA-1 glass photographic plate through a grating monochrometer. The transmission of the calcium line at 393.3666 nm on the plate was measured by using a microphotometer. The right and left preparations were shot alternately. The diameter of a crater which was formed by a laser shot was also measured in order to check the difference of the matrix effects and staining between the right and left preparations, and to confirm the stability of each sampling.
Table 1. Effect of staining of sliced tissues on the laser sampling. Methylene Blue solution (1 g/100 ml) was used for staining. The optical densities were measured at 393.3666 nm (calcium emission line). The data were calculated from the experiment of 5 laser shots of 1 sample slice. Mean _+ SE Without staining
With staining
Output Energy
1 joule
2 joules
1 joule
Optical Density
0.0_+0.0
0.15_+0.06
0.82+0.80
Laser Microprobe
325
Fig. 1. Photograph of non-stained sample shot with an energy of 1 joule. The arrow indicates the site of the shot. Scale: 0.5 m m Fig. 2. Photograph of stained sample shot with an energy of 1 joule. The arrow indicates the site of the shot. Scale: 0.5 m m
326
H. Gotoh et al.
Results
Effect of Staining on the Laser Sampling When the sliced samples which were not stained were shot with the laser beam, the light emission was very weak. However, the staining of the samples sufficiently intensified the emission line of calcium. The results are shown in Table 1. When the output energy of the laser shot was 1 joule and the slice was not stained, we could not detect the emission line of calcium. Only when the energy was raised to 2 joules, could we detect a trace quantity of calcium. The laser shot, however, did not make a crater in this case (Fig. 1). When the stained slices were shot, the emission line of calcium was very intense. Actually the shot area was seen to be effectively vaporized and a distinct crater was produced (Fig. 2). Little calcium was detected from the dye itself in the present experimental condition.
Intensity-Transmittance Relationship In order to compare calcium content in tissues, it is necessary to obtain t-he relationship between the transmittance (7) of the recorded line on the plate and the elemental content (C) in the tissue. The intensity (/) of the characteristic light emission is related to T by the following equation (see Appendix)
I = K T -1/~
(1)
where K is the proportionality constant, and 7 is the contrast. Because I is porportional to C, Eq. (1) can be rewritten
C= CoT- 1/~
(2)
where Co is the proportionality constant. Thus, the elemental content is compared on the basis of T - 1/~ and 7 can be estimated from the I - T relationship. In order to determine this relationship, we shot a standard sample of the alluminium alloy with the laser beam varying the intensity of exposure by covering a part of the focusing lens. The results are shown in Fig. 3. The contrast 7 was calculated to be 0.31 from the slopes of regression lines. Therefore, we employ T-3.2 as the index for the calcium content.
Experiments We compared the mean values of T - 3.2 in the right and left ventricular preparations and the results are shown in Fig. 4. The right subepicardial muscle contains significantly more calcium than the left. The crater diameters were almost the same in the right and left ventricular preparations. This indicates that almost the same quantity was sampled in these two preparations. These results were reproducible. Comparison of the mean values suggests that the right subepicadial muscle contains approximately 1.9 times as much calcium as the left.
Laser Microprobe
327
200 1.01
-
T3.2
T O.6 0.4
100"l,.
oe
0.2
0.1 0.1
' 0.2
'
' ' ' ' ''J 0.4 0.6 1.0
0-
L R
I Fig. 3
Fig. 4
Fig. 3. Intensity-transmittance relationship. A plate of aluminium alloy was shot. The solid line is the regression line which is calculated on the basis of the emission of manganese with a wavelength of 405.6 nm; the interrupted line, for a wavelength of 405.8 nm. Io is the light intensity in the case without a covering sheet on the focusing lens. The value of y is calculated to be 0.31 by the use of least mean squares. However, it should be mentioned that in the range of very low transmittance the value of y deviates from 0.31 Fig. 4. Comparison of mean values of T 3.2 of the right and left subepicardial muscles. The results were calculated from the experiment of 20 shots with the laser beam using 7 sample slices. ~L=0.20+0.08 ram. 45R=0.19+0.05 ram. Here, ~L and 4~R are the crater diameters in the left and right preparations respectively and their values are presented as mean _+ SE. Each bar in the graph represents a 90% confidence interval Discussion
S a m p l i n g efficiency was i m p r o v e d by M e t h y l e n e Blue staining o f the b i o l o g i c tissues. H o w e v e r , the a b s o r p t i o n p e a k s (667.8, 609.3 nm) o f M e t h y l e n e Blue d o n o t coincide with the emission p e a k (1,060 nm) o f the n e o d y m i u m - g l a s s laser used in the present study. Thus, one w o u l d expect the efficiency o f the a b s o r p t i o n o f the laser b e a m b y the b i o l o g i c m a t e r i a l s to be a u g m e n t e d by the staining. Nevertheless, staining as t h i c k as in the p r e s e n t case d i d l e a d to greater a b s o r p t i o n o f laser light energy with s u b s e q u e n t v a p o r i z a t i o n . Besides M e t h y l e n e Blue, we tested s o m e o t h e r dyes such as Berlin Blue, B r o m o t h y m o l Blue a n d E r i o c h r o m e Black T. W e f o u n d t h a t M e t h y l e n e Blue was the only one t h a t i m p r o v e d the efficiency o f the laser sampling. In the case o f electron m i c r o s c o p i c a l analysis such as X - r a y m i c r o a n a l y s i s , the s o a k i n g o f the s a m p l e s for fixation, d e h y d r a t i o n or staining s o m e t i m e s c h a n g e s t h e i r e l e m e n t a l c o n t e n t s so severely as to m a k e the e l e m e n t a l analysis impossible. D o e s o u r p r e s e n t p r o c e d u r e have a similar p r o b l e m ? In o r d e r to e x a m i n e the reliability o f o u r a n a l y t i c a l p r o c e d u r e , we e x t e n d e d the e s t i m a t i o n o f c a l c i u m c o n t e n t to the following two cases: (1) C a l c i u m c o n t e n t s in the sliced s a m p l e s o f 100 g m thickness a n d t h a t o f a 200 g m t h i c k sample. T h e latter was f o u n d to have a c a l c i u m c o n t e n t a p p r o x i m a t e l y 2.7 times as m u c h as the former. The d e v i a t i o n o f 2.7 f r o m 2 (the r a t i o o f the s a m p l e thicknesses)
328
H. Gotoh et al.
is probably due to the non-linear dependence of the laser sampling efficiency on the depth; the laser beam samples the tissue in a cone shaped crater, not in a cylindrical form. (2) Calcium content in deep areas of the right and left ventricular muscles. The results indicated that the right and the left contain almost the same quantity of calcium. Ordinary flame photometry is applicable to such a non-local analysis and we reached the same conclusion with this technique. The unequal distribution of calcium in the right and left subepicardial muscles is possibly due to the difference in their extracellular spaces. However, further study is necessary to explain the present result. Acknowledgements. We are grateful to Dr. Tohru Yoshioka for his technical comments and sugges-
tions. Methylene Blue was kindly provided by Mrs. T. Ogawa of Daiwa Kakousho. We wish to thank Mr. David Harris and Mr. William Patterson for correcting our English.
Appendix For light of any given spectral composition, the density D of the image developed on an exposed plate, varies with the light intensity I and with the time of exposure t, approximately according to the relationship D = Y(log I t ' - log i)
(A 1)
where 7 is a constant, the "gamma" or contrast of the plate, p is Schwarzchild's constant of the plate, and i is another constant of the plate, the inertia (Walsh, 1953). By definition, transmittance T of the characteristic emission line on the plate is related to D as 1 D = log ~ .
(A 2)
Combining Eqs. (A1) and (A2), and taking it into consideration that tp is a constant in our system, we can obtain Eq. (1) I = K T -z/7
(1)
References Hillenkamp, F., Kaufmann, R., Nitsche, R., Remy, E., Uns61d, E. : Recent results in the development of a laser microprobe. In: Microprobe analysis as applied to cells and tissues. Hall, T., Echlin, P. (eds.), pp. 1-14, London: Academic Press 1974 Glick, D. : The laser microprobe. Its use for elemental analysis in histochemistry. J. Histochem. Cytochem. 14, 862 868 (1966) Kamiyama, A., Saeki, Y.: Myocardial action potentials of right- and left-subepicardial muscles in the canine ventricle an effects of manganese ions. Proc. Jpn. Acad. 50, 771-774 (1974) Marich, K.W., Carr, P.W., Treytle, W.J., Glick, D.: Effect of matrix material on laser-induced elemental spectral emission. Anal. Chem. 42, 1775-1779 (1970) Rosan, R.C., Healy, M.K., McNary, W.F.Jr. : Spectroscopic ultra microanalysis with a laser. Science 142, 236 237 (1963) Sherman, D.B., Ruben, M.P., Goldman, H.M.: The application of laser for the spectrochemical analysis of calcified tissues. Ann. N.Y. Acad. Sci. 122, 767-772 (1965) Walsh, J.W.T. : in: Photochemistry, 2nd ed., p. 364. London: Constable & Co. Lts. 1953 Wechsung, V.R., Hillenkamp, F., Kaufmann, R., Nitsche, R., Vogt, H.: Laser-Mikrosonden-Massen-Analysator. " L A M M A " : Ein neues Analysenverfahren ffir Forschung und Technologie. Mikroskopie 34, 47 54 (1978) Received September 22, 1.978 Accepted July 13, 1979
Histochemistry 9 by Springer-Verlag 1979
Staining and Relative Determination of Elemental Contents by the Laser Microprobe H. G o t o h 1 ,, M. Murota 2, and A. Kamiyama 3 Department of Physiology,Yokohama City University School of Medicine, Yokohama 232, Japan, 2 Research Fellow of Japan Electron Optics Laboratory, Akishima City, Tokyo, Japan, and 3 Department of Physiology, Teikyo University School of Medicine, Itabashiku, Tokyo, Japan
Summary. An improved laser microprobe procedure is developed and applied to the measurement of calcium content in microareas of right and left subepicardial muscles. The staining of canine cardiac muscles by Methylene Blue solution (1% w/v) was found to improve sampling efficiency. Elemental content is proportional to T - 1/,~, where T is the transmittance of the characteristic emission line of the element of the photographic plate and 7 is its contrast. In the present system, the calcium content is analyzed using T -32. We find that the staining of samples and the determination of T 1/~ are useful procedures in the application of laser microprobe to the study of elemental content in biologic microareas.
Introduction The laser microprobe is a modern apparatus which is useful in determining the elemental contents of many kinds of materials (Rosan et al., 1963; Sherman etal., 1965; Glick, 1966). With this instrument, a microsite of usually 10~300 gm in diameter or a microgram in dry weight is vaporized by a laser shot. The laser beam can deal with single cells or even subcellular bodies. Thus, the area of destruction is so minute that the preparation may be retained for other types of investigation. Also from the viewpoint of preparation of samples, the laser microprobe has some advantages compared with other instruments; i.e., living or wet samples can be analyzed without fixation, dehydration or ashing. However, the laser microprobe has two problems. The first is its low efficiency in vaporizing biological tissues. Recently, the laser microprobe has been found to be very useful when combined with a mass analyzer (Hillenkamp * Please send offprint request to . Dr, H. Gotoh. Departments of Physiology,College of Physicians and Surgeons of Columbia University, 630 West 168th Street, NewYork, N.Y., 10032, USA
0301-5564/79/0063/0323/$01.40
324
H. Gotoh et al.
eta!., 1974; Wechsung et al., 1978). This approach combines the advantages of high lateral resolution (< 1 gm) and high detection probability (detection limit, 10 - i s to 10 . 2 0 nag). However, it requires tedious handling of the mass analyzer, and this limits its applicability. Moreover, it cannot analyze wet biologic tissues because of its vacuum chamber for samples. Marich et al. (1970) stained samples with Methyl Blue to intensify the laser energy absorption by the samples, but this resulted in little improvement. We report here the successful use of Methylene Blue for staining of biologic tissues. The second problem with the laser microprobe is the difficulty of obtaining suitable reference standards. Nonetheless, the relationship between the emission intensity and the transmittance of the recorded characteristic emission line of an element permits an estimation of the relative content of the element. One of us has reported differences in electrophysiological response due to differences in the calcium environment or as the result of blockage of the calcium current in the canine right and left subepicardial muscles (Kamiyama and Saeki, 1974). In the present study, laser microprobe is used to compare the calcium content in local regions of canine subepicardial muscles so as to clarify physiocochemical characteristics of these muscles.
Materials and Methods Mongrel dogs were anethetized by an intravenous injection of pentobarbital sodium, 30 mg/Kg body weight, and the hearts were immediately removed from thoracic cavities. The free walls of the ventricles were frozen at - 2 0 ~ and excised perpendicularly to the wails using a freezing microtome (Yamoto, EFM-A). Sample slices of 100 gm thickness were obtained. The slices were placed on a plate of vicor glass and stained by immersing them in Methylene Blue solution (1% w/v) for 40 s. A microarea at 0.2 mm depth from the ventricular surface was shot with the laser beam. The laser microprobe of Japan Electron Optics Laboratory, JLL-200 was used. The wavelength of the laser beam was 1.06 gm from neodymium-glass. The fluctuation of the laser output energy was less than 5%. The sample vapor was cross-excited by a spark. The light emission was recorded on a Kodak SA-1 glass photographic plate through a grating monochrometer. The transmission of the calcium line at 393.3666 nm on the plate was measured by using a microphotometer. The right and left preparations were shot alternately. The diameter of a crater which was formed by a laser shot was also measured in order to check the difference of the matrix effects and staining between the right and left preparations, and to confirm the stability of each sampling.
Table 1. Effect of staining of sliced tissues on the laser sampling. Methylene Blue solution (1 g/100 ml) was used for staining. The optical densities were measured at 393.3666 nm (calcium emission line). The data were calculated from the experiment of 5 laser shots of 1 sample slice. Mean _+ SE Without staining
With staining
Output Energy
1 joule
2 joules
1 joule
Optical Density
0.0_+0.0
0.15_+0.06
0.82+0.80
Laser Microprobe
325
Fig. 1. Photograph of non-stained sample shot with an energy of 1 joule. The arrow indicates the site of the shot. Scale: 0.5 m m Fig. 2. Photograph of stained sample shot with an energy of 1 joule. The arrow indicates the site of the shot. Scale: 0.5 m m
326
H. Gotoh et al.
Results
Effect of Staining on the Laser Sampling When the sliced samples which were not stained were shot with the laser beam, the light emission was very weak. However, the staining of the samples sufficiently intensified the emission line of calcium. The results are shown in Table 1. When the output energy of the laser shot was 1 joule and the slice was not stained, we could not detect the emission line of calcium. Only when the energy was raised to 2 joules, could we detect a trace quantity of calcium. The laser shot, however, did not make a crater in this case (Fig. 1). When the stained slices were shot, the emission line of calcium was very intense. Actually the shot area was seen to be effectively vaporized and a distinct crater was produced (Fig. 2). Little calcium was detected from the dye itself in the present experimental condition.
Intensity-Transmittance Relationship In order to compare calcium content in tissues, it is necessary to obtain t-he relationship between the transmittance (7) of the recorded line on the plate and the elemental content (C) in the tissue. The intensity (/) of the characteristic light emission is related to T by the following equation (see Appendix)
I = K T -1/~
(1)
where K is the proportionality constant, and 7 is the contrast. Because I is porportional to C, Eq. (1) can be rewritten
C= CoT- 1/~
(2)
where Co is the proportionality constant. Thus, the elemental content is compared on the basis of T - 1/~ and 7 can be estimated from the I - T relationship. In order to determine this relationship, we shot a standard sample of the alluminium alloy with the laser beam varying the intensity of exposure by covering a part of the focusing lens. The results are shown in Fig. 3. The contrast 7 was calculated to be 0.31 from the slopes of regression lines. Therefore, we employ T-3.2 as the index for the calcium content.
Experiments We compared the mean values of T - 3.2 in the right and left ventricular preparations and the results are shown in Fig. 4. The right subepicardial muscle contains significantly more calcium than the left. The crater diameters were almost the same in the right and left ventricular preparations. This indicates that almost the same quantity was sampled in these two preparations. These results were reproducible. Comparison of the mean values suggests that the right subepicadial muscle contains approximately 1.9 times as much calcium as the left.
Laser Microprobe
327
200 1.01
-
T3.2
T O.6 0.4
100"l,.
oe
0.2
0.1 0.1
' 0.2
'
' ' ' ' ''J 0.4 0.6 1.0
0-
L R
I Fig. 3
Fig. 4
Fig. 3. Intensity-transmittance relationship. A plate of aluminium alloy was shot. The solid line is the regression line which is calculated on the basis of the emission of manganese with a wavelength of 405.6 nm; the interrupted line, for a wavelength of 405.8 nm. Io is the light intensity in the case without a covering sheet on the focusing lens. The value of y is calculated to be 0.31 by the use of least mean squares. However, it should be mentioned that in the range of very low transmittance the value of y deviates from 0.31 Fig. 4. Comparison of mean values of T 3.2 of the right and left subepicardial muscles. The results were calculated from the experiment of 20 shots with the laser beam using 7 sample slices. ~L=0.20+0.08 ram. 45R=0.19+0.05 ram. Here, ~L and 4~R are the crater diameters in the left and right preparations respectively and their values are presented as mean _+ SE. Each bar in the graph represents a 90% confidence interval Discussion
S a m p l i n g efficiency was i m p r o v e d by M e t h y l e n e Blue staining o f the b i o l o g i c tissues. H o w e v e r , the a b s o r p t i o n p e a k s (667.8, 609.3 nm) o f M e t h y l e n e Blue d o n o t coincide with the emission p e a k (1,060 nm) o f the n e o d y m i u m - g l a s s laser used in the present study. Thus, one w o u l d expect the efficiency o f the a b s o r p t i o n o f the laser b e a m b y the b i o l o g i c m a t e r i a l s to be a u g m e n t e d by the staining. Nevertheless, staining as t h i c k as in the p r e s e n t case d i d l e a d to greater a b s o r p t i o n o f laser light energy with s u b s e q u e n t v a p o r i z a t i o n . Besides M e t h y l e n e Blue, we tested s o m e o t h e r dyes such as Berlin Blue, B r o m o t h y m o l Blue a n d E r i o c h r o m e Black T. W e f o u n d t h a t M e t h y l e n e Blue was the only one t h a t i m p r o v e d the efficiency o f the laser sampling. In the case o f electron m i c r o s c o p i c a l analysis such as X - r a y m i c r o a n a l y s i s , the s o a k i n g o f the s a m p l e s for fixation, d e h y d r a t i o n or staining s o m e t i m e s c h a n g e s t h e i r e l e m e n t a l c o n t e n t s so severely as to m a k e the e l e m e n t a l analysis impossible. D o e s o u r p r e s e n t p r o c e d u r e have a similar p r o b l e m ? In o r d e r to e x a m i n e the reliability o f o u r a n a l y t i c a l p r o c e d u r e , we e x t e n d e d the e s t i m a t i o n o f c a l c i u m c o n t e n t to the following two cases: (1) C a l c i u m c o n t e n t s in the sliced s a m p l e s o f 100 g m thickness a n d t h a t o f a 200 g m t h i c k sample. T h e latter was f o u n d to have a c a l c i u m c o n t e n t a p p r o x i m a t e l y 2.7 times as m u c h as the former. The d e v i a t i o n o f 2.7 f r o m 2 (the r a t i o o f the s a m p l e thicknesses)
328
H. Gotoh et al.
is probably due to the non-linear dependence of the laser sampling efficiency on the depth; the laser beam samples the tissue in a cone shaped crater, not in a cylindrical form. (2) Calcium content in deep areas of the right and left ventricular muscles. The results indicated that the right and the left contain almost the same quantity of calcium. Ordinary flame photometry is applicable to such a non-local analysis and we reached the same conclusion with this technique. The unequal distribution of calcium in the right and left subepicardial muscles is possibly due to the difference in their extracellular spaces. However, further study is necessary to explain the present result. Acknowledgements. We are grateful to Dr. Tohru Yoshioka for his technical comments and sugges-
tions. Methylene Blue was kindly provided by Mrs. T. Ogawa of Daiwa Kakousho. We wish to thank Mr. David Harris and Mr. William Patterson for correcting our English.
Appendix For light of any given spectral composition, the density D of the image developed on an exposed plate, varies with the light intensity I and with the time of exposure t, approximately according to the relationship D = Y(log I t ' - log i)
(A 1)
where 7 is a constant, the "gamma" or contrast of the plate, p is Schwarzchild's constant of the plate, and i is another constant of the plate, the inertia (Walsh, 1953). By definition, transmittance T of the characteristic emission line on the plate is related to D as 1 D = log ~ .
(A 2)
Combining Eqs. (A1) and (A2), and taking it into consideration that tp is a constant in our system, we can obtain Eq. (1) I = K T -z/7
(1)
References Hillenkamp, F., Kaufmann, R., Nitsche, R., Remy, E., Uns61d, E. : Recent results in the development of a laser microprobe. In: Microprobe analysis as applied to cells and tissues. Hall, T., Echlin, P. (eds.), pp. 1-14, London: Academic Press 1974 Glick, D. : The laser microprobe. Its use for elemental analysis in histochemistry. J. Histochem. Cytochem. 14, 862 868 (1966) Kamiyama, A., Saeki, Y.: Myocardial action potentials of right- and left-subepicardial muscles in the canine ventricle an effects of manganese ions. Proc. Jpn. Acad. 50, 771-774 (1974) Marich, K.W., Carr, P.W., Treytle, W.J., Glick, D.: Effect of matrix material on laser-induced elemental spectral emission. Anal. Chem. 42, 1775-1779 (1970) Rosan, R.C., Healy, M.K., McNary, W.F.Jr. : Spectroscopic ultra microanalysis with a laser. Science 142, 236 237 (1963) Sherman, D.B., Ruben, M.P., Goldman, H.M.: The application of laser for the spectrochemical analysis of calcified tissues. Ann. N.Y. Acad. Sci. 122, 767-772 (1965) Walsh, J.W.T. : in: Photochemistry, 2nd ed., p. 364. London: Constable & Co. Lts. 1953 Wechsung, V.R., Hillenkamp, F., Kaufmann, R., Nitsche, R., Vogt, H.: Laser-Mikrosonden-Massen-Analysator. " L A M M A " : Ein neues Analysenverfahren ffir Forschung und Technologie. Mikroskopie 34, 47 54 (1978) Received September 22, 1.978 Accepted July 13, 1979
