- Email: [email protected]
Local elastic properties of cells studied by SFM
Applied Surface Science 141 Ž1999. 345–349
Local elastic properties of cells studied by SFM M. Lekka
a,)
, J. Lekki a , M. Marszałek a , P. Golonka a , Z. Stachura a , B. Cleff b, A.Z. Hrynkiewicz a
a
b
Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Cracow, Poland Institute of Nuclear Physics, Wilhelm-Klemm-Str. 9, UniÕersity of Munster, Munster, Germany ¨ ¨ Received 29 May 1998; accepted 12 August 1998
Abstract Scanning force microscopy ŽSFM. can be used in nano-indentation experiments for very soft samples of elastic, organic materials. The present paper describes the methodology of device calibration and Young’s moduli determination, to evaluate the elastic properties of living cells in their culture conditions. Two similar lines of normal cells ŽHu609. and cancerous ones ŽT24. were measured. A significant difference in Young’s modulus for normal and cancerous cells was detected. q 1999 Elsevier Science B.V. All rights reserved. PACS: 07.79; 87.45.y k; 87.80 q s Keywords: Scanning force microscopy ŽSFM.; Methodology; Elasticity; Normal cells; Cancerous cells
1. Introduction Scanning force microscopy ŽSFM. can be used in nano-indentation experiments for very soft samples of soft, organic materials w1,2x. Nevertheless, in order to obtain reliable results some preparation of the device is needed before starting the SFM measurements. The main points which should be taken into account are: the determination of the cantilever spring constant, control of the tip shape and curvature radius, and corrections of scanner voltage signals removing the scanner nonlinearity and hysteresis. The present paper describes the methodology of device calibration and Young’s moduli determina-
)
Corresponding author. Tel.: q48-12-6370222 ext. 271; Fax: q48-12-6371881; E-mail: [email protected]
tion, to evaluate the elastic properties of living cells in their natural growing conditions. Two similar lines of healthy, normal cells ŽHu609. and cancerous ones ŽT24. were measured. Determination of elastic properties of the normal and cancerous cells was applied to characterisation of changes due to oncogenic transformation.
2. Materials and methods Two similar cell lines were chosen for the SFM measurements: Hu609—non-malignant ureter Žnormal., and T24—bladder transitional cell carcinoma Žcancerous.. Cell cultures were grown at 378C in 95% airr5% CO 2 in an incubator ŽASSAB type.. They were grown in the RPMI 1640 medium ŽpH 7.4. containing 10% fetal calf serum ŽFCS. provid-
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 5 2 2 - 4
346
M. Lekka et al.r Applied Surface Science 141 (1999) 345–349
ing cells nutrition. The cells were taken after the same time of growing, and then they were cultured on a glass coverslip with poly-L-lysine as an adhesive factor, since the SFM measurements required that the cells be strongly attached to the substrate. Strong adhesion of such system is caused by the interaction of negatively charged cell membrane and positively charged NH 2 groups present in poly-Llysine. A home-built scanning force microscope was used in the experiment w3x. It was equipped with a scanner with a maximum XY scan range of 20 mm = 20 mm, and a Z range of "1.5 mm Ž1.25-inch piezotube was purchased at Staveley Sensor.. Commercial, sharpened Si 3 N4 cantilevers were used ŽPark Scientific Instruments, Geneva.. A plexiglass ‘liquid cell’ without the ‘o-ring’ sealing was used. Cell measurements were performed in the RPMI medium containing 10% FCS serum at room temperature. The coverslip with cells Žstill immersed in the RPMI medium. was put into the ‘liquid cell’ placed on an SFM scanner. The measured cell was localised using an optical microscope, and the tip was moved towards the cell centre in order to avoid or minimise the influence of the substrate and the neighbouring cells. Every culture was measured several times using a new tip and fresh medium. Data analysis requires very careful controlling of the SFM set-up properties and experimental conditions. The most important factors are the spring constant of the cantilever, its tip geometry and scan-
ner nonlinearity. The value of the spring constant is crucial for the estimation of the force applied to an investigated surface. Spring constants of commercial cantilevers are distributed around a nominal value, which introduces uncertainty of loading force value. In order to obtain the true force calibration, it is necessary to determine the real spring constant. In our microscope this task is an option of data acquisition software and is performed by the measurement of resonant frequency of a thermally excited cantilever at room temperature. Fig. 1 presents the resonant spectra obtained for two types of cantilevers, with spring constants k s 0.1 Nrm and k s 0.03 Nrm. These spectra were obtained by applying FFT ŽFast Fourier Transform. to an average of six noise spectra, each of 4096 points, collected with a frequency of about 80 kHz from quadrant photodiode output. The calculated resonant frequencies are lower than the nominal values: 11.8 kHz instead of 15 kHz and 27.7 kHz instead of 35 kHz, but these values were well reproducible for most of the cantilevers in a wafer. For example, for k s 0.03 Nrm the resonant frequency varied from 11.8 kHz to 12.2 kHz. The procedure of the spring constant determination was performed following the method described by Sader et al. w4x. Models describing the behaviour of the surface under load assume the knowledge of tip-surface geometry. Therefore the tip curvature radius was checked using a TGT01 standard ŽNMDT, Molecular Devices and Tools for Nanotechnology, Moscow.,
Fig. 1. Noise spectra of two types of cantilevers: Ža. spring constant k s 0.03 Nrm and Žb. k s 0.1 Nrm.
M. Lekka et al.r Applied Surface Science 141 (1999) 345–349
347
Fig. 2. Ža. SFM image of a scanning tip and Žb. its cross-section.
capable of estimating tip radii down to 10 nm. The standard consists of a set of very sharp needles, much sharper than the cantilever tip. The collected image of such a standard represents the scanning tip topography. The tips used in experiment were characterised by tip radii of about 20–30 nm ŽFig. 2.. The correction of the piezo scanner nonlinearity and hysteresis was performed using the interference method w5x, adapted to our SFM set-up. This method is exact, easy to perform and does not require additional equipment ŽFig. 3.. Force curves reflecting the interaction between the probing tip and the cell surface were collected
using the laser deflection technique. A separate force calibration using reference hard material Žglass. was performed for each sequence of curves taken in the same session both before and after the measurement. In the range of the applied force Žup to 20 nN. there is no significant glass deformation caused by the SFM tip, and therefore the glass sample was used as a calibration reference, assuming its infinitesimal stiffness. Cell indentation was obtained by comparing the experimental curve with the corresponding curve for the reference glass sample. The elastic behaviour of cells can be described using Sneddon’s mechanics w6x. In order to evaluate deformability of
Fig. 3. Ža. The principle of the interference method and Žb. the interference signal Žboth approaching and retracting curves are shown..
M. Lekka et al.r Applied Surface Science 141 (1999) 345–349
348
cells, Young’s modulus was determined assuming paraboloidal tip shape with a radius of curvature R at the apex. Following this model, sample indentation D z depends on acting force F, geometry of the tip and material properties according to the equation: F s 43 P 'R EX P D z 1.5 ,
Ž 1.
where EX is a reduced Young’s modulus of the tip–cell sample system: 1 E
X s
2 1 y mtip
q
Etip
2 1 y mcell
Ecell
,
Ž 2.
and Etip , Ecell are Young’s moduli of the tip and the cell, while mtip , mcell are Poisson’s ratios. Taking into account that Etip 4 Ecell , one finally obtains: EX s
Ecell 1 y m2cell
,
Ž 3.
In order to obtain statistically reliable data multiple measurements were performed. Next, Young’s moduli were determined by fitting Eq. Ž1. to the experimental curves and finally data histograms were created and a Gaussian distribution was fitted to each histogram.
3. Results and discussion The calculated Young’s moduli values, fitted to a population of 200–300 force curves are presented in Fig. 4. Results for a normal, epithelial cell line coming from non-malignant ureter ŽHu609. differ significantly from the data obtained for cancerous cell line T24. The higher Young’s moduli indicates that normal cells are significantly stiffer than the latter ones, while curves taken from several different cultures of the same cell line show reasonable reproducibility. Low stiffness of cancerous cells may be caused by a partial loss of actin filaments andror microtubules, and therefore by lower density of the cellular scaffold w7,8x. The measurement of Young’s modulus of the cells can help to determine the range of cytoskeleton changes.
Fig. 4. Histograms and their Gaussian fits created for measured normal ŽHu609. and cancerous ŽT24. cell lines.
Measurement errors originate from the following factors: – Discrepancies arising from inhomogeneities of cell membrane and of cell age. – Distributions in tip radius R and spring force constants k. – Uncertainty of the Poisson ratio, the real value of which for cells is difficult to estimate. Nevertheless, we do not expect significant differences in Poisson’s ratio for similar types of cells. – Approximations due to the applied physical model of indentation.
4. Conclusions SFM was used for the quantitative description of living cell deformability. The presented simple and convenient calibration methods allowed for exact control of instrument operation. The measured Young’s moduli values were in the range 1–10 kPa, with the natural Gaussian width not exceeding 40%. The observed differences for normal and cancerous cells were very significant and may be explained by cytoskeleton degradation caused by oncogenic transformation.
Acknowledgements Work partially supported by the State Committee for Scientific Research ŽKBN. Grant No. 2 P03B 033 12.
M. Lekka et al.r Applied Surface Science 141 (1999) 345–349
References w1x M. Radmacher, R.W. Tillmann, H.E. Gaub, Biophys. J. 64 Ž1993. 735. w2x D. Ricci, M. Tedesco, M. Grattarola, Microsc. Res. Technique 36 Ž1997. 165. w3x M. Lekka, J. Lekki, A.P. Schoulyarenko, B. Cleff, J. Stachura, Z. Stachura, Polish J. Pathol. 47 Ž2. Ž1996. 51.
349
w4x J.E. Sader, I. Larson, P. Mulvaney, L.R. White, Rev. Sci. Instr. 66 Ž7. Ž1995. 3789. w5x M. Jaschke, H.J. Butt, Rev. Sci. Instr. 68 Ž2. Ž1995. 1258. w6x I. Sneddon, Int. J. Eng. Sci. 3 Ž1965. 47. w7x A. Ben-Ze’ev, Biochimica Biophys. Acta 780 Ž1985. 197. w8x A. Ben-Ze’ev, Curr. Opin. Cell Biol. 9 Ž1997. 99.
Local elastic properties of cells studied by SFM M. Lekka
a,)
, J. Lekki a , M. Marszałek a , P. Golonka a , Z. Stachura a , B. Cleff b, A.Z. Hrynkiewicz a
a
b
Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Cracow, Poland Institute of Nuclear Physics, Wilhelm-Klemm-Str. 9, UniÕersity of Munster, Munster, Germany ¨ ¨ Received 29 May 1998; accepted 12 August 1998
Abstract Scanning force microscopy ŽSFM. can be used in nano-indentation experiments for very soft samples of elastic, organic materials. The present paper describes the methodology of device calibration and Young’s moduli determination, to evaluate the elastic properties of living cells in their culture conditions. Two similar lines of normal cells ŽHu609. and cancerous ones ŽT24. were measured. A significant difference in Young’s modulus for normal and cancerous cells was detected. q 1999 Elsevier Science B.V. All rights reserved. PACS: 07.79; 87.45.y k; 87.80 q s Keywords: Scanning force microscopy ŽSFM.; Methodology; Elasticity; Normal cells; Cancerous cells
1. Introduction Scanning force microscopy ŽSFM. can be used in nano-indentation experiments for very soft samples of soft, organic materials w1,2x. Nevertheless, in order to obtain reliable results some preparation of the device is needed before starting the SFM measurements. The main points which should be taken into account are: the determination of the cantilever spring constant, control of the tip shape and curvature radius, and corrections of scanner voltage signals removing the scanner nonlinearity and hysteresis. The present paper describes the methodology of device calibration and Young’s moduli determina-
)
Corresponding author. Tel.: q48-12-6370222 ext. 271; Fax: q48-12-6371881; E-mail: [email protected]
tion, to evaluate the elastic properties of living cells in their natural growing conditions. Two similar lines of healthy, normal cells ŽHu609. and cancerous ones ŽT24. were measured. Determination of elastic properties of the normal and cancerous cells was applied to characterisation of changes due to oncogenic transformation.
2. Materials and methods Two similar cell lines were chosen for the SFM measurements: Hu609—non-malignant ureter Žnormal., and T24—bladder transitional cell carcinoma Žcancerous.. Cell cultures were grown at 378C in 95% airr5% CO 2 in an incubator ŽASSAB type.. They were grown in the RPMI 1640 medium ŽpH 7.4. containing 10% fetal calf serum ŽFCS. provid-
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 5 2 2 - 4
346
M. Lekka et al.r Applied Surface Science 141 (1999) 345–349
ing cells nutrition. The cells were taken after the same time of growing, and then they were cultured on a glass coverslip with poly-L-lysine as an adhesive factor, since the SFM measurements required that the cells be strongly attached to the substrate. Strong adhesion of such system is caused by the interaction of negatively charged cell membrane and positively charged NH 2 groups present in poly-Llysine. A home-built scanning force microscope was used in the experiment w3x. It was equipped with a scanner with a maximum XY scan range of 20 mm = 20 mm, and a Z range of "1.5 mm Ž1.25-inch piezotube was purchased at Staveley Sensor.. Commercial, sharpened Si 3 N4 cantilevers were used ŽPark Scientific Instruments, Geneva.. A plexiglass ‘liquid cell’ without the ‘o-ring’ sealing was used. Cell measurements were performed in the RPMI medium containing 10% FCS serum at room temperature. The coverslip with cells Žstill immersed in the RPMI medium. was put into the ‘liquid cell’ placed on an SFM scanner. The measured cell was localised using an optical microscope, and the tip was moved towards the cell centre in order to avoid or minimise the influence of the substrate and the neighbouring cells. Every culture was measured several times using a new tip and fresh medium. Data analysis requires very careful controlling of the SFM set-up properties and experimental conditions. The most important factors are the spring constant of the cantilever, its tip geometry and scan-
ner nonlinearity. The value of the spring constant is crucial for the estimation of the force applied to an investigated surface. Spring constants of commercial cantilevers are distributed around a nominal value, which introduces uncertainty of loading force value. In order to obtain the true force calibration, it is necessary to determine the real spring constant. In our microscope this task is an option of data acquisition software and is performed by the measurement of resonant frequency of a thermally excited cantilever at room temperature. Fig. 1 presents the resonant spectra obtained for two types of cantilevers, with spring constants k s 0.1 Nrm and k s 0.03 Nrm. These spectra were obtained by applying FFT ŽFast Fourier Transform. to an average of six noise spectra, each of 4096 points, collected with a frequency of about 80 kHz from quadrant photodiode output. The calculated resonant frequencies are lower than the nominal values: 11.8 kHz instead of 15 kHz and 27.7 kHz instead of 35 kHz, but these values were well reproducible for most of the cantilevers in a wafer. For example, for k s 0.03 Nrm the resonant frequency varied from 11.8 kHz to 12.2 kHz. The procedure of the spring constant determination was performed following the method described by Sader et al. w4x. Models describing the behaviour of the surface under load assume the knowledge of tip-surface geometry. Therefore the tip curvature radius was checked using a TGT01 standard ŽNMDT, Molecular Devices and Tools for Nanotechnology, Moscow.,
Fig. 1. Noise spectra of two types of cantilevers: Ža. spring constant k s 0.03 Nrm and Žb. k s 0.1 Nrm.
M. Lekka et al.r Applied Surface Science 141 (1999) 345–349
347
Fig. 2. Ža. SFM image of a scanning tip and Žb. its cross-section.
capable of estimating tip radii down to 10 nm. The standard consists of a set of very sharp needles, much sharper than the cantilever tip. The collected image of such a standard represents the scanning tip topography. The tips used in experiment were characterised by tip radii of about 20–30 nm ŽFig. 2.. The correction of the piezo scanner nonlinearity and hysteresis was performed using the interference method w5x, adapted to our SFM set-up. This method is exact, easy to perform and does not require additional equipment ŽFig. 3.. Force curves reflecting the interaction between the probing tip and the cell surface were collected
using the laser deflection technique. A separate force calibration using reference hard material Žglass. was performed for each sequence of curves taken in the same session both before and after the measurement. In the range of the applied force Žup to 20 nN. there is no significant glass deformation caused by the SFM tip, and therefore the glass sample was used as a calibration reference, assuming its infinitesimal stiffness. Cell indentation was obtained by comparing the experimental curve with the corresponding curve for the reference glass sample. The elastic behaviour of cells can be described using Sneddon’s mechanics w6x. In order to evaluate deformability of
Fig. 3. Ža. The principle of the interference method and Žb. the interference signal Žboth approaching and retracting curves are shown..
M. Lekka et al.r Applied Surface Science 141 (1999) 345–349
348
cells, Young’s modulus was determined assuming paraboloidal tip shape with a radius of curvature R at the apex. Following this model, sample indentation D z depends on acting force F, geometry of the tip and material properties according to the equation: F s 43 P 'R EX P D z 1.5 ,
Ž 1.
where EX is a reduced Young’s modulus of the tip–cell sample system: 1 E
X s
2 1 y mtip
q
Etip
2 1 y mcell
Ecell
,
Ž 2.
and Etip , Ecell are Young’s moduli of the tip and the cell, while mtip , mcell are Poisson’s ratios. Taking into account that Etip 4 Ecell , one finally obtains: EX s
Ecell 1 y m2cell
,
Ž 3.
In order to obtain statistically reliable data multiple measurements were performed. Next, Young’s moduli were determined by fitting Eq. Ž1. to the experimental curves and finally data histograms were created and a Gaussian distribution was fitted to each histogram.
3. Results and discussion The calculated Young’s moduli values, fitted to a population of 200–300 force curves are presented in Fig. 4. Results for a normal, epithelial cell line coming from non-malignant ureter ŽHu609. differ significantly from the data obtained for cancerous cell line T24. The higher Young’s moduli indicates that normal cells are significantly stiffer than the latter ones, while curves taken from several different cultures of the same cell line show reasonable reproducibility. Low stiffness of cancerous cells may be caused by a partial loss of actin filaments andror microtubules, and therefore by lower density of the cellular scaffold w7,8x. The measurement of Young’s modulus of the cells can help to determine the range of cytoskeleton changes.
Fig. 4. Histograms and their Gaussian fits created for measured normal ŽHu609. and cancerous ŽT24. cell lines.
Measurement errors originate from the following factors: – Discrepancies arising from inhomogeneities of cell membrane and of cell age. – Distributions in tip radius R and spring force constants k. – Uncertainty of the Poisson ratio, the real value of which for cells is difficult to estimate. Nevertheless, we do not expect significant differences in Poisson’s ratio for similar types of cells. – Approximations due to the applied physical model of indentation.
4. Conclusions SFM was used for the quantitative description of living cell deformability. The presented simple and convenient calibration methods allowed for exact control of instrument operation. The measured Young’s moduli values were in the range 1–10 kPa, with the natural Gaussian width not exceeding 40%. The observed differences for normal and cancerous cells were very significant and may be explained by cytoskeleton degradation caused by oncogenic transformation.
Acknowledgements Work partially supported by the State Committee for Scientific Research ŽKBN. Grant No. 2 P03B 033 12.
M. Lekka et al.r Applied Surface Science 141 (1999) 345–349
References w1x M. Radmacher, R.W. Tillmann, H.E. Gaub, Biophys. J. 64 Ž1993. 735. w2x D. Ricci, M. Tedesco, M. Grattarola, Microsc. Res. Technique 36 Ž1997. 165. w3x M. Lekka, J. Lekki, A.P. Schoulyarenko, B. Cleff, J. Stachura, Z. Stachura, Polish J. Pathol. 47 Ž2. Ž1996. 51.
349
w4x J.E. Sader, I. Larson, P. Mulvaney, L.R. White, Rev. Sci. Instr. 66 Ž7. Ž1995. 3789. w5x M. Jaschke, H.J. Butt, Rev. Sci. Instr. 68 Ž2. Ž1995. 1258. w6x I. Sneddon, Int. J. Eng. Sci. 3 Ž1965. 47. w7x A. Ben-Ze’ev, Biochimica Biophys. Acta 780 Ž1985. 197. w8x A. Ben-Ze’ev, Curr. Opin. Cell Biol. 9 Ž1997. 99.