Ultramicroscopy 0 North-Holland
3 (1978) 273-281 Publishing Company
Andreas ENGEL Dept. of Microbiology,
Biozentrum
of the University Basel, KIingelbergstrasse 70, CH-4056 Base& Switzerland
Received 1 August 1978
A scanning transmission electron microscope is employed to determine the mass of biological macromolecules. Elastically scattered electrons are collected by an annular detector that is capable of counting single electrons Off-line processing of these dark field micrographs stored on a magnetic tape is accomplished by a mini-computer. It allows the number of electrons scattered by spherical or filamentous proteins to be evaluated. The calibration factor relating the number of scattered electrons to the mass of the protein is derived from scattering theory and is experimentally determined from biological macromolecules of known mass. Mass-loss kinetics of biological specimens due to the electron beam are measured for various protein strut tures. The application of this method is illustrated by determination of the mass of an oligomeric protein (major phageT4 head protein) and the mass per unit length of a filamentous protein aggregate (F-pili). The unique possibilities of this new techniqueas well as its limitations are discussed.
1. Introduction
2. Theory
Molecular weight is one of the crucial parameters for the characterization of a biological macromolecule. Although severaltechniquesfor the determination of molecular weights have been developed, thesemethods either require relatively largeamounts of highly purified material (e.g. sedimentationequilibrium) or lead to complete dissociationof the structures of interest (e.g. SDS gel electrophoresis).Furthermore, there are almost no techniquesthat allow the determination of the mass of supramolecularstructures. Elastic scattering of electrons by matter is a well-understood processwhich is largely independent of the chemicalbonds associating atomsinto molecules[ 11. Electrons therefore provide an excellent probe to measurethe masswithin the irradiated specimenarea.While a convenient method for the measurementof molecular weight employing the conventional electron microscopewas developed more than a decadeago [2], recent results suggestthat the scanningtransmissionelectron microscope(STEM) is very well suited to assess quantitatively the massof large structures at low dose [3-51,
The theoretical background for molecular weight determination by meansof electron microscopy has been established[2,3], and elastic aswell asinelastic scattering hasbeen thoroughly investigated both theoretically and experimentally usingscanningtransmission electron microscopy [ 1,6-81. Assumingthin specimens (i.e. neglectingmultiple scattering), the number N, of electrons elastically scattered by proteinous matter is given by
273
N, = (o,>nNo/A
(1)
where ( ue ) = averageelastic scattering crosssection of atoms that constitute a protein *, n = total number of atoms within the irradiated areaA;N,-, = total number of electrons incident on areaA. The singlescattering approximation is satisfiedif n( a,)
3 (1978) 273-281 Publishing Company
Andreas ENGEL Dept. of Microbiology,
Biozentrum
of the University Basel, KIingelbergstrasse 70, CH-4056 Base& Switzerland
Received 1 August 1978
A scanning transmission electron microscope is employed to determine the mass of biological macromolecules. Elastically scattered electrons are collected by an annular detector that is capable of counting single electrons Off-line processing of these dark field micrographs stored on a magnetic tape is accomplished by a mini-computer. It allows the number of electrons scattered by spherical or filamentous proteins to be evaluated. The calibration factor relating the number of scattered electrons to the mass of the protein is derived from scattering theory and is experimentally determined from biological macromolecules of known mass. Mass-loss kinetics of biological specimens due to the electron beam are measured for various protein strut tures. The application of this method is illustrated by determination of the mass of an oligomeric protein (major phageT4 head protein) and the mass per unit length of a filamentous protein aggregate (F-pili). The unique possibilities of this new techniqueas well as its limitations are discussed.
1. Introduction
2. Theory
Molecular weight is one of the crucial parameters for the characterization of a biological macromolecule. Although severaltechniquesfor the determination of molecular weights have been developed, thesemethods either require relatively largeamounts of highly purified material (e.g. sedimentationequilibrium) or lead to complete dissociationof the structures of interest (e.g. SDS gel electrophoresis).Furthermore, there are almost no techniquesthat allow the determination of the mass of supramolecularstructures. Elastic scattering of electrons by matter is a well-understood processwhich is largely independent of the chemicalbonds associating atomsinto molecules[ 11. Electrons therefore provide an excellent probe to measurethe masswithin the irradiated specimenarea.While a convenient method for the measurementof molecular weight employing the conventional electron microscopewas developed more than a decadeago [2], recent results suggestthat the scanningtransmissionelectron microscope(STEM) is very well suited to assess quantitatively the massof large structures at low dose [3-51,
The theoretical background for molecular weight determination by meansof electron microscopy has been established[2,3], and elastic aswell asinelastic scattering hasbeen thoroughly investigated both theoretically and experimentally usingscanningtransmission electron microscopy [ 1,6-81. Assumingthin specimens (i.e. neglectingmultiple scattering), the number N, of electrons elastically scattered by proteinous matter is given by
273
N, = (o,>nNo/A
(1)
where ( ue ) = averageelastic scattering crosssection of atoms that constitute a protein *, n = total number of atoms within the irradiated areaA;N,-, = total number of electrons incident on areaA. The singlescattering approximation is satisfiedif n( a,)
