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Use of STM for analysis of surfaces of biological samples
Applied Surface Science 144–145 Ž1999. 146–150
Use of STM for analysis of surfaces of biological samples N.K. Permjakov, M.A. Ananyan, P.N. Luskinovich ) , V.I. Sorokovoi, S.V. Saveliev Institute of Human Morphology of the Russian Academy of Medical Sciences, Tsurupi st., 3, Moscow 117418, Russian Federation ICF Institute for Nanotechnologies, B. Tatarskaya, 38, Moscow 113184, Russian Federation
Abstract Scanning tunnelling microscopy ŽSTM. was used to image the cell surfaces of the olfactory organ of the shark Carcharhinus longimanus and ectoderm of the frog Xenopus laeÕis blastulae of 1024 stages, as well as human low-density lipoproteins surface. The samples from two of these objects were prepared by using traditional techniques for scanning electron microscopy ŽSEM.. The lipoprotein samples were prepared by drying in the air. A comparison of the STM images with the earlier obtained SEM images indicates that there are some earlier unknown details of the surface structures of receptor microvilli and support cell membranes of the olfactory organ of the shark. There was found a fold of membrane on the surface of the ectodermal frog embryo cells, which covered yolk granules. STM images of the lipoprotein surface were obtained without increasing conductivity treatment. q 1999 Elsevier Science B.V. All rights reserved. PACS: 87.64.D; 02.40.S Keywords: STM; Cell; Microwilli; Lipoprotein
1. Introduction In the present work we used the method of scanning tunnelling microscopy ŽSTM. as a new instrumental stage in morphology for the investigation of the cell-surface structures of shark olfactory organ and the frog Xenopus laeÕis blastulae of 1024 stages as well as human low-density lipoproteins. Detailed studies of the first object are necessary for the development of our knowledge of the structure of receptoral cells in general and behaviour of a shark in particular w1,2x. Fine investigations of a frog em-
)
Corresponding author. ICF Institute for Nanotechnologies, B. Tatarskaya, 38, Moscow 113184, Russian Federation. Tel.: q 7-95-953-53-94; F ax: q 7-95-953-53-82; E -m ail: [email protected]
bryo’s developing cells are permanently interesting for embryologists w3x. The results of the study of the surface structure of human lipoproteins may be used for clinical diagnosis in the near future.
2. Materials and methods The samples of shark olfactory organ and frog blastulae were fixed with 2% glutaraldehyde, dehydrated throughout ethanol series, dried in critical point, gold-coated and observed in the open using gold-coated glass as a support. Low-density lipoproteins with normal concentration of triglycerides Žless than 200 mgrdl. were isolated from human serum by ultracentrifugation w4x, then dialysed against 20 mM NH 4 CO 3 , pH 8.0, placed on nickel-coated glass, air-dried and observed in the open. The scanning
0169-4332r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 4 3 3 2 Ž 9 8 . 0 0 7 9 1 - 0
N.K. PermjakoÕ et al.r Applied Surface Science 144–145 (1999) 146–150
147
Fig. 1. Investigation of biological samples by the scanning tunnelling microscope. ŽA. A surface of chemoreceptive areas of the olfactory organ of the shark Carcharhinus longimanus. ŽB. Outlet of differentiating receptive cell to the surface of olfactory organ. ŽC. Lateral surface of support cell microvilli. ŽD. Apical surface of support cell microvilli. ŽE. Lateral surface of receptive cell microvilli. ŽF. Apical surface of receptive cell microvilli. ŽG. Ectodermal cell surface of the frog Xenopus laeÕis animal pole blastulae of 1024 stages. ŽH. Ectodermal cell membrane fold on the border of two yolk granules. ŽI, J. The borders of ectodermal cell. ŽK, L. A surface of human low-density lipoproteins.
148
N.K. PermjakoÕ et al.r Applied Surface Science 144–145 (1999) 146–150
Fig. 1 Žcontinued..
tunnelling microscope used in our work was manufactured by ICF Institute for Nanotechnologies, Moscow, Russia and gave a possibility of wide-range
scanning in three dimensions 30 mkm = 30 mkm = 30 mkm and quick-action scanning in three dimensions 160 nm = 240 nm = 700 nm.
N.K. PermjakoÕ et al.r Applied Surface Science 144–145 (1999) 146–150
Fig. 1 Žcontinued..
149
150
N.K. PermjakoÕ et al.r Applied Surface Science 144–145 (1999) 146–150
3. Results Investigation of shark olfactory organ cells proved to be highly difficult as chemoreceptive and support cells have very complex-shaped surfaces for STM w2x. However, we discovered as a result of STM studies of chemoreceptive areas of olfactory epithelial cells some details which could not be observed using scanning electron microscopy ŽSEM. in a previous study w2x. Using large scans Žlower magnification. we obtained finely resolved receptive cell borders ŽFig. 1A. as well as outlets of differentiated receptive cells ŽFig. 1B.. Using small scans Žhigher magnification. it was possible to reveal structural inhomogeneity of lateral and apical surfaces of support cells ŽFig. 1C, D. as well as receptive cell microvilli ŽFig. 1E, F.. The STM study of the animal pole of frog blastulae cell surfaces also revealed earlier unknown details. On the surface of ectodermal cells we found a membrane fold which covered yolk granules. This microfold of the cell surface had the size of 20–50 nm in diameter and about 100 nm in length ŽFig. 1G, H. and was absent on the border of cell yolk granules ŽFig. 1I, J.. Finally we investigated human low-density lipoproteins which had the size of about 20 nm ŽFig. 1K, L. without increasing conductivity treatment. However, in our case these particles were obtained mostly as flattened ones evidently as a consequence of surface stretch force action. When this difficulty is
overcome then STM would have a good chance to be used in clinical investigation of lipoproteins.
4. Summary Our results showed that STM is capable of giving images of biological samples with higher resolution in comparison to traditional SEM methods and that STM requires simpler probe treatment.
Acknowledgements The authors are thankful to A.D. Dergunov, PhD, for the human lipoproteins preparations. We are also grateful to O.A. Obyedkov, A.N. Kosyakov, A.V. Kotenkov and V.N. Yakovlev for STM apparatus creation.
References w1x E. Zeiske, B. Theisen, S. Gruber, Can. J. Zool. 65 Ž1987. 2406. w2x S.V. Saveliev, V.P. Chernikov, Vopr. Ichthyology 34 Ž1994. 219. w3x V. Balinski, Introduction to Embryology, Saunders, Amsterdam, 1989. w4x R.J. Havel, H.A. Eder, J.H. Bragdon, J. Clin. Invest. 34 Ž1955. 1345.
Use of STM for analysis of surfaces of biological samples N.K. Permjakov, M.A. Ananyan, P.N. Luskinovich ) , V.I. Sorokovoi, S.V. Saveliev Institute of Human Morphology of the Russian Academy of Medical Sciences, Tsurupi st., 3, Moscow 117418, Russian Federation ICF Institute for Nanotechnologies, B. Tatarskaya, 38, Moscow 113184, Russian Federation
Abstract Scanning tunnelling microscopy ŽSTM. was used to image the cell surfaces of the olfactory organ of the shark Carcharhinus longimanus and ectoderm of the frog Xenopus laeÕis blastulae of 1024 stages, as well as human low-density lipoproteins surface. The samples from two of these objects were prepared by using traditional techniques for scanning electron microscopy ŽSEM.. The lipoprotein samples were prepared by drying in the air. A comparison of the STM images with the earlier obtained SEM images indicates that there are some earlier unknown details of the surface structures of receptor microvilli and support cell membranes of the olfactory organ of the shark. There was found a fold of membrane on the surface of the ectodermal frog embryo cells, which covered yolk granules. STM images of the lipoprotein surface were obtained without increasing conductivity treatment. q 1999 Elsevier Science B.V. All rights reserved. PACS: 87.64.D; 02.40.S Keywords: STM; Cell; Microwilli; Lipoprotein
1. Introduction In the present work we used the method of scanning tunnelling microscopy ŽSTM. as a new instrumental stage in morphology for the investigation of the cell-surface structures of shark olfactory organ and the frog Xenopus laeÕis blastulae of 1024 stages as well as human low-density lipoproteins. Detailed studies of the first object are necessary for the development of our knowledge of the structure of receptoral cells in general and behaviour of a shark in particular w1,2x. Fine investigations of a frog em-
)
Corresponding author. ICF Institute for Nanotechnologies, B. Tatarskaya, 38, Moscow 113184, Russian Federation. Tel.: q 7-95-953-53-94; F ax: q 7-95-953-53-82; E -m ail: [email protected]
bryo’s developing cells are permanently interesting for embryologists w3x. The results of the study of the surface structure of human lipoproteins may be used for clinical diagnosis in the near future.
2. Materials and methods The samples of shark olfactory organ and frog blastulae were fixed with 2% glutaraldehyde, dehydrated throughout ethanol series, dried in critical point, gold-coated and observed in the open using gold-coated glass as a support. Low-density lipoproteins with normal concentration of triglycerides Žless than 200 mgrdl. were isolated from human serum by ultracentrifugation w4x, then dialysed against 20 mM NH 4 CO 3 , pH 8.0, placed on nickel-coated glass, air-dried and observed in the open. The scanning
0169-4332r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 4 3 3 2 Ž 9 8 . 0 0 7 9 1 - 0
N.K. PermjakoÕ et al.r Applied Surface Science 144–145 (1999) 146–150
147
Fig. 1. Investigation of biological samples by the scanning tunnelling microscope. ŽA. A surface of chemoreceptive areas of the olfactory organ of the shark Carcharhinus longimanus. ŽB. Outlet of differentiating receptive cell to the surface of olfactory organ. ŽC. Lateral surface of support cell microvilli. ŽD. Apical surface of support cell microvilli. ŽE. Lateral surface of receptive cell microvilli. ŽF. Apical surface of receptive cell microvilli. ŽG. Ectodermal cell surface of the frog Xenopus laeÕis animal pole blastulae of 1024 stages. ŽH. Ectodermal cell membrane fold on the border of two yolk granules. ŽI, J. The borders of ectodermal cell. ŽK, L. A surface of human low-density lipoproteins.
148
N.K. PermjakoÕ et al.r Applied Surface Science 144–145 (1999) 146–150
Fig. 1 Žcontinued..
tunnelling microscope used in our work was manufactured by ICF Institute for Nanotechnologies, Moscow, Russia and gave a possibility of wide-range
scanning in three dimensions 30 mkm = 30 mkm = 30 mkm and quick-action scanning in three dimensions 160 nm = 240 nm = 700 nm.
N.K. PermjakoÕ et al.r Applied Surface Science 144–145 (1999) 146–150
Fig. 1 Žcontinued..
149
150
N.K. PermjakoÕ et al.r Applied Surface Science 144–145 (1999) 146–150
3. Results Investigation of shark olfactory organ cells proved to be highly difficult as chemoreceptive and support cells have very complex-shaped surfaces for STM w2x. However, we discovered as a result of STM studies of chemoreceptive areas of olfactory epithelial cells some details which could not be observed using scanning electron microscopy ŽSEM. in a previous study w2x. Using large scans Žlower magnification. we obtained finely resolved receptive cell borders ŽFig. 1A. as well as outlets of differentiated receptive cells ŽFig. 1B.. Using small scans Žhigher magnification. it was possible to reveal structural inhomogeneity of lateral and apical surfaces of support cells ŽFig. 1C, D. as well as receptive cell microvilli ŽFig. 1E, F.. The STM study of the animal pole of frog blastulae cell surfaces also revealed earlier unknown details. On the surface of ectodermal cells we found a membrane fold which covered yolk granules. This microfold of the cell surface had the size of 20–50 nm in diameter and about 100 nm in length ŽFig. 1G, H. and was absent on the border of cell yolk granules ŽFig. 1I, J.. Finally we investigated human low-density lipoproteins which had the size of about 20 nm ŽFig. 1K, L. without increasing conductivity treatment. However, in our case these particles were obtained mostly as flattened ones evidently as a consequence of surface stretch force action. When this difficulty is
overcome then STM would have a good chance to be used in clinical investigation of lipoproteins.
4. Summary Our results showed that STM is capable of giving images of biological samples with higher resolution in comparison to traditional SEM methods and that STM requires simpler probe treatment.
Acknowledgements The authors are thankful to A.D. Dergunov, PhD, for the human lipoproteins preparations. We are also grateful to O.A. Obyedkov, A.N. Kosyakov, A.V. Kotenkov and V.N. Yakovlev for STM apparatus creation.
References w1x E. Zeiske, B. Theisen, S. Gruber, Can. J. Zool. 65 Ž1987. 2406. w2x S.V. Saveliev, V.P. Chernikov, Vopr. Ichthyology 34 Ž1994. 219. w3x V. Balinski, Introduction to Embryology, Saunders, Amsterdam, 1989. w4x R.J. Havel, H.A. Eder, J.H. Bragdon, J. Clin. Invest. 34 Ž1955. 1345.