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Applications of scanning electron microscopy in immunology
Micron. 1980, Vo].: I I, pp. 369-370 Pergamon Press Ltd. Printed in Great Britain
APPLICATIONS OF SCANNING ELECTRON MICROSCOPY IN IMMUNOLOGY
H. W. CARTER St. Vincent Charity Hospital, Department of Laboratory Medicine, Cleveland, OH 44115, U.S.A. Fluorescent microscopy (FM), transmission electron microscopy (TEM) and scannlng electron microscopy (SEM) are used to view and record the localization of specific molecules on cell surfaces. FM has the advantages of being usable on viable cells and its efficiency. TEM can be used on sectioned material for the localization of markers with intracellular structure and has the highest resolution. The SEM's large specimen capability, depth of focus, and imaging modes lets one avoid tedious procedures required to evaluate results of similar studies using TEM. SEM is more sensitive than FM and multiple labels are more easily studied, however, combined studies may be more informative than either technique by itself. 1 Labeling techniques commonly used in FM and TEM are adaptable for use in the SEM and a variety of markers are now available. Large markers (100-300 NM) include synthetic polymers, tobacco mosaic virus and bacteriophage T-4 and are useful for studies at low magnification. Intermediate markers (30-50 NM) such as hemocyanln, bushy stunt virus and synthetic polymers are visible even after coating with metals. Small markers (10-15 NM) such as ferritln are at the limit of resolution of the SEM. Resolving ferrltin greatly expanded the SEM's potential for immunologic applications. Non-specific binding of ferrltin is avoided by using the ferritin bridge and penetration into cells is enhanced by the use of saponins. 2 Ferritln can be increased in size (approx. two-fold) and intensified in electron density by staining with bismuth subnitrate. 3 The ferritln bismuth unit should be more easily seen in the SEM, particularly in the backscattered electron imaging (BEI) mode. The BE1 mode adds the potential for detecting specific molecules beneath the surface of the cell. 4 Gold granules may be prepared in several sizes (5-150 NM) and are suitable for multiple labeling experiments. The use of gold granules as a marker has been evaluated.5 Gold labels should be easily detected in the BEI mode and would certainly distinguish them from low molecular weight particulate material. The cathodluminescent mode of SEM has not made any significant and presently is not an improvement over FM.
contribution
The analytical modes of SEM may be useful when studying heavy metal combined with BEI.
to immunology
labels particularly
when
Immunology is a field with very advanced and sophisticated techniques for the detection of the presence of specific molecules. Is there a need for SEM? I think SEM adds to the present visual methods and there is a need to localize as precisely as possible specific molecules. Through visualization methods, we can determine their number, their distribution and their redistribution to changes in their environment. I think the riddle posed by Lerch 6 is relevant to microscopy's role in immunology. "Given the power to see the unseen, will we learn anything important? At first blush, we are compelled to answer yes, but it really depends on how prepared we are to understand the meaning of our new vision." i. Molday, R.S. Cell surface labeling techniques for SEM. In: Scanning Electron Microscopy/1977/ll. Joharl, O. and Becker, R.P. (Eds), liT Research Institute, Chicago, IIi., 60616, 59-74. 2. Willingham, Antigens in Ann. Meeting Baton Rouge,
M.C. Electron Microscopic Immunocytochemlcal localization of Intracellular cultured cells: The EGS and Ferritin bridge procedures. In: Proc. 37th Electron Micr. Soc. Amer., Bailey, G.W. (Ed.) Claltor's Publishing Div., La. 104-107.
370
H.W. Carter
3. Hayat, M.A., Positive Staining for Electron Microscopy, Van Nostrand Reinhold Co., New York, N.Y., 1975, p. 217, 4. R.P. Becker and M. Sogard. Visualization of Subsurface structures in cells and tissues by Backscattered Electron Imaging. In: Scanning Electron Microscopy/1979/ll, Johari, O and Becket, R.P. (Eds.), Sem Inc., AMF O'Hare, 111., 60666, U.S.A., 835-870. 5. Goodman, S.L., Hodges, G.M., et al. Colloidal Gold Probe - A further evaluation. In: Scanning Electron Mlcroscopy/1979/lll, Johari, O,, and Becket, R.P. (Eds.), Sem Inc., AMF O'Hare, Ill., 60666, U.S.A., 619-628. 6. Lerch, l.A., Beyond the CT Scanner: In search of a point of light. published by the New York Academy of Sciences, Feb. 1980, pp. 6-9.
In:
The Sciences
APPLICATIONS OF SCANNING ELECTRON MICROSCOPY IN IMMUNOLOGY
H. W. CARTER St. Vincent Charity Hospital, Department of Laboratory Medicine, Cleveland, OH 44115, U.S.A. Fluorescent microscopy (FM), transmission electron microscopy (TEM) and scannlng electron microscopy (SEM) are used to view and record the localization of specific molecules on cell surfaces. FM has the advantages of being usable on viable cells and its efficiency. TEM can be used on sectioned material for the localization of markers with intracellular structure and has the highest resolution. The SEM's large specimen capability, depth of focus, and imaging modes lets one avoid tedious procedures required to evaluate results of similar studies using TEM. SEM is more sensitive than FM and multiple labels are more easily studied, however, combined studies may be more informative than either technique by itself. 1 Labeling techniques commonly used in FM and TEM are adaptable for use in the SEM and a variety of markers are now available. Large markers (100-300 NM) include synthetic polymers, tobacco mosaic virus and bacteriophage T-4 and are useful for studies at low magnification. Intermediate markers (30-50 NM) such as hemocyanln, bushy stunt virus and synthetic polymers are visible even after coating with metals. Small markers (10-15 NM) such as ferritln are at the limit of resolution of the SEM. Resolving ferrltin greatly expanded the SEM's potential for immunologic applications. Non-specific binding of ferrltin is avoided by using the ferritin bridge and penetration into cells is enhanced by the use of saponins. 2 Ferritln can be increased in size (approx. two-fold) and intensified in electron density by staining with bismuth subnitrate. 3 The ferritln bismuth unit should be more easily seen in the SEM, particularly in the backscattered electron imaging (BEI) mode. The BE1 mode adds the potential for detecting specific molecules beneath the surface of the cell. 4 Gold granules may be prepared in several sizes (5-150 NM) and are suitable for multiple labeling experiments. The use of gold granules as a marker has been evaluated.5 Gold labels should be easily detected in the BEI mode and would certainly distinguish them from low molecular weight particulate material. The cathodluminescent mode of SEM has not made any significant and presently is not an improvement over FM.
contribution
The analytical modes of SEM may be useful when studying heavy metal combined with BEI.
to immunology
labels particularly
when
Immunology is a field with very advanced and sophisticated techniques for the detection of the presence of specific molecules. Is there a need for SEM? I think SEM adds to the present visual methods and there is a need to localize as precisely as possible specific molecules. Through visualization methods, we can determine their number, their distribution and their redistribution to changes in their environment. I think the riddle posed by Lerch 6 is relevant to microscopy's role in immunology. "Given the power to see the unseen, will we learn anything important? At first blush, we are compelled to answer yes, but it really depends on how prepared we are to understand the meaning of our new vision." i. Molday, R.S. Cell surface labeling techniques for SEM. In: Scanning Electron Microscopy/1977/ll. Joharl, O. and Becker, R.P. (Eds), liT Research Institute, Chicago, IIi., 60616, 59-74. 2. Willingham, Antigens in Ann. Meeting Baton Rouge,
M.C. Electron Microscopic Immunocytochemlcal localization of Intracellular cultured cells: The EGS and Ferritin bridge procedures. In: Proc. 37th Electron Micr. Soc. Amer., Bailey, G.W. (Ed.) Claltor's Publishing Div., La. 104-107.
370
H.W. Carter
3. Hayat, M.A., Positive Staining for Electron Microscopy, Van Nostrand Reinhold Co., New York, N.Y., 1975, p. 217, 4. R.P. Becker and M. Sogard. Visualization of Subsurface structures in cells and tissues by Backscattered Electron Imaging. In: Scanning Electron Microscopy/1979/ll, Johari, O and Becket, R.P. (Eds.), Sem Inc., AMF O'Hare, 111., 60666, U.S.A., 835-870. 5. Goodman, S.L., Hodges, G.M., et al. Colloidal Gold Probe - A further evaluation. In: Scanning Electron Mlcroscopy/1979/lll, Johari, O,, and Becket, R.P. (Eds.), Sem Inc., AMF O'Hare, Ill., 60666, U.S.A., 619-628. 6. Lerch, l.A., Beyond the CT Scanner: In search of a point of light. published by the New York Academy of Sciences, Feb. 1980, pp. 6-9.
In:
The Sciences