Ultramicrt,scopy 45 (1992) 199-2113 North-t Iolland
Scanning tunneling microscopy of an ionic crystal: ferritin core Jic Yang, K u n i o Takcyasu, A n d r c w P. Somlyo and Z h i f c n g Shao * Bio-SI'M l.ahoratot3'. Department of t'hysioh~gy, Uml'ersity of [/il;l~inia. Box 449, Chark, tte.~'t'ilh,. VA 2290& USA Received 23 April 1~,~92
Ferrilin molecules wcrc imaged directly in air by scanning lunneling microscopy (STM). The lateral dimensions were close to the values determined by electron microscopy, and the vertical dimension was much reduced. Several clusters of partially naked ferritin cores displayed a hexagonal structure of lattice constant 4.9 ± 0.5 A. It is thus shown that the STM can be used to image thin ionic cl.'ystals ill high resolution.
1. Introduction
Many biological specimens, ranging from organic compounds [I-4] and DNA [5-10] to large molecules like glycogen, phosphorylasc, microtubules [11-13], and polyglucosc [14] have bccn imaged by scanning tunneling microscopy (STM) in air, undcl oil or water at various resolutions, although the underlying contrast mechanisms arc not well understood [2,3,15-19]. In this report, we present results of direct STM imaging of ferritin molecules adsorbed on thc graphite surface. Fcrritin plays an important role in iron metabolism with a size ranging between 12 and 16 nm [20-26], depending on the molecular orientation [24-26], and it has a crystalline iron corc (fcrrihydratc) of up to 7 nm containing 40005(100 irons [27-33]. Our results show that thc STM not only can image the intact whole molecules on a conducting substrate, but also can reveal atomic structure of the iron-containing core when the protein shell is absent.
[14]. Suitable concentrations for STM studies were determined by electron microscopy. A solution of 2 m g / m l ferritin from Sigma Chemicals, St. Louis, (12.8mM NaCi), dried on freshly cleaved highly oricntcd pyrolytic graphite (HOPG), was studied by STM (Nanoscope 11, Digit.al Instruments inc., Santa Barhara, CA), at ambient conditions. As a control, buffer solution alone was also applied to HOPG and dried in air, and observed in STM, and did not show any features similar to either fcrritin ~r its core in structure. 3. Results and discussion
At low resolution, many individual particles as well as small clusters were readily observed and occasionally a large cluster was broken apart by the scanning tip. Fig. I shows four typical ferritin molecules observed by STM. Large deformation in shape, maybe due to the effect of non-uniform adhesion to the HOPG surface, and dragging by the tunneling tip, gives the molecules a somehow I I 1 ~..~.,.~U It.Oil
2. Material and methods The experimental procedure and instrumentation wcrc similar to those previously described :+ To wh~ml correspondence shot, ld bc addressed.
KI[,.J'~.,t,A,I
¢dl, i I K . ~ ,
~"l~k# t i l l , . ,
il|~./l%,~Ulltlt,
Itlt
It~,¢ I l l ~ l i
tion cannot be directly determined from these observations. Thc hcigM dctcrmincd by STM is only 3 - 4 nm, much smaller than expected. The lateral dimension determined from high resolution STM images of wholc molcculcs (total of 21 ) is in thc range of 13-19 nm. The reduced height and c,largcd lateral dirncnsion may be a result of
(131)4-3991/'t.~2/$(15.(t() ~,~, 1992 -Elscvicr Science Publishers B.¥. All righls reserved
2011
J. Yang et aL / Sc'am:ing tunneling micros~'opy of an ioni¢" cO'stal: ferritin core
the drying process (surface interaction) a n d / o r thc spccimcn being squcczcd by the STM tip [15]. The reduced hcight in STM images of large macromolecules has been observed [0,11], but its mechanism is not understood. Ferritin molecules often lose their protein shells during specimen preparation [34], and the STM tip can also peel off the top portion of a molecule accidentally (occasionally observable in experiments), if the scanning speed is too fast or the current is too high. In our STM study, several
partially bare core clusters were also found among intact ferritin molecules. Each core can be recognized with boundaries with a size between 5 and 9 nm. Surprisingly, high resolution STM imaging of these core clusters revealed atomic resolution images of the core structure, as shown in fig. 2. At the upper right corner, the boundary of the core is cicarly recognizable by the sharp contrast between the regularly arranged atomic structure (indicating the ferritin c~re) and the irregular structure of the protein shell. This image was very
Fig. I. STM images of four t~pical ferrilin molecules oht;lined :~! ;,, hia,~ voltage eft N(~ m V and tunneling current of 1).5 nA. Image size for c;tch is 31) nm 3~3() nm, The l;iter;d tlimen~i¢}n i.~ close t(~ the v;due determined by "[EM "~itli ;ippareaii def,.rmati(m. The height i:, hel~seen 3 ;ind 4 nm.
J. Yang et al. / Scanning tunneling microscopy o f an ion,' cO'stul: li'rritin con'
stable over repeated scans at various speeds, and scaled accordingly when the image size was changed. By moving the scanning lip a short distance away from the core cluster, the graphite surface structure was also clearly visualized at atomic resolution, which showed a different symmetry axis orientation and different lattice constant from that of the ferritin core. Furthermore, two ferritin cores imaged in close proximity have the same lattice stntcture, but different symmetry. axis orientations. These differences in symmetry. orientations eliminate any possible artifact due to graphite surface structure. The hexagonal lattice constant, determined from nearest neighbor interdistances of two cores, is a = 4.9 + (I.5 A (S,D.), consistent with the Towe-Bradley model [27-,~, ]. The alternative iron-dextran model of the ferritin core [35-38], with hexagonal lattices of 3.3 ,~ for iron and 1.9 /k for O ' - / O H - , has no resemblance to the structure observed in this study.
2()1
/
.
o: %o
"N
o .
o "
n IOl I'1
_v;,; • A + "*t
J
Fig. 3. The lattice .-,lrudure o[ the ['owe-Bradley model when pmjecled on the basa I ane. "[he rhomboids represent the projection ¢~f ( ) : /I:e ~' complex, q'he symbols and positions of It ,(), ()~' , and t:e ~' are shm,~n in Ihe chart at lower right cnrner. W h e r e lhere is fllOl-e th[lll Olle alom in the SilllI¢ column, the symbols are simply ¢~verlaid. The exacl position ¢;I' Fe ~' is between the adjacent layers of ()" / ! t , O (e.g. (ot represents Fe ~' belween Is| and 2nd layers of ()~ / t ! ~ O , see ref. [21]), Nolice the simihtrily between the rhomboid lattice of 5.1 A aml the observed STM image {fig. 5). Belween the 4th and 5th (lsl) layers of ( ) : / ! t , O , the occupancy nf Fe ~* is oplional, wilh additiona! II ' to balance Ihe charge of the unit cell. Only tree possible arrangemen! ol Fe ~' in lifts layer is given here. Even wilh this ambiguity, the structure ~:lld charge sequence (as discus.~cd in Ihe text) between the rhornhoidal volume and that conlaining water are strikingly differcnl,
Fig, 2. A typical STM image ¢~b!ai.n.ed from ": partially shed fcrritin molecule (unfillered), which shows the atomic details of a ferritin core. The boundary at the upper right corner is clearly seen. The bias voltage is 174 mY, and the tunneling current is 0.49 hA, Scanning area: 5 nm x 5 nm. The hexagonal lattice of 4.9 + 0.5 .~ is clearly resolved. At¢~mic resolution graphite surface structure was also observed outside the boundary wi|h a different lattice axis orientation which indicated that the observed core structure was not graphite artilaets.
Earlier .studies of ferritin core by transmission dectron microscopy (TEM) and scanning transmission electron microscopy (STEM)have shown that the core has a hexagonal structure ,vith a lattice constant of 5.1 A in the basal plane [29,31)], where the image basically is a projection along the electron beam direction. Since the heavy element (Fe) contributes the most to contrast formation in electron microscopy, the structure determined by TEM and STEM should correspond primarily to Fe atoms. Therefore, the similarity between S T E M / T E M and STM images would seem to suggest that the lattice structure observed by STM is mainly due to the Fe ~+ ions inside the ferritin core. However, a close e:;amination of the Towe-Bradley model [27,28] shows a° hexagonal packing lattice structure with a = 5.1 A, c = 9.4 A, and a unit cell containing 4 layers, in which ome oxygen ions also arrange themselves in the same hexagonal structure on the basal plane projection. As shown in fig. 3, the
.li_
~
J. fang et al. / Scanning tunneling microscopy. of an ionic crystal: [erritin core -
c-axis with a period of 9.4 A. An alternative possibility, that the observed contrast is cntircly duc to the surfacc laycr of thc corc, is unlikely, bccausc the four laycrs within the unit cell have differcnt planar structurcs, and would not yield idcntical structurcs of diffcrent molccules, unlcss the ferritin core ha:; a unique surface structure.
4. Conclusion
Fig. 4. Filtered STM image of the core lattice. The average size of the site measured is !.5 ,,% ( _+11.2,4,) x 3.11 ,,% ( + 11.3 A), not sufficient to reveal whether O z a n d / o r Fc ~+ is responsible fl~r the observed hittite. Image size is 1.8 nm x 1.8 nm.
projcction of thc 0 2 / F c 3+ complcx rcprescntcd by the rhomboid also forms a hcxagonal lattice, but water molecules, occupying two layers (1 and 4) within the unit ccll. have a projection structure incompatible with the observed STM image. Fig. 4 shows tile filtered STM image of the core lattice. Since the O: and Fc 3' ;Arc in close proximity on the projection, and tile whole group terms a hcxagonal lattice of 5.1 A, the obscrvcd lattice by STM can a l s o be attributcd to the oxygen ions, or the O 2 / F c 3+ complex within the core. Unfortunately. the resolution wc were able to obtain was not sufficient to resolve this finc detail. It may bc noted that. within the volume of the rhoraboid, there is an atomic sequence of ()~" - F c 3 + - O 2 -(2Fc 3+)-O: Fc 3+(): - F c 3 +. along the c-axis, corresponding to a charge sequence of 2e. + qe, 2e, + 6e. "~ +3c, -'~_e. +3e, hut for the columns containing |t :(). the se~!uencc is composed of | | ~0 and O : only, which is lotally differcnl from lilt2 olle ;.lb~we. Wc can spc~:ulate thait this structural difference nazi,,' be the '~asis of t h e obser-ved contrasl: I~ tunneling electrons, the polen!ial within the rhomboid (corresponding to the obscrvcd hittite sites) presenls a sawlooth structure along the o
We demonstrate that ferritin molecules can be imagcd dircctly in air by STM. Although the lateral dimcnsion dctcrmined is ciosc to known values, the vertic::! dimension is much reduced. Thc oxygcn/iron sitc hcxagonal latticc of partially shcd fcrritin corcs was also imagcd, with a lattice constant of 4.9 + 0.5 A, consistent with the Towc-Bradlcy modcl. A theoretical understanding of this high rcsolution STM imagc of nonconducting ionic crystal will be important, and may providc a rational basis for ncw applications of STM to other non-conducting materials. o
Acknowledgements J.Y. would like to thank l)rs. W.H. Massover and K.M. Towe for useful discussion. We wish to thank Dr. A.W. Moore of Union Carbide for a generous gift of HOPG. This work is supported by grants from the Whi!aker Foundation for Biomedical Engineering Research (Z.S.), Digital Instruments, inc. (Z.S. and A.P.S,), US Army Research Office granl DAAL03-92-G-002 (J.Y., Z.S. and K,'l'.), NIIt grants t1L15835 to the Pennsylvania Muscle Institute (A.P,S.) and GM 44373 (K.T,).
References I l l ~i.C'. Mc(ionigal. R.II. B c l n h a l d l and I).]. 'lhonl.,,on. Appl. Phys. l+cll. 57 (ItJt,tll) 2N. i_~l .t.i
Scanning tunneling microscopy of an ionic crystal: ferritin core Jic Yang, K u n i o Takcyasu, A n d r c w P. Somlyo and Z h i f c n g Shao * Bio-SI'M l.ahoratot3'. Department of t'hysioh~gy, Uml'ersity of [/il;l~inia. Box 449, Chark, tte.~'t'ilh,. VA 2290& USA Received 23 April 1~,~92
Ferrilin molecules wcrc imaged directly in air by scanning lunneling microscopy (STM). The lateral dimensions were close to the values determined by electron microscopy, and the vertical dimension was much reduced. Several clusters of partially naked ferritin cores displayed a hexagonal structure of lattice constant 4.9 ± 0.5 A. It is thus shown that the STM can be used to image thin ionic cl.'ystals ill high resolution.
1. Introduction
Many biological specimens, ranging from organic compounds [I-4] and DNA [5-10] to large molecules like glycogen, phosphorylasc, microtubules [11-13], and polyglucosc [14] have bccn imaged by scanning tunneling microscopy (STM) in air, undcl oil or water at various resolutions, although the underlying contrast mechanisms arc not well understood [2,3,15-19]. In this report, we present results of direct STM imaging of ferritin molecules adsorbed on thc graphite surface. Fcrritin plays an important role in iron metabolism with a size ranging between 12 and 16 nm [20-26], depending on the molecular orientation [24-26], and it has a crystalline iron corc (fcrrihydratc) of up to 7 nm containing 40005(100 irons [27-33]. Our results show that thc STM not only can image the intact whole molecules on a conducting substrate, but also can reveal atomic structure of the iron-containing core when the protein shell is absent.
[14]. Suitable concentrations for STM studies were determined by electron microscopy. A solution of 2 m g / m l ferritin from Sigma Chemicals, St. Louis, (12.8mM NaCi), dried on freshly cleaved highly oricntcd pyrolytic graphite (HOPG), was studied by STM (Nanoscope 11, Digit.al Instruments inc., Santa Barhara, CA), at ambient conditions. As a control, buffer solution alone was also applied to HOPG and dried in air, and observed in STM, and did not show any features similar to either fcrritin ~r its core in structure. 3. Results and discussion
At low resolution, many individual particles as well as small clusters were readily observed and occasionally a large cluster was broken apart by the scanning tip. Fig. I shows four typical ferritin molecules observed by STM. Large deformation in shape, maybe due to the effect of non-uniform adhesion to the HOPG surface, and dragging by the tunneling tip, gives the molecules a somehow I I 1 ~..~.,.~U It.Oil
2. Material and methods The experimental procedure and instrumentation wcrc similar to those previously described :+ To wh~ml correspondence shot, ld bc addressed.
KI[,.J'~.,t,A,I
¢dl, i I K . ~ ,
~"l~k# t i l l , . ,
il|~./l%,~Ulltlt,
Itlt
It~,¢ I l l ~ l i
tion cannot be directly determined from these observations. Thc hcigM dctcrmincd by STM is only 3 - 4 nm, much smaller than expected. The lateral dimension determined from high resolution STM images of wholc molcculcs (total of 21 ) is in thc range of 13-19 nm. The reduced height and c,largcd lateral dirncnsion may be a result of
(131)4-3991/'t.~2/$(15.(t() ~,~, 1992 -Elscvicr Science Publishers B.¥. All righls reserved
2011
J. Yang et aL / Sc'am:ing tunneling micros~'opy of an ioni¢" cO'stal: ferritin core
the drying process (surface interaction) a n d / o r thc spccimcn being squcczcd by the STM tip [15]. The reduced hcight in STM images of large macromolecules has been observed [0,11], but its mechanism is not understood. Ferritin molecules often lose their protein shells during specimen preparation [34], and the STM tip can also peel off the top portion of a molecule accidentally (occasionally observable in experiments), if the scanning speed is too fast or the current is too high. In our STM study, several
partially bare core clusters were also found among intact ferritin molecules. Each core can be recognized with boundaries with a size between 5 and 9 nm. Surprisingly, high resolution STM imaging of these core clusters revealed atomic resolution images of the core structure, as shown in fig. 2. At the upper right corner, the boundary of the core is cicarly recognizable by the sharp contrast between the regularly arranged atomic structure (indicating the ferritin c~re) and the irregular structure of the protein shell. This image was very
Fig. I. STM images of four t~pical ferrilin molecules oht;lined :~! ;,, hia,~ voltage eft N(~ m V and tunneling current of 1).5 nA. Image size for c;tch is 31) nm 3~3() nm, The l;iter;d tlimen~i¢}n i.~ close t(~ the v;due determined by "[EM "~itli ;ippareaii def,.rmati(m. The height i:, hel~seen 3 ;ind 4 nm.
J. Yang et al. / Scanning tunneling microscopy o f an ion,' cO'stul: li'rritin con'
stable over repeated scans at various speeds, and scaled accordingly when the image size was changed. By moving the scanning lip a short distance away from the core cluster, the graphite surface structure was also clearly visualized at atomic resolution, which showed a different symmetry axis orientation and different lattice constant from that of the ferritin core. Furthermore, two ferritin cores imaged in close proximity have the same lattice stntcture, but different symmetry. axis orientations. These differences in symmetry. orientations eliminate any possible artifact due to graphite surface structure. The hexagonal lattice constant, determined from nearest neighbor interdistances of two cores, is a = 4.9 + (I.5 A (S,D.), consistent with the Towe-Bradley model [27-,~, ]. The alternative iron-dextran model of the ferritin core [35-38], with hexagonal lattices of 3.3 ,~ for iron and 1.9 /k for O ' - / O H - , has no resemblance to the structure observed in this study.
2()1
/
.
o: %o
"N
o .
o "
n IOl I'1
_v;,; • A + "*t
J
Fig. 3. The lattice .-,lrudure o[ the ['owe-Bradley model when pmjecled on the basa I ane. "[he rhomboids represent the projection ¢~f ( ) : /I:e ~' complex, q'he symbols and positions of It ,(), ()~' , and t:e ~' are shm,~n in Ihe chart at lower right cnrner. W h e r e lhere is fllOl-e th[lll Olle alom in the SilllI¢ column, the symbols are simply ¢~verlaid. The exacl position ¢;I' Fe ~' is between the adjacent layers of ()" / ! t , O (e.g. (ot represents Fe ~' belween Is| and 2nd layers of ()~ / t ! ~ O , see ref. [21]), Nolice the simihtrily between the rhomboid lattice of 5.1 A aml the observed STM image {fig. 5). Belween the 4th and 5th (lsl) layers of ( ) : / ! t , O , the occupancy nf Fe ~* is oplional, wilh additiona! II ' to balance Ihe charge of the unit cell. Only tree possible arrangemen! ol Fe ~' in lifts layer is given here. Even wilh this ambiguity, the structure ~:lld charge sequence (as discus.~cd in Ihe text) between the rhornhoidal volume and that conlaining water are strikingly differcnl,
Fig, 2. A typical STM image ¢~b!ai.n.ed from ": partially shed fcrritin molecule (unfillered), which shows the atomic details of a ferritin core. The boundary at the upper right corner is clearly seen. The bias voltage is 174 mY, and the tunneling current is 0.49 hA, Scanning area: 5 nm x 5 nm. The hexagonal lattice of 4.9 + 0.5 .~ is clearly resolved. At¢~mic resolution graphite surface structure was also observed outside the boundary wi|h a different lattice axis orientation which indicated that the observed core structure was not graphite artilaets.
Earlier .studies of ferritin core by transmission dectron microscopy (TEM) and scanning transmission electron microscopy (STEM)have shown that the core has a hexagonal structure ,vith a lattice constant of 5.1 A in the basal plane [29,31)], where the image basically is a projection along the electron beam direction. Since the heavy element (Fe) contributes the most to contrast formation in electron microscopy, the structure determined by TEM and STEM should correspond primarily to Fe atoms. Therefore, the similarity between S T E M / T E M and STM images would seem to suggest that the lattice structure observed by STM is mainly due to the Fe ~+ ions inside the ferritin core. However, a close e:;amination of the Towe-Bradley model [27,28] shows a° hexagonal packing lattice structure with a = 5.1 A, c = 9.4 A, and a unit cell containing 4 layers, in which ome oxygen ions also arrange themselves in the same hexagonal structure on the basal plane projection. As shown in fig. 3, the
.li_
~
J. fang et al. / Scanning tunneling microscopy. of an ionic crystal: [erritin core -
c-axis with a period of 9.4 A. An alternative possibility, that the observed contrast is cntircly duc to the surfacc laycr of thc corc, is unlikely, bccausc the four laycrs within the unit cell have differcnt planar structurcs, and would not yield idcntical structurcs of diffcrent molccules, unlcss the ferritin core ha:; a unique surface structure.
4. Conclusion
Fig. 4. Filtered STM image of the core lattice. The average size of the site measured is !.5 ,,% ( _+11.2,4,) x 3.11 ,,% ( + 11.3 A), not sufficient to reveal whether O z a n d / o r Fc ~+ is responsible fl~r the observed hittite. Image size is 1.8 nm x 1.8 nm.
projcction of thc 0 2 / F c 3+ complcx rcprescntcd by the rhomboid also forms a hcxagonal lattice, but water molecules, occupying two layers (1 and 4) within the unit ccll. have a projection structure incompatible with the observed STM image. Fig. 4 shows tile filtered STM image of the core lattice. Since the O: and Fc 3' ;Arc in close proximity on the projection, and tile whole group terms a hcxagonal lattice of 5.1 A, the obscrvcd lattice by STM can a l s o be attributcd to the oxygen ions, or the O 2 / F c 3+ complex within the core. Unfortunately. the resolution wc were able to obtain was not sufficient to resolve this finc detail. It may bc noted that. within the volume of the rhoraboid, there is an atomic sequence of ()~" - F c 3 + - O 2 -(2Fc 3+)-O: Fc 3+(): - F c 3 +. along the c-axis, corresponding to a charge sequence of 2e. + qe, 2e, + 6e. "~ +3c, -'~_e. +3e, hut for the columns containing |t :(). the se~!uencc is composed of | | ~0 and O : only, which is lotally differcnl from lilt2 olle ;.lb~we. Wc can spc~:ulate thait this structural difference nazi,,' be the '~asis of t h e obser-ved contrasl: I~ tunneling electrons, the polen!ial within the rhomboid (corresponding to the obscrvcd hittite sites) presenls a sawlooth structure along the o
We demonstrate that ferritin molecules can be imagcd dircctly in air by STM. Although the lateral dimcnsion dctcrmined is ciosc to known values, the vertic::! dimension is much reduced. Thc oxygcn/iron sitc hcxagonal latticc of partially shcd fcrritin corcs was also imagcd, with a lattice constant of 4.9 + 0.5 A, consistent with the Towc-Bradlcy modcl. A theoretical understanding of this high rcsolution STM imagc of nonconducting ionic crystal will be important, and may providc a rational basis for ncw applications of STM to other non-conducting materials. o
Acknowledgements J.Y. would like to thank l)rs. W.H. Massover and K.M. Towe for useful discussion. We wish to thank Dr. A.W. Moore of Union Carbide for a generous gift of HOPG. This work is supported by grants from the Whi!aker Foundation for Biomedical Engineering Research (Z.S.), Digital Instruments, inc. (Z.S. and A.P.S,), US Army Research Office granl DAAL03-92-G-002 (J.Y., Z.S. and K,'l'.), NIIt grants t1L15835 to the Pennsylvania Muscle Institute (A.P,S.) and GM 44373 (K.T,).
References I l l ~i.C'. Mc(ionigal. R.II. B c l n h a l d l and I).]. 'lhonl.,,on. Appl. Phys. l+cll. 57 (ItJt,tll) 2N. i_~l .t.i
