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Observation of unstained biological macromolecules with the STEM
Ultamicroscopy 5 (1980) 317-323 © North-Holland Publishing Company
OBSERVATION OF UNSTAINED BIOLOGICAL MACROMOLECULES WITH THE STEM M. OHTSUKI The Enrico Fermi Institute, The University of Chicago, Chicago, Illinois 60637, USA
Received 13 February 1980
We discuss the observation of an unstained single biological macromoleeule of human serum low density lipoprotein in a scanning transmission electron microscope (STEM). Using a eaxbon substrate with 16 A average thickness and an electron dose of 19 electrons/A 2 the image obtained from the unstained molecule shows a similar surface substructure as that seen in a negatively stained conventional transmission electron microscope (CTEM) micrograph.
1. Introduction
for the observation of an unstained single biological macromolecule in which an image of a single molecule can be obtained with a sufficient signal (contrast) at a resolution comparable to that which can be obtained using a negatively stained molecule.
The very high collection efficiency of the annular detector (elastic signal) dark field mode of operation in the STEM has been discussed by Crewe et al. [1]. This particular advantage enables us to obtain micrographs with far fewer electrons than is possible in a micrograph taken with a CTEM. This means that direct observation of an unstained single biological molecule should be possible. Unstained CTEM observation of single biological molecules on the other hand is somewhat more difficult because it requires a substantial number of electrons per unit area (e.g. 500 electrons A 2 for a resolution of 5 A at 50000 × [2]) and then the effects of electron radiation damage are so high that one will obtain only a ghost image of the molecule. Unwin and Henderson [2] have overcome this difficulty by observing unstained, glucose-preserved crystals of catalase and purple membrane in which the combined information from many molecules using very low dose images along with corresponding electron diffraction patterns were analysed with a resolution of below 10 A. Observation of unstained biological molecules with the STEM is a relatively new area and only a few reports have been published as a rnicrograph of an Epon-embedded sectioned catalase crystal by Engel et al. [31. In this paper, we discuss the utility of the STEM
2. Specimen preservation and radiation damage The observation of an unstained specimen involves solving some important problems: (1) the specimen must preserve its structure after dehydration; (2) the sensitivity to electron irradiation is higher than that o f a stained particle due to the absence of a protecti~,e (e.g. staining) agent. For this reason one must take a micrograph with a low dosage of electrons. The first problem is illustrated in fig. 1. Negatively stained (1% (w/v) uranyl formate) glutamine synthetase from anabaena 7120 was dried in air: In (a) the staining agent was applied while the sample was wet; while in (b) the staining was carried out after the sample was completely dried. It appears that the staining agent has kept the structure of the molecule intact when the molecule was stained before the sampie was dried; however, when the molecule was stained after it was air-dried, collapse of the particle appears to have occurred presumably due to the surface tension of the solution. The problem of electron radiation damage has been discussed elsewhere by many authors (e.g., refs. 317
318
M. Oh tsuki / Observation o f unstained biological macromolecules with STEM
[2,4--6]) and this is the main limit o f resolution in biological microscopy. In a S T E M , as n o t e d before, one can obtain an image with sufficient signal using less electrons striking the specimen. Fig. 2 shows, as an e x a m p l e , micrographs o f negatively stained (1% (w/v) uranyl acetate) h e m o g l o b i n from h t m b r i c u s terrestris [7] taken with two different electron doses, 10 electrons )k ~ (a) and 0.3 electrons A ~ (b) in which the signal to noise ratio is sufficient to give a statistically meaningful image o f a stained single molecule [8].
.a
200.a.
b
Fig. 1. CTEM micrographs of negatively stained (1% uranyl formate) glutamine synthetase from anabaena 7120. The molecule consists of double disc of two opposing hecamers. (a) Uranyl acetate was applied while the sample was still wet in which the staining agent kept the molecule intact. (b) The sample was washed 3 times with water on the grid, then dried in air. After the sample was completely dried, the staining agent was applied for 2 min. The collapsed molecules are seen presumably due to the surface tension of the solution. The sample was kindly provided by Drs. R. Haselkorn and J. Orr of The University of Chicago.
Fig. 2. STEM micrograph (darkfield) of negatively stained (1% uranyl acetate) hemoglobin from Lumbricus terrestris. Like glutamine synthetase, the molecule contains 12 subunits in two opposing hexamers. The micrograph (a) was taken with electron dosage of 10 electrons]A 2 and (b) was with 0.3 electrons/A 2. The low dose micrograph (b) shows the substantial contrast in which fine details of the subunit are seen (see arrow). This illustrates the very high efficiency of the STEM annular detector dark field imaging. The sample was kindly provided by Drs. S.N. Vinogradov and O.H. Kapp of Wayne State University.
319
M. Ohtsuki / Observation of unstained biological macromolecules with STEM
These numbers may appear to be surprisingly low and therefore it may be important to indicate how we calculate the dose. Essentially we must rely upon the known characteristics of the solid-state detector and the anaplifier for the electron dose (D) in the STEM we use,
!
D = IBT/A = 2.4VsT/VoGrd (X1013 electrons/A2),
(1) where I B is the beam current at the specimen, T is the scanning t i m e , A is the area of the field o f view, Vs is the signal voltage, Vo is the accelerating voltage and G v is the gain in the preamplifier of the STEM. For example, when we use T = 17 s, Vo = 37500 eV, Gp = 20 a n d A = (2250 A) 2 with Vs = 0.1 V, we will obtain D = 10.7 electrons/A 2. 3. Contrast of an unstained biological macromolecule In the following, we use the STEM annular detector elastic signal to produce the image. A carbon film with an average thickness o f approximately 16 A was used as a substrate as seen in fig. 3 [9]. A typical CRT trace of a carbon substrate is shown in fig. 4 where we observe a thickness variation of about 30%. The elastic scattering intensities from a molecule and a substrate can be written as I M ~ Z ~ 2 NMTMIo,
(2)
1s ~ Z~ 12 NsTsIo,
(3)
where 1 M and I s are the intensities from a molecule and the substrate, respectively. Z M and Z s are the atomic numbers. N M and N s are the atom densities in atoms/A 3 • TMand T s are the thicknesses (in A) of the areas through which the beam passes, lo is the total beam striking the specimen. A generally accepted criterion of image visibility is that one can observe a molecule with 100% or more contrast when 1M >/IS. Then we must have Z ~ 2 N M T M >1Z~ 12 N s T s.
Fig. 3. Dark field STEM micrograph of very clean, 16 A average thickness carbon film. A similar carbon film was used as a aubstrate for the observation of an unstained molecule of LDL.
and obtain a thickness T M ~ 28 A, which is required to give more than 100% contrast. In the case of a particle o f diameter of about 200 A, the intensity is so great that we can expect to see small differences of contrast within the particle from small variations of thickness or density. The calculated resolution is illustrated in fig. 5 in which we use a probe size o f 2.5 A and a convergence half angle of 15 mrad. When we focus the beam at the center of a particle of 200 A, we will get a resolution o f about 6 A on both sides of the surface. This indicates that any information larger than 6 A with sufficient contrast can theoretically be observed.
4. Observation of an unstained single molecule of human serum low density lipoprotein In order to demonstrate the ability of the STEM to image an unstained biological macromolecule, par-
(4)
For our substrates we can use Z s = 6 , N s = 0.11 atoms/A 3 and T s = 16 3,. Since most of biological specimens consist of low Z atoms, we can assume an average atomic number o f 6. For the biological specimen (see below) we assume N M ~ 0.06 atoms/A 3,
khole
H° 20A
Fig. 4. Typical CRT trace of elastic signal (/el) from 16 A average thickness carbon film similar to the one shown in fig. 3. About 30% thickness fluctuations are seen.
320
M. Ohtsuki / Observation of unstained biological macromolecules with STEM
tides of human serum low density lipoprotein (LDL) were examined. Human serum LDL (d = 1.04 g/ml, MW = 2.7 × 104) has an outside diameter of about 216 A; its function is to transport lipids and regulate the cholesterol metabolism [ 10]. Electron microscope studies of this particle have been reported in the past with inconsistent results: Examination of the particle by Pollard et al. [ 11 ] concluded that the LDL had 12 surface subunits dodecahedraUy placed; Forte and Nichols reported that the particle showed a granular structure [12] ; later Forte and Nordhausen concluded from their electron microscopy that there were no substructures in the LDL [13] and claimed that the earlier observation of the granularity was due to phase effects in the objective lens. It should be noted that unordered particles such as LDL are somewhat difficult to investigate in the CTEM because without knowing the exact defocus value of the objective lens, one may not be able to make reliable observations of fine structure at high resolution. Deutsch and Sogard [14] realized these problems and studied negatively stained LDL using the STEM and CTEM in which they showed STEM micrographs without phase contrast effects [ 1]. Their investigation led to the conclusion that there were about 60 surface substructures with a diameter of 20 to 35 A which seemed to be evenly distributed on the surface of LDL particles. Our unstained observations were performed in an attempt to confirm this conclusion.
freeze-dried preparation; we had no success using airdried specimens. 4.2. Observation o f unstained LDL As a first step we obtained CTEM micrographs of negatively stained LDL. The micrographs were taken in a careful manner in which the defocus of the objective lens was kept at about - 1 0 0 0 A so that the theoretical phase contrast resolution was reduced to about 3 A without any reverse contrast problems [13]. Fig. 6 shows negatively stained LDL (with 1% sodium phosphotungstate, pH = 7.0) in which the presence of surface substructure is obvious. For comparison we then used the STEM to obtain images of unstained LDL with an electron dosage of 19 electrons/A 2. Fig. 7a shows an unstained particle in which the intensity of the central region is very high. Since we are observing the total elastic cross section of the specimen, we expect to get larger scattering from the thicker central region of the particle. Fig. 7b was obtained
INCIDENT BEAM
4.1. Specimen preservation In order to preserve the structure of the molecule, the freeze-dried method was employed, The particles were diluted with 5 mM NH4HCO3 (pH = 8.0) to 150/ag/ml just before being deposited on a 16 A thick (average) carbon f'tlm. The sample was washed a few times with 1 mM NH4HCO3 before freezing. It was frozen by inserting the grid into liquid nitrogen. After about 30 s the grid was removed from the nitrogen and placed on a precooled (liquid nitrogen temperature) stainless-steel block. This block was then inserted into a vacuum chamber for about 12 h to sublime the ice. This specimen conservation treatment is very important for the visualization of a three-dimensional unstained particle such as LDL and our success in imaging only occurred with the
Fig. 5. Schematic diagram showing the electron probe penet_rating a molecule of thickness TM. We obtain ti X = ,~TM + 60 where ~ is the convergence half angle, 60 is the probe size at infocus and 5 X is the probe size at the surface of both sides of the molecule assuming the beam was focused at the center of the molecule. When 6 o = 2.5 A, t~ = 15 mrad, and TM = 216 A are given, we will obtain ~X = 5.7 A.
M. Oh tsuki / Observation o f unstained biological macromolecules with STEM
321
from the same negative shown in 7a, but printed in such a manner that the central region was exposed more in order to bring up information from that area. The micrograph 7b exhibits clear substructures o f about 20 to 35 A in the central region which have a very similar appearance and dimensions to that o f the surface substructure of the negatively stained particle shown in fig. 6. In addition, the y - m o d u l a t i o n (pseudo three-dimensional) technique was applied for observation of a single unstained molecule as seen in fig. 7d. The thickness variations can be observed. Fig. 6. Bright field CTEM micrograph of LDL stained with 1% sodium phosphotungstate, pH = 7.0. The presence of surface substrates is obvious. The sample was kindly provided by Dr. A.M. Scanu of The University of Chicago.
5. Discussion In the case o f negatively stained biological particles, a substructure can clearly be seen when both
f
d Fig. 7. Dark field STEM micrographs of unstained LDL molecule taken with the annular detector (elastic) signal: (a) and (b) were printed from the same negative in which (a) was printed with even exposure..The exposure for the central region was increased to obtain the fine details of the substructure which show similarity compared with the stained particle shown in fig. 6; (c) shows another LDL particle which was printed the same way as (b); (d) the y-modulation (pseudo three-dimensional modulation) of a single unstained molecule of LDL shows the thickness variation.
322
M. Ohtsuki / Observation of unstained biological macromolecules with STEM
sides of the surface are in register and are superimposed in the direction of the incident beam. For an unstained molecule using the STEM annular detector signal, the contrast is proportional to the total elastic cross section of the molecule. The STEM micrograph of an unstained LDL particle shown in fig. 7 indicates that the shape of the substructures is rather uniform, particularly in the central region of the particle. This may suggest the superposition of similar surface information from both sides of the particle. Also the appearance of the substructure is very similar to that of the substructure of'negatively stained LDL shown in fig. 6. These results may indicate that the structural details shown in the unstained particle are surface substructure and the particle presumably contains an amorphous region in the center of the molecule. Shen et al. [15] proposed a model based on their theoretical calculation. This model for various lipoproteins consists of a hydrophobic sphere forming an amorphous core of about 76 A in diameter which is made of triglycerol and cholesterol ester and the core is surrounded by the outer layer composed of free cholesterol, phospholipid and protein. In addition, Ohtsuki et al. [16] have experimentally confirmed the presence of the core and surface substructure in the HDL3 particle using complementary observation techniques of freeze,etching, negative staining, STEM and CTEM..All lipoproteins including HDLa and LDL presumably have similar structures [ 15]. As noted before, since the LDL particle scatters a substantial amount of elastic signal, we can expect to see very small differences of contrast within the molecule. Assuming we can detect 20% of the contrast difference (ACM), then we will be able to observe thickness differences (ATM) from one side of the molecule (assuming this is surface substructure), AT M = ACMTM/2.For LDL, TM = 216 ,~ when the incident beam passes through the center of the particle, we obtain ATM = 22 A. The thickness variation of 22 A or larger is well within the range of the model proposed by Shen et al. [15] in which the thickness of the outer layer surrounding the central core is about 70 A.
6. Conclusion In this experiment, we used an electron dosage of 19 electrons/A 2. This is higher than the limit given by
Unwin and Henderson [2] for the observation of unstained biological specimens. They suggested.0.5 electrons/A 2 is the upper limit for observation of unstained particles without incurring some damage to the molecule. Nevertheless, our comparison of a micro graph from a negatively stained preparation with the micrograph of the unstained molecule, taken with the electron dose of 19 electrons/h 2, shows very similar structural appearance. This indicates that we can obtain a micrograp.h of an unstained single biological macromolecule using the STEM at a comparable resolution to that obtained with a uranyl acetate stained particle using the CTEM.
Acknowledgements The author wishes to express appreciation to Dr. A.V. Crewe for his helpful comments and discussions and use of his STEM facility at the University of Chicago. The author is grateful to Dr. A.M. Scanu of The University of Chicago for providing low density lipoprotein, Drs. R. Haselkorn and J. Orr of The University of Chiacgo for providing the glutamine synthetase and Drs. S.N. Vinogradov and O.H. Kapp of Wayne State University for providing hemoglobin. The author also thanks M. Slauson and A. Johns for their assistance in photography. This work was supported by grants from the Biotechnology Resources Brance of the National Institute of Health, and the United States Department of Energy.
References [1] A.V. Crewe, J.P. Langmore and M.S. Isaacson, Resolution and Contrast in the Scanning Transmission Electron Microscope, in: Physical Aspects of Electron Microscopy and Microbeam Analysis, Eds. B.M. Siegel and D.R. Beaman (Wiley, New York, 1975) p. 47. [2] P.N.T. Unwin and R. Henderson, J. Mol. Biol. 94 (1974) 425. [3] A. Engel, J. Dubochet and E. Kellenberger, in: Scanning Electron Microscopy/1977, Ed. O. J ohari (IITRI, Chicago, 1977) Vol. 1, p. 371. [4] M.S. lsaacson, Specimen Damage in the Electron Microscope, in: Principles and Techniques of Electron Microscopy, Ed. M.A. Hayat (Van Nostrand-Reinhold, New York, 1977) Vol. 7, p. 1. [5] R.C. Williams and H.W. Fisher, J. Mol. Biol. 52 (1970) 121.
M. Ohtsuki / Observation of unstained biological macromolecules with STEM [6] M. Ohtsuki, in: Proc. 37th Electron Microscopy Society of America Meeting, San Antonio, TX, 1979, Ed. G.W. Bailey (Claitor's, Baton Rouge, LA, 1979) p. 130. [7] S.N. Vinogradov, J.M. Shlom, B.C. Hall, O.H. Kapp and H. Mizukami, Biochimica et Biophysica Acta 432 (1977) 136. [8] M. Ohtsuki and A.V. Crewe, Proc. Natl. Acad. Sci. USA 77 (1980). [9] M. Ohtsuki, M.S. lsaacson and A.V. Crewe, in: Scanning Electron Microscopy/1979, Ed. O. Johari (SEM, Inc., 1979) Vol. 2,p. 375. [10] A.V. Nichols, Proc. Nail. Acad. Sci. USA 64 (1969) 1128.
323
[ 11] H. Pollard, A.M. Scanu and E.W. Taylor, in: Proc. Natl. Acad. Sci. USA 64 (1969) 304. [12] T. Forte and A.N. Nichols, Advan. Lipid Res. 10 (1972) 1. [131 T. Forte and R. Nordhausen, Circulation Suppl. II 54 (1976) 94. [141 D.G. Deutsch and M. Sogard, Micro 10 (1979) 25. [15] B.W. Shen, A.M. Scanu and F.J. Kezdy, Proc. Natl Acad. Sci. USA 74 (1977) 837. [16] M. Ohtsuki, C. Edelstein, M. Sogard and A.M. Scanu, Proc. Natl. Acad. Sci. USA 74 0977) 5001.
OBSERVATION OF UNSTAINED BIOLOGICAL MACROMOLECULES WITH THE STEM M. OHTSUKI The Enrico Fermi Institute, The University of Chicago, Chicago, Illinois 60637, USA
Received 13 February 1980
We discuss the observation of an unstained single biological macromoleeule of human serum low density lipoprotein in a scanning transmission electron microscope (STEM). Using a eaxbon substrate with 16 A average thickness and an electron dose of 19 electrons/A 2 the image obtained from the unstained molecule shows a similar surface substructure as that seen in a negatively stained conventional transmission electron microscope (CTEM) micrograph.
1. Introduction
for the observation of an unstained single biological macromolecule in which an image of a single molecule can be obtained with a sufficient signal (contrast) at a resolution comparable to that which can be obtained using a negatively stained molecule.
The very high collection efficiency of the annular detector (elastic signal) dark field mode of operation in the STEM has been discussed by Crewe et al. [1]. This particular advantage enables us to obtain micrographs with far fewer electrons than is possible in a micrograph taken with a CTEM. This means that direct observation of an unstained single biological molecule should be possible. Unstained CTEM observation of single biological molecules on the other hand is somewhat more difficult because it requires a substantial number of electrons per unit area (e.g. 500 electrons A 2 for a resolution of 5 A at 50000 × [2]) and then the effects of electron radiation damage are so high that one will obtain only a ghost image of the molecule. Unwin and Henderson [2] have overcome this difficulty by observing unstained, glucose-preserved crystals of catalase and purple membrane in which the combined information from many molecules using very low dose images along with corresponding electron diffraction patterns were analysed with a resolution of below 10 A. Observation of unstained biological molecules with the STEM is a relatively new area and only a few reports have been published as a rnicrograph of an Epon-embedded sectioned catalase crystal by Engel et al. [31. In this paper, we discuss the utility of the STEM
2. Specimen preservation and radiation damage The observation of an unstained specimen involves solving some important problems: (1) the specimen must preserve its structure after dehydration; (2) the sensitivity to electron irradiation is higher than that o f a stained particle due to the absence of a protecti~,e (e.g. staining) agent. For this reason one must take a micrograph with a low dosage of electrons. The first problem is illustrated in fig. 1. Negatively stained (1% (w/v) uranyl formate) glutamine synthetase from anabaena 7120 was dried in air: In (a) the staining agent was applied while the sample was wet; while in (b) the staining was carried out after the sample was completely dried. It appears that the staining agent has kept the structure of the molecule intact when the molecule was stained before the sampie was dried; however, when the molecule was stained after it was air-dried, collapse of the particle appears to have occurred presumably due to the surface tension of the solution. The problem of electron radiation damage has been discussed elsewhere by many authors (e.g., refs. 317
318
M. Oh tsuki / Observation o f unstained biological macromolecules with STEM
[2,4--6]) and this is the main limit o f resolution in biological microscopy. In a S T E M , as n o t e d before, one can obtain an image with sufficient signal using less electrons striking the specimen. Fig. 2 shows, as an e x a m p l e , micrographs o f negatively stained (1% (w/v) uranyl acetate) h e m o g l o b i n from h t m b r i c u s terrestris [7] taken with two different electron doses, 10 electrons )k ~ (a) and 0.3 electrons A ~ (b) in which the signal to noise ratio is sufficient to give a statistically meaningful image o f a stained single molecule [8].
.a
200.a.
b
Fig. 1. CTEM micrographs of negatively stained (1% uranyl formate) glutamine synthetase from anabaena 7120. The molecule consists of double disc of two opposing hecamers. (a) Uranyl acetate was applied while the sample was still wet in which the staining agent kept the molecule intact. (b) The sample was washed 3 times with water on the grid, then dried in air. After the sample was completely dried, the staining agent was applied for 2 min. The collapsed molecules are seen presumably due to the surface tension of the solution. The sample was kindly provided by Drs. R. Haselkorn and J. Orr of The University of Chicago.
Fig. 2. STEM micrograph (darkfield) of negatively stained (1% uranyl acetate) hemoglobin from Lumbricus terrestris. Like glutamine synthetase, the molecule contains 12 subunits in two opposing hexamers. The micrograph (a) was taken with electron dosage of 10 electrons]A 2 and (b) was with 0.3 electrons/A 2. The low dose micrograph (b) shows the substantial contrast in which fine details of the subunit are seen (see arrow). This illustrates the very high efficiency of the STEM annular detector dark field imaging. The sample was kindly provided by Drs. S.N. Vinogradov and O.H. Kapp of Wayne State University.
319
M. Ohtsuki / Observation of unstained biological macromolecules with STEM
These numbers may appear to be surprisingly low and therefore it may be important to indicate how we calculate the dose. Essentially we must rely upon the known characteristics of the solid-state detector and the anaplifier for the electron dose (D) in the STEM we use,
!
D = IBT/A = 2.4VsT/VoGrd (X1013 electrons/A2),
(1) where I B is the beam current at the specimen, T is the scanning t i m e , A is the area of the field o f view, Vs is the signal voltage, Vo is the accelerating voltage and G v is the gain in the preamplifier of the STEM. For example, when we use T = 17 s, Vo = 37500 eV, Gp = 20 a n d A = (2250 A) 2 with Vs = 0.1 V, we will obtain D = 10.7 electrons/A 2. 3. Contrast of an unstained biological macromolecule In the following, we use the STEM annular detector elastic signal to produce the image. A carbon film with an average thickness o f approximately 16 A was used as a substrate as seen in fig. 3 [9]. A typical CRT trace of a carbon substrate is shown in fig. 4 where we observe a thickness variation of about 30%. The elastic scattering intensities from a molecule and a substrate can be written as I M ~ Z ~ 2 NMTMIo,
(2)
1s ~ Z~ 12 NsTsIo,
(3)
where 1 M and I s are the intensities from a molecule and the substrate, respectively. Z M and Z s are the atomic numbers. N M and N s are the atom densities in atoms/A 3 • TMand T s are the thicknesses (in A) of the areas through which the beam passes, lo is the total beam striking the specimen. A generally accepted criterion of image visibility is that one can observe a molecule with 100% or more contrast when 1M >/IS. Then we must have Z ~ 2 N M T M >1Z~ 12 N s T s.
Fig. 3. Dark field STEM micrograph of very clean, 16 A average thickness carbon film. A similar carbon film was used as a aubstrate for the observation of an unstained molecule of LDL.
and obtain a thickness T M ~ 28 A, which is required to give more than 100% contrast. In the case of a particle o f diameter of about 200 A, the intensity is so great that we can expect to see small differences of contrast within the particle from small variations of thickness or density. The calculated resolution is illustrated in fig. 5 in which we use a probe size o f 2.5 A and a convergence half angle of 15 mrad. When we focus the beam at the center of a particle of 200 A, we will get a resolution o f about 6 A on both sides of the surface. This indicates that any information larger than 6 A with sufficient contrast can theoretically be observed.
4. Observation of an unstained single molecule of human serum low density lipoprotein In order to demonstrate the ability of the STEM to image an unstained biological macromolecule, par-
(4)
For our substrates we can use Z s = 6 , N s = 0.11 atoms/A 3 and T s = 16 3,. Since most of biological specimens consist of low Z atoms, we can assume an average atomic number o f 6. For the biological specimen (see below) we assume N M ~ 0.06 atoms/A 3,
khole
H° 20A
Fig. 4. Typical CRT trace of elastic signal (/el) from 16 A average thickness carbon film similar to the one shown in fig. 3. About 30% thickness fluctuations are seen.
320
M. Ohtsuki / Observation of unstained biological macromolecules with STEM
tides of human serum low density lipoprotein (LDL) were examined. Human serum LDL (d = 1.04 g/ml, MW = 2.7 × 104) has an outside diameter of about 216 A; its function is to transport lipids and regulate the cholesterol metabolism [ 10]. Electron microscope studies of this particle have been reported in the past with inconsistent results: Examination of the particle by Pollard et al. [ 11 ] concluded that the LDL had 12 surface subunits dodecahedraUy placed; Forte and Nichols reported that the particle showed a granular structure [12] ; later Forte and Nordhausen concluded from their electron microscopy that there were no substructures in the LDL [13] and claimed that the earlier observation of the granularity was due to phase effects in the objective lens. It should be noted that unordered particles such as LDL are somewhat difficult to investigate in the CTEM because without knowing the exact defocus value of the objective lens, one may not be able to make reliable observations of fine structure at high resolution. Deutsch and Sogard [14] realized these problems and studied negatively stained LDL using the STEM and CTEM in which they showed STEM micrographs without phase contrast effects [ 1]. Their investigation led to the conclusion that there were about 60 surface substructures with a diameter of 20 to 35 A which seemed to be evenly distributed on the surface of LDL particles. Our unstained observations were performed in an attempt to confirm this conclusion.
freeze-dried preparation; we had no success using airdried specimens. 4.2. Observation o f unstained LDL As a first step we obtained CTEM micrographs of negatively stained LDL. The micrographs were taken in a careful manner in which the defocus of the objective lens was kept at about - 1 0 0 0 A so that the theoretical phase contrast resolution was reduced to about 3 A without any reverse contrast problems [13]. Fig. 6 shows negatively stained LDL (with 1% sodium phosphotungstate, pH = 7.0) in which the presence of surface substructure is obvious. For comparison we then used the STEM to obtain images of unstained LDL with an electron dosage of 19 electrons/A 2. Fig. 7a shows an unstained particle in which the intensity of the central region is very high. Since we are observing the total elastic cross section of the specimen, we expect to get larger scattering from the thicker central region of the particle. Fig. 7b was obtained
INCIDENT BEAM
4.1. Specimen preservation In order to preserve the structure of the molecule, the freeze-dried method was employed, The particles were diluted with 5 mM NH4HCO3 (pH = 8.0) to 150/ag/ml just before being deposited on a 16 A thick (average) carbon f'tlm. The sample was washed a few times with 1 mM NH4HCO3 before freezing. It was frozen by inserting the grid into liquid nitrogen. After about 30 s the grid was removed from the nitrogen and placed on a precooled (liquid nitrogen temperature) stainless-steel block. This block was then inserted into a vacuum chamber for about 12 h to sublime the ice. This specimen conservation treatment is very important for the visualization of a three-dimensional unstained particle such as LDL and our success in imaging only occurred with the
Fig. 5. Schematic diagram showing the electron probe penet_rating a molecule of thickness TM. We obtain ti X = ,~TM + 60 where ~ is the convergence half angle, 60 is the probe size at infocus and 5 X is the probe size at the surface of both sides of the molecule assuming the beam was focused at the center of the molecule. When 6 o = 2.5 A, t~ = 15 mrad, and TM = 216 A are given, we will obtain ~X = 5.7 A.
M. Oh tsuki / Observation o f unstained biological macromolecules with STEM
321
from the same negative shown in 7a, but printed in such a manner that the central region was exposed more in order to bring up information from that area. The micrograph 7b exhibits clear substructures o f about 20 to 35 A in the central region which have a very similar appearance and dimensions to that o f the surface substructure of the negatively stained particle shown in fig. 6. In addition, the y - m o d u l a t i o n (pseudo three-dimensional) technique was applied for observation of a single unstained molecule as seen in fig. 7d. The thickness variations can be observed. Fig. 6. Bright field CTEM micrograph of LDL stained with 1% sodium phosphotungstate, pH = 7.0. The presence of surface substrates is obvious. The sample was kindly provided by Dr. A.M. Scanu of The University of Chicago.
5. Discussion In the case o f negatively stained biological particles, a substructure can clearly be seen when both
f
d Fig. 7. Dark field STEM micrographs of unstained LDL molecule taken with the annular detector (elastic) signal: (a) and (b) were printed from the same negative in which (a) was printed with even exposure..The exposure for the central region was increased to obtain the fine details of the substructure which show similarity compared with the stained particle shown in fig. 6; (c) shows another LDL particle which was printed the same way as (b); (d) the y-modulation (pseudo three-dimensional modulation) of a single unstained molecule of LDL shows the thickness variation.
322
M. Ohtsuki / Observation of unstained biological macromolecules with STEM
sides of the surface are in register and are superimposed in the direction of the incident beam. For an unstained molecule using the STEM annular detector signal, the contrast is proportional to the total elastic cross section of the molecule. The STEM micrograph of an unstained LDL particle shown in fig. 7 indicates that the shape of the substructures is rather uniform, particularly in the central region of the particle. This may suggest the superposition of similar surface information from both sides of the particle. Also the appearance of the substructure is very similar to that of the substructure of'negatively stained LDL shown in fig. 6. These results may indicate that the structural details shown in the unstained particle are surface substructure and the particle presumably contains an amorphous region in the center of the molecule. Shen et al. [15] proposed a model based on their theoretical calculation. This model for various lipoproteins consists of a hydrophobic sphere forming an amorphous core of about 76 A in diameter which is made of triglycerol and cholesterol ester and the core is surrounded by the outer layer composed of free cholesterol, phospholipid and protein. In addition, Ohtsuki et al. [16] have experimentally confirmed the presence of the core and surface substructure in the HDL3 particle using complementary observation techniques of freeze,etching, negative staining, STEM and CTEM..All lipoproteins including HDLa and LDL presumably have similar structures [ 15]. As noted before, since the LDL particle scatters a substantial amount of elastic signal, we can expect to see very small differences of contrast within the molecule. Assuming we can detect 20% of the contrast difference (ACM), then we will be able to observe thickness differences (ATM) from one side of the molecule (assuming this is surface substructure), AT M = ACMTM/2.For LDL, TM = 216 ,~ when the incident beam passes through the center of the particle, we obtain ATM = 22 A. The thickness variation of 22 A or larger is well within the range of the model proposed by Shen et al. [15] in which the thickness of the outer layer surrounding the central core is about 70 A.
6. Conclusion In this experiment, we used an electron dosage of 19 electrons/A 2. This is higher than the limit given by
Unwin and Henderson [2] for the observation of unstained biological specimens. They suggested.0.5 electrons/A 2 is the upper limit for observation of unstained particles without incurring some damage to the molecule. Nevertheless, our comparison of a micro graph from a negatively stained preparation with the micrograph of the unstained molecule, taken with the electron dose of 19 electrons/h 2, shows very similar structural appearance. This indicates that we can obtain a micrograp.h of an unstained single biological macromolecule using the STEM at a comparable resolution to that obtained with a uranyl acetate stained particle using the CTEM.
Acknowledgements The author wishes to express appreciation to Dr. A.V. Crewe for his helpful comments and discussions and use of his STEM facility at the University of Chicago. The author is grateful to Dr. A.M. Scanu of The University of Chicago for providing low density lipoprotein, Drs. R. Haselkorn and J. Orr of The University of Chiacgo for providing the glutamine synthetase and Drs. S.N. Vinogradov and O.H. Kapp of Wayne State University for providing hemoglobin. The author also thanks M. Slauson and A. Johns for their assistance in photography. This work was supported by grants from the Biotechnology Resources Brance of the National Institute of Health, and the United States Department of Energy.
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M. Ohtsuki / Observation of unstained biological macromolecules with STEM [6] M. Ohtsuki, in: Proc. 37th Electron Microscopy Society of America Meeting, San Antonio, TX, 1979, Ed. G.W. Bailey (Claitor's, Baton Rouge, LA, 1979) p. 130. [7] S.N. Vinogradov, J.M. Shlom, B.C. Hall, O.H. Kapp and H. Mizukami, Biochimica et Biophysica Acta 432 (1977) 136. [8] M. Ohtsuki and A.V. Crewe, Proc. Natl. Acad. Sci. USA 77 (1980). [9] M. Ohtsuki, M.S. lsaacson and A.V. Crewe, in: Scanning Electron Microscopy/1979, Ed. O. Johari (SEM, Inc., 1979) Vol. 2,p. 375. [10] A.V. Nichols, Proc. Nail. Acad. Sci. USA 64 (1969) 1128.
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[ 11] H. Pollard, A.M. Scanu and E.W. Taylor, in: Proc. Natl. Acad. Sci. USA 64 (1969) 304. [12] T. Forte and A.N. Nichols, Advan. Lipid Res. 10 (1972) 1. [131 T. Forte and R. Nordhausen, Circulation Suppl. II 54 (1976) 94. [141 D.G. Deutsch and M. Sogard, Micro 10 (1979) 25. [15] B.W. Shen, A.M. Scanu and F.J. Kezdy, Proc. Natl Acad. Sci. USA 74 (1977) 837. [16] M. Ohtsuki, C. Edelstein, M. Sogard and A.M. Scanu, Proc. Natl. Acad. Sci. USA 74 0977) 5001.