Short Scanning Electron Microscope Observations of ... · Plant Physiol. (1973) 51,415-420 Short...
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Plant Physiol. (1973) 51, 415-420 Short Communication Scanning Electron Microscope Observations of Herbicide Dispersal Using Cathodoluminescence as the Detection Mode' Received for publication February 14, 1972 BRUCE Y. ONG,2 RICHARD H. FALK, AND DAVID E. BAYER Department of Botany, University of California, Davis, California 95616 When high energy electrons strike a specimen in an elec- tron microscope a number of phenomena occur. Some elec- trons are reflected with little or no energy loss. Others actually penetrate the specimen to some degree and excite molecules within a volume of the solid. Some molecules divest them- selves of their surplus energy by allowing excited electrons to escape from their orbitals. If these electrons are able to escape from the specimen, they are known as secondary electrons. If the excited electrons do not leave the molecule but instead fall back to their ground state, they must transfer their surplus energy to other molecules or emit it as discrete packages of energy. The emission can be heat, infrared to visible light, or x-rays (for further details of these processes see 1, 4, 7, 8). We will confine ourselves to a consideration of the infrared to visible light production and reflected electron phenomena in this communication. Researchers working with herbicides frequently have a need to know the spatial location of an herbicide in or upon a plant. A common method for obtaining such information is that of radioautography (9). Radioautography, while certainly adequate for many studies, often requires weeks of exposure time to produce a sufficiently intense film image and also in- volves the expense of radioactively labeled compounds. A number of compounds will fluoresce3 under electron bom- bardment (3). We have used a scanning electron microscope equipped to detect this fluorescence (cathodoluminescence) and wish to report a new, rapid method for spatially localizing herbicides on leaf surfaces. MATERIALS AND METHODS The technical grade of 17 herbicides was used in this study as detailed in Table I. Saturated solutions of the herbicides were prepared in either absolute ethanol or in distilled water. Aliquots of these satu- rated solutions were then placed on aluminum specimen stubs, evaporated to dryness, and examined for cathodoluminescence properties. In other studies the dry herbicide powder was examined 'This study was supported in part by grants from the Rice Re- search Board and a University of California Faculty Research Grant. 'National Science Foundation summer undergraduate trainee. 'Fluorescence as used in this communication will refer to that radiation emitted only during specimen excitation. Cathodolumi- nescence (CL) is defined as the emission of visible radiation due to electron bombardment and includes fluorescence as used in this com- munication. directly for UV excited fluorescence with a Leitz Ortholux microscope. A xenon lamp (BG-12 excitation filter and K 530 barrier filter) was used for illumination. Following the above studies, undiluted commercial formula- tions of herbicides were applied to leaf surfaces with an atom- izer. After allowing the liquid phases to evaporate, small pieces of treated leaves were glued to specimen stubs and quickly examined without further treatment (2, 6). Specimens were examined in a Cambridge Stereoscan Mark IIa scanning electron microscope. Electron beam potentials were varied from 2 to 30 kv. A beam current of about 200 ,uamp was maintained by appropriate filament biasing. The secondary or reflected electron detector was operated in the A B FIG. 1. Signal level responses as seen on a monitor video. A: Penultimate signal clipping; B: signal clipping. I , I 20 . - 16. 12 z 8 Z 4 0 0.1 0.2 0.3 0.4 0.5 0.6 0. 7 WAVELENGTH- MICRONS FIG. 2. Advertised spectral response of EMI 6255B photomulti- plier tube. 415 www.plantphysiol.org on May 26, 2020 - Published by Downloaded from Copyright © 1973 American Society of Plant Biologists. All rights reserved.
Transcript of Short Scanning Electron Microscope Observations of ... · Plant Physiol. (1973) 51,415-420 Short...
Plant Physiol. (1973) 51, 415-420
Short Communication
Scanning Electron Microscope Observations of HerbicideDispersal Using Cathodoluminescence as the Detection Mode'
Received for publication February 14, 1972
BRUCE Y. ONG,2 RICHARD H. FALK, AND DAVID E. BAYERDepartment of Botany, University of California, Davis, California 95616
When high energy electrons strike a specimen in an elec-tron microscope a number of phenomena occur. Some elec-trons are reflected with little or no energy loss. Others actuallypenetrate the specimen to some degree and excite moleculeswithin a volume of the solid. Some molecules divest them-selves of their surplus energy by allowing excited electrons toescape from their orbitals. If these electrons are able to escapefrom the specimen, they are known as secondary electrons. Ifthe excited electrons do not leave the molecule but instead fallback to their ground state, they must transfer their surplusenergy to other molecules or emit it as discrete packages ofenergy. The emission can be heat, infrared to visible light, orx-rays (for further details of these processes see 1, 4, 7, 8).We will confine ourselves to a consideration of the infraredto visible light production and reflected electron phenomena inthis communication.
Researchers working with herbicides frequently have a needto know the spatial location of an herbicide in or upon aplant. A common method for obtaining such information isthat of radioautography (9). Radioautography, while certainlyadequate for many studies, often requires weeks of exposuretime to produce a sufficiently intense film image and also in-volves the expense of radioactively labeled compounds.A number of compounds will fluoresce3 under electron bom-
bardment (3). We have used a scanning electron microscopeequipped to detect this fluorescence (cathodoluminescence)and wish to report a new, rapid method for spatially localizingherbicides on leaf surfaces.
MATERIALS AND METHODS
The technical grade of 17 herbicides was used in this studyas detailed in Table I.
Saturated solutions of the herbicides were prepared in eitherabsolute ethanol or in distilled water. Aliquots of these satu-rated solutions were then placed on aluminum specimen stubs,evaporated to dryness, and examined for cathodoluminescenceproperties.
In other studies the dry herbicide powder was examined
'This study was supported in part by grants from the Rice Re-search Board and a University of California Faculty ResearchGrant.
'National Science Foundation summer undergraduate trainee.'Fluorescence as used in this communication will refer to that
radiation emitted only during specimen excitation. Cathodolumi-nescence (CL) is defined as the emission of visible radiation due toelectron bombardment and includes fluorescence as used in this com-munication.
directly for UV excited fluorescence with a Leitz Ortholuxmicroscope. A xenon lamp (BG-12 excitation filter and K 530barrier filter) was used for illumination.
Following the above studies, undiluted commercial formula-tions of herbicides were applied to leaf surfaces with an atom-izer. After allowing the liquid phases to evaporate, small piecesof treated leaves were glued to specimen stubs and quicklyexamined without further treatment (2, 6).
Specimens were examined in a Cambridge Stereoscan MarkIIa scanning electron microscope. Electron beam potentialswere varied from 2 to 30 kv. A beam current of about 200,uamp was maintained by appropriate filament biasing. Thesecondary or reflected electron detector was operated in the
A B
FIG. 1. Signal level responses as seen on a monitor video. A:Penultimate signal clipping; B: signal clipping.
I ,I
20 .-
16.
12z
8
Z 4
0
0.1 0.2 0.3 0.4 0.5 0.6 0. 7WAVELENGTH- MICRONS
FIG. 2. Advertised spectral response of EMI 6255B photomulti-plier tube.
415
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ONG, FALK, AND BAYER
Table I. Tabulation of Herbicides and Their Fluorescence Properties
UV-EXCITEDFLIJORESCENCEEMISSIONCOLOR
ELECTRONEXCITATION
KV RESPONSE
0
C-OHC-N-H
Cl
HVH6,3111
H Hy *k< H
CH3 -C-* C N 2H5CH3
Cl Cl
CHC3CO2 co2CH3Cl Cl
yellow
greenish
2-30 excellent
10-30 fair
green-yellow 10-30 very good
green 2-30 very good
0 CH
gC C-C-NC
\~~~CH3
green-yellow 5-30 very good
H O
Ci N-C-N(CH3)2
C
yellow 10-30 excellent
H O
DO~N-C-N(CH3)2green-yellow 3-30 excellent
HERBICIDE
Naptalam
Amitrole
Atrazine
DCPA
Diphenamid
Diuron
Fenuron
416 Plant Physiol. Vol. 51, 1973
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HERBICIDE CATHODOLUMINESCENCE
Table I.-Continued
F.ndothal
0
?i~t-C-OH-OHC-OH
0
green 10-30 veryr -ood1
OH
HVJCt H
C2H5- -t -i-"25green-yellow
yellow
green
C1
& COOH
ci bCH
c1 C1 0
Cl IlOC-OHC1 0-CH3
Paraquat yellow2[
10-30 good
2-30 excellent
10-30 very good
10-30 good
0C1Q -CH2-C..OH
CH3C3
green-yellow 3-30 excellent
Cl C-CH2-C-OH
Cl
CL OCl -O-CH2-C-OH
Cl
yellow
yellow
20-30 very good
10-30 very good
Hydroxy-simazine
Dicamba
Tricamba
MCPA
2,4-D
Plant Physiol. Vol. 51, 1973 417
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ONG, FALK, AND BAYER
Propanil
Trifluralin
Table I.-Continued
ClONH-C-CH2-CHCl
ITO
CF3 N-( 3H7)2
NO2
yellow
green
20-30 fair
5-30 poor
A 3000 A electron beam spot size was used; all other spot sizes were 2000 A.
Table It. Effect of Various Substituietits otn Benzene Fluorescence
Substituent Effect on Intensity
Alkyl Very slight increase or decrease-OH, -OCH3, -OC2H5 Increase-COOH Large increase-NH2, -NHR, NR2 Increase-NO2,-NO Total quench-CN Increase-SH Decrease-F Decrease-Cl Decrease-Br Decrease-I Decrease-SO3H None
' Modified from Hercules (5).
reflected mode to minimize fluorescence contributions fromthe light guide of this detector.
Qualitative measurements of fluorescence yield were madeby advancing the gain on the photomultiplier of the CL de-tector until the signal monitor video tube indicated penultimatesignal clipping (Fig. 1). Values were then read from the photo-multiplier gain potentiometer and categorized relative to eachother as excellent, very good, good, fair, poor, or no response.The CL photomultiplier tube used in this study was an EMI
type 6255B. Figure 2 is a reproduction of its advertised spec-tral response.'
RESULTS AND DISCUSSION
The herbicides examined in this study illustrate the widevariety of compounds that can be excited to fluorescence inthe scanning electron microscope. Variously substituted fiveand six membered nitrogen heterocycles, anilines, and benzenesare represented among the herbicides listed in Table I. Allfluoresced to some degree.
Fluorescence is a property usually associated with mobile -relectrons. Since aromatic compounds contain r electrons, mostof them can be induced to fluoresce. The intensity of fluores-cence is dependent on the relative freedom of 7r electrons inthe compound. Various substituent groups can thus drastically
4 EMI Photomultiplier Tube and Accessory Catalog. GencomDivision, Varian/EMI, 80 Express St., Plainview, New York 11803.
affect fluorescence intensity. Table II lists some common sub-stituent groups and their effects on fluorescence intensity.These effects are generally true for simple compounds such asbenzene but may not hold for more complex molecules (10).
Substituents that were found to affect fluorescence inten-sities may be readily seen by examining the results shown inTable I. Propanil' and Diuron serve to compare the effects ofa substituted amino group (-N(CH2)2) and an alkyl group(-CH2CH2). Diuron, which has the substituted amino group,fluoresced quite strongly as compared to Propanil. ComparingDicamba and Tricamba reveals the quenching effect of an ad-ditional -Cl group. On the other hand 2,4-D and 2,4,5-Thave similar fluorescence intensities, the additional -Cl groupon the latter did not cause a noticeable change in quenching.We usually found that the more highly substituted an amino
group, the greater the fluorescence intensity. Atrazine andhydroxysimazine support these findings.We find that we can exploit the fluorescent properties of
herbicides to excellent advantage for spatially localizing themon the surfaces of leaves. Figure 3 shows the CL image of arather heavy application of MCPA on a sugar beet leaf. Thecorresponding reflected electron image (Fig. 6) shows the leafsurface detail. Figure 4 shows the CL image of a very lightapplication of Dicamba on a sugar beet leaf. Figure 7 is thecorresponding reflected electron image. Figure 5 shows theCL image of a very heavy application of commercial blankMCPA (commercial formulation containing no MCPA). Thephotomultiplier gain was advanced to the point of producingsubstantial electric noise in this case (Fig. 5), yet no CL imagewas apparent. Figure 8 is the corresponding reflected electronimage, and it may be noted the solvent application was indeedheavy.We are aware of the possibility of insecticides fluorescing
in a similar manner and suggest that examination of leaves bythe method described might well be used to estimate the per-
' Chemical names of herbicides: Naptalam: N-1-naphthylphthal-amic acid; Amitrole: 3-amino-s-triazole; Atrazine: 2-chloro-4-(ethyl-amino)-6-(isopropylamino)-s-triazine; DCPA: dimethyl tetrachloro-terephthalate; Diphenamid: N ,N-dimethyl-2, 2-diphenylacetamide;Diuron (DCMU): 3-(3 ,4-dichlorophenyl)-1, 1-dimethylurea; Fe-nuron: 1,1-dimethyl-3-phenylurea; Endothal: 7-oxabicyclo [2.2.1]heptane-2, 3-dicarboxylic acid; Hydroxysimazine: 2-hydroxy-4, 6-bis(ethylamino)-s-triazine; Dicamba: 3, 6-dichloro-o-anisic acid;Tricamba: 3,5, 6-trichloro-o-anisic acid; Paraquat: 1, 1'-dimethyl-4, 4'-bipyridinium dichloride; MCPA: [(4-chloro-o-tolyl)oxy]aceticacid; Propanil: 3',4'-dichloropropionanilide; Trifluralin: ae,a, a-tri-fluoro-2, 6-dinitro-N, N-dipropyl-p-toluidine.
418 Plant Physiol. Vol. 51, 1973
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-
- .
.C W .,...... .R:' £w_
w.J.Y .! :z._
*^.
-:.:, :N_' _FM:w.: :. .. _. .... 16 s: . .... .. :_Ji' diiMiL: 'S°.iw w
FIGs. 3, 4, and 5. Cathodoluminescence images of herbicide FIGS. 6, 7, and 8. Reflected electron images of sugar beet leavesdispersal patterns. corresponding to the CL images seen in Figures 3, 4 and 5, respec-
FIG. 3. A heavy application of MCPA on a sugar beet leaf. x 130. tively.FIG. 4. A very light application of Dicamba on a sugar beet leaf. FIG. 6. Leaf surface upon which MCPA was sprayed. X 130.
x 130. FIG. 7. Leaf surface upon which Dicamba was sprayed. X 130.FIG. 5. A very heavy application of commercial blank MCPA FIG. 8. Leaf surface upon which MCPA blank was applied.
(herbicide solvent but no MCPA). The small specks represents am- x 130.plifier electric noise. X 130.
419
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w :40-. I
-- -
- e.
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420 ONG, FALK,
sistence of insecticides in treated fields. This possibility is underinvestigation at present.
LITERATURE CITED
1. ANDERSON, C. A. 1967 Introduction to the electron microprobe and its ap-plication to biochemistry. In: D. Glick, ed., Methods of BiochemicalAnalysis, Vol. 15. Interscience, New York. pp. 147-270.
2. FALK, R. H., E. M. GIFFORD, JR., AND E. G. CUTTER. 1970. Scanning electronmicroscopy of developing plant organs. Science 168: 1471-1474.
3. GARLICK, G. F. J. 1966. Cathodo- and radioluminescence. In: P. Goldberg,ed., Luminescence of Inorganic Solids. Academic Press, New York. pp.685-731.
4. GOLDBERG, P. 1966. Luminescence of Inorganic Solids. Academic Press, NewYork.
AND BAYER Plant Physiol. Vol. 51, 1973
5. HERCULES, D. M. 1966. Theory of luminescence processes. In: D. MI.Hercules, ed., Fltuorescence and Phosphorescence Analysis. Wiley, New%-York. pp. 2-40.
6. HESLOP-HARRISON, Y. 1970. Scanning electron microscopy of fresh leaves ofPinguicula. Science 167: 172-174.
7. IVEY, H. F. 1963. Electroluminescence and related effects. In: L. Mlorton,ed., Advances in Electronics and Electron Physics. Academic Press, NewYork. pp. 1-240.
8. KAY, D. H. 1965. Techniques for Electron Microscopy. F. A. Davies, Phila-delphia.
9. PICKERING, E. R. 1966. Autoradiography of mobile, C'4-labeled herbicides insections of leaf tissue. Stain Tech. 41: 131-137.
10. WEHRY, E. L. AND L. B. RODGERS. 1966. Fluorescence and phosphorescenceof organic molecules. In: D. MI. Hercules, ed., Fluorescence and Phos-phoresceeice Analysis. Wiley, New York. pp. 81-150.
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