Vol. June

177,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

14, 1991

636-643

AN APPROACH TO IMAGING OF LIVING CELL SURFACETOPOGRAPHY BY SCANNINGTUNNELING MICROSCOPY Etsuro

ITO' , Tetsuo TAKAHASH12, Wataru MIZUTANI3, Hajime

'Advanced

Research

2Department

Center

for

of Human Basic

Waseda University, 3Frontier

Technology

April

17,

YOSHIOKA1'2"' ON03

Human Sciences,

University,

Sciences,

Division,

Waseda

School

Tokorozawa,

Tsukuba, Received

Kiyoshi HAMA1p2, Tohru SHIMIZU3, and Masatoshi

of Human Sciences,

Saitama

359,

Electrotechnical

Ibaraki

305,

Japan Laboratory,

Japan

1991

SUMMARY: Since a scanning tunneling microscope (STM) was developed, an observation of living cell surface has been one of the final aims in the biological application of STM. By developing a new style of STM which was combined with an optical microscope and with a novel system for the centering got the STM images of living cell surface of of both images, we successfully T2.!+ cells (human bladder cancer cell line) and CHO cells (Chinese hamster ovary fibroblast) cultured on highly oriented pyrolytic graphite under the appropriate condition (V > 8.0 V, I < 0.2 nA). Unexpectedly, the living T21, cell showed a slightly uneven surface with a steep foot slope. The CHO cell showed more rough surface with steeper slope of cell foot. Although the STM system had a fine spatial resolution less than 3 nm, the profile of living cell surface covered with electrolyte was clear only when the scanning area 0 1991 AcademrcPESS, 1°C. was more than 10 urn square.

The scanning structure

of various

semiconductors. attempted protein conductive information

tunneling

(1

microscope

surfaces

(STM)

in atomic

The application

size

was developed including

of STM to biology

(4), metal-covered

metals

bacteriophage

I-13))

lipid

bilayer

(17).

The STM has an advantage to provide tip, which traces the surface profile

plate

by probing

constant

current

ambient

pressure

mode operation. and aqueous

To whom correspondence

should

(14-16)

Furthermore, environments.

(5-7)

and native

and dried

the

the

and

has also

(I-3)

for

membrane

to study

been DNA

cells three

(8-IO),

on the dimensional

of sample

STM can be operated

But nobody

could

get

in in

an high

be addressed.

ABBREVIATIONS CHO:Chinese hamster ovary fibroblast; HOPG:highly oriented pyrolytic graphite; 0M:optical microscope; PZT:Pb(Zr,Ti)O?, titanate zirconate lead; STM:scanning tunneling microscope; T24:human bladder cancer cell line. 0006-291X/91

$1.50

636

the

vol.

177,

No.

resolution of it

2, 1991

image

because

and generally report,

of living

a living

cell

surface an optical

and CHO cells oriented

covered

cell

with

(Chinese the

surface which

images

BIOPHYSICAL

and even

(OM).

of the

could

by using fibroblast)

MATERIALS

by using

the on its

surface In this

of non-conductive bladder

were

living

of STM combined cancer

cultured

combined

cell

line)

on highly

to STM observations. this

topography

certainty.

a new style

(human

(HOPG) and submitted cell

with

topography

COMMUNICATIONS

identify

characteristics

T21, cells ovary

STM-observed

RESEARCH

can be refered

electrolyte

hamster

graphite

AND

has no conductive

got

microscope

pyrolytic identify

cell

has no landmarks

we successfully

with

could

BIOCHEMICAL

We

OM.

AND METHODS

T24 cells (human bladder cancer cell line) or --Cell Culture: (Chinese hamster ovary fibroblast) maintained in cell-culture F-12 medium (Gibco, USA) supplemented with 10 % fetal bovine CO /a.r at 37" C were plated onto a HOPG in the culture dish of2105 cells/dish. This HOPG used in the culture had already adhesive tape and cleaned by 70 % ethanol and then linsed by solution (Hanks' BSS, Gibco, USA) and by the culture medium. maintained for 2 days prior to the STM observation.

CHO cells medium (Ham's serum) under 5 % at a density been cleaved by balanced salt The culture was

Positioning: Adjustment between Center of Region Observed & --STM and That & ~__OM: Before the sample observation by the STM, the parameters of an imagepositioning system for adjustment between the center of region observed by STM and that of OM were set by use of a special lithography pattern on a silicon wafer. The discrepancy between an observed area of STM and that of OM was minimized within 1 urn and it became possible to identify the spot on living cell surface observed by the STM. OM Observation: The OM view was observed with an objective lens (x50, Olympus, Japan), a CCD camera (CS5330, Tokyo Electronic Industry, Japan) and a TV monitor (CX-IOIM, Victor, Japan) under control of a personal computer system (PC-386, EPSON, Japan). Then the photograph of this TV picture was taken by a usual camera. Although the most of medium on cells were removed before the measaurements with the OM and the STM, the adequate amount of culture medium was constantly supplied onto the HOPG in order to maintain the cell surface wet during the measurements. STM Observation: The STM system used in this experiment was developed by Seiko Instrument Inc., Japan (e.g. see ref. 23 about the STM with OM for large sample measurement). The features of this STM are as follows: (1) this STM has an ability of the adjustment of centering for sample by the combination with an OM, (2) the maximum scanning area is 18.0um x 18.6um. This STM system consists of a tunnel unit with objective lenses and a CCD camera, a STM controller, a PZT driver, a stage driver, a camera controller and a TV monitor under control of a personal computer system. The probing tip was made of a tungsten wire (d = 0.30 mm) purchased from NILACO, Japan and electrochemically etched by curselves. I I (a) ___Resolution: The test sample purchased from Seiko Instruments Inc., Japan was a semiconductor plate evaporated with platinum powder. The bias voltage and the tunnel current were set at 0.10 V and 0.5 nA, respectively. The scanning spleed was 0.1 see/raster and the detecting points were 256 x 128 pixels. (b) Observation of T24 Cells -~or CHO Cells: The bias voltage and the tunnel --current were 8.00 V and 0.2 nA, respectively. The scanning speed was 2 set/raster for T24 cells or 5 see/raster for CHO cells. Each of data was taken at 256 x 64 pixels. (c) Fluctuation of ~~ Tunnel Current between Probing Tip and One Point on Sample Surface: The freshly cleaved surface of dried HOPG, the cleaved surface of HOPG covered with electrolyte and the living CHO cell surface on HOPG covered 637

Vol.

177,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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with electrolyte were used as sample surfaces. The tip was fixed on one point on the sample surface, and the fluctuation of tunnel current between those was measured as operational-amplifier conveted voltage (IO8 V/A). The bias voltage and tunnel current were 8.0 V and 0.2 nA, respectively. The distance between tip and sample surface was roughly estimated by using the relation among the values of tunnel current, bias voltage and PZT constant (8 rim/V) in z direction. Since the feed-back circuit was worked, the obtained data showed the limitation of measurement of this low magnification STM for each sample. The measurement time (300 see) was almost same as that spent to get a STM image of living cell surface. RESULTS Prior the

to the

STM used

platinum

in

powder

nm to 15 nm. resolution

observation the present

Therefore 2a shows

the bottom) of the

urn.

The gentle

high

bias

STM images

part

image

voltage (17,18),

was examined.

lateral

As shown

particles

reasonable image

resolution in Fig.

varing

to decide

T2,!+-cell

In this

that

of

1, the

in diameter this

living unevenness

image

figure

the

T24 cell

system

from

3

has a

surface was appeared

cells

were

not

damaged

Figure

in Fig. surface.

current as far

region

was shown (the

highest

the

The apparent Although

(< 0.2 as the

and 1.85

l.lOum

we here

nA) to get

clear

OM view.

1. Test of the STM performance. The sample is platinum powder evaporated on semiconductor plate and the image is shown as black-and-white expression. Scanning area is 125 nm x 125 nm and the maximum brightness shows the thickness of 15.6 nm. Since the smallest size of particle is 3.0 nm, the resolving power of this STM is estimated at least 3.0 nm, if we observe it in the air.

Fig.

638

as

was expressed

2c shows

2b.

T24

the

thickest

to be between

on cell tunnel

guiding

of cell

to the

was found

confluent

T24 cells

thickness

on HOPG.

for

for

of living

as shown

(> 8.0 V) and small the

was used

corresponds

same region

cell

microscope

STM image

region

of living

of the

of optical

2b the

The brightest

stereoscopic thickness

on the In Fig.

image.

as brightness.

is

surface,

3 nm.

line

a black-and-white

cell

as separate

a typical

of STM observation.

from

it

than

The cross

cells.

study

was displayed

better

Figure

of living

used

Vol.

177,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pi~.;J~IConparison of images of T24 cells with the optical microscope and with . (a) Optical micrograph. Calibration bar = 10 urn. The cross point of white lines is used as an index for the adjustment of the center for both images of the optical microscope and of the STM. (b) Black-and-white expression of the STM image. The scanning area is 18.0 urn x 18.0 urn and the maximum expression

brightness of Fig.

Figure

3 shows

cells. identified

Similar

corresponds 2b.

the

to the

by cross-line

to

optical case

the

thickness

micrograph

and the

of T24 cells,

on the

optical

of

the

urn.

(c)

STM images

STM images

micrograph. 639

1.85

Stereoscopic

of living

showed

The diameter

the

CHO

same cell

of this

Vol.

177,

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

microscope and with wTMComparison of images of CHO cells with the optical . (a) Optical micrograph. Calibration bar = 10 urn. (b) Black-andwhite expression of the STM image. The scanning area is 18.0um x 18.0 pm and the maximum brightness shows the thickness of 1.75 IJIII. (c) Stereoscopic expression of Fig. 3b.

targeting

cell

Urn to 1.751.lm. cell.

The shape

was about The surface of these

1611111

in diameter

of the cells

cell

and its was found

was hemispherical

640

thickness

varied

to be more rough similar

to that

from than

1.00

T24

of a bowl

Vol.

177,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

a YW*CIl...‘-i.

“’ 1, -

‘1”. 100

II

mV

b

C

I

I

0

60

I

I

120 Measurement

160 Time WC)

I

I

240

300

Fig. 4. Fluctuation of tunnel current between probing tip and one point on sample surface during 300 sec. Tunnel current shown in this figure are expressed as voltage converted by an operational amplifier. (a) Freshly cleaved surface of dried HOPG. The maximum fluctuation was 20 mV, and the resolution in z direction was estimated less than 0.7 nm. (b) Cleaved surface of HOPG covered with electrolyte. The resolution in z direction was estimated less than 3.2 nm. (c) Living CHO cell surface on HOPG covered with electrolyte. The resolution in z direction was within 10.0 nm.

put

upside-down

was seemed

on the

Although platinum

by waving

(Fig.

this

assumption

that

it

cell

in Fig.

rigid

in

than

matter

during

environment. times)

such

as shown

cell

as

the

current

STM

in Figs.

2

observation

In order

of tunnel 10

type

3 nm by our

structure

fluctuation

fluctuation

bowl

in average).

of at least

of fine

aqueous

(more

of this 40"

and conductive

current

the

8=

resolution

surface

enhanced

of foot

l.Oum,

images

tunnel

we measured

as shown

the

to get

due to the

was greatly

the

with

1)

of living

The slope = 0.8um/

observe

was impossible

and 3, probably

surface

(tan0

we could

powder it

system,

substrate.

to be steep

to confirm

and we found

when we observe

living

cell

i. DISCUSSION

As was described topography surface

of living

as high

as 109-

(LB)

CdAch molecules

= 40 mV and they than

under

the

expected

was obtained explained

this

under

R-cm

lOI

film were

estimated value. the

by a simple

an initial

The reasons

by Mann and Kuhn that

Langmuir-Blodgett (14),

we took

cells.

was observable

estimated is

above,

why the

condition the

step are

resistivity

STM image still

to the

of Cadmium-arachidate observable the Baro

condition tunnelling

under

resistivity et al.

not

of the

According

(19).

of STM imaging

the

condition

reported

phenomenum 641

that

STM

cm, which (4).

They

cell

revealed.

It

bilayer

observation

as IO3 R-cm,

of p = l.OR.

of living

lipid

(CdAch)

of surface was

membrane for

by Smith

et al.

of I = 2.0

nA and V

which

was much lower

image

of bacteriophage

can also proposed

not

be

a novel

Vol.

177,

No.

mechanism

2, 1991

of electron

bacteriophage they

(conduction they

distribution

levels

higher

membrane

by the

Therefore

it

surface

likely

this

by the purpose

is

of biological

detailed

deformed

force,

with

for

for

electron)

and regions energy

of the

resistivity leaky

living

of lipid

current

cell

(16).

by using

ionic

ion-conductance

surface

Only

supposed

protein the

and the in

the

to make

fibrinogen

to be difficult

of such

on the

lower

case

(21)

and

to observe

shape

of 10 -9 N, exerted

observation,

to be

of STM for

has been used

by AFM, since

order

was found

alternative

force

samples

sample leads

of STM is

to

expected

to

AFM. compelled for

to say that observation

the

to the

in crystalized

living

isolared

cell.

form

with

scanning

of living

of STM to biomolecule than

be observed

cell

(AFM) which

The deformation

suitable

application

living current.

such as human blood

of the

for

we are

necessarily

for

material

tip

than

be more significant will

tunnel

lower

case of a scanning

however,

so far.

To sum up, the

is,

probing

resolution

Namely

the

of

of membrane,

the kinetic

conduction

microscopy

of soft

by the

by the

force

It

topography

not

of the

than

STM image

of tunnel

samples

(22).

be much smaller is

of STM imaging atomic

cells

poorer

the

resistance

electrons

energy lower

COMMUNICATIONS

resistance

the

explained

to the

fluctuation

images

surface

a low

kinetic

of surface

similarly

such a low

where

levels however,

RESEARCH

(20).

living is

the

to obtain

The resolution limited

than

et al.,

conductance

microscopy

of regions

introduction

is

to explain

(conduction

Mizutani

BIOPHYSICAL

to explain

In order

the

propagate

electron).

AND

transportation

membrane.

assumed

where

BIOCHEMICAL

An ion

probiong

sample.

from

or cell

organ

channel

reasonably

microscopy

biological

high

may

or a receptor resolution.

ACKNOWLEDGMENTS T24 and CHO cells University) Messrs. Inoue

were

and Prof. M. Shigeno,

(Seiko

This from

O224llOl)

E. Tomita,

Instruments

discussion.

kind

gifts

M. Mishina

from

(Niigata K. Ishihara,

Inc.)

for

their

research

was supported

the Ministry

of Education,

Dr.

Y. Kubota

University), C. Miyata, technical

Y. Shikakura

supports

by Grant-in-Aids Science

(Yokohama

City

respectively.

We thank and A.

and valuable (6388032

and Culture

and

of Japan.

REFERENCES Guckenberger, R., Kosslinger, C., and Baumeister, W. (1988) J. Vat. Sci. Technol. A6, 383-385. 2. Crowther, R.A. (1989) Nature 339, 426-427. 3. Zasadzinski, J.A.N. (1989) Biotech. 7, 174-187. 4. Baro, A.M., Miranda, R., Alaman, J., Garcia, N., Binnig, G., Rohrer, H., Gerber, Ch., and Carrascosa, J.L. (1985) Nature 315, 253-254. 5. Travaglini, G., Rohrer, H., Amrein, M., and Gross, H. (1987) Surf. Sci. 1.

181, 6.

380-390.

Amrein, Science

M., Stasiak, 240, 514-516.

A.,

Gross,

H.,

Stoll,

642

E.,

and Travaglini,

G. (1988)

Vol.

177,

No.

7. Amrein, Science 8. Dunlap, 9. Cricen-ti, W., and 10. Drisco.11, 346, 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

23.

M., 243,

BIOCHEMICAL

Durr,

R.,

Stasiak,

AND

A.,

BIOPHYSICAL

Gross,

H.,

RESEARCH

COMMUNICATIONS

and Travaglini,

G. (1989)

1708-1711.

D.D. and Bunstamante, C. (1989) Nature 342, 204-206. A., Selci, S., Felici, A.C., Generosi, R., Gori, E., Chiarotti, G. (1989) Science 245, 1226-1227. R., Youngquist, M.G., and Baldeschwieler, J.D. (1990)

Djaczenko, Nature

294-296.

Simic-Krstic, Y., Kelley, M., Schneiler, C., Krasovich, M., MacCuskey, R., Koruga, D., and Hameroff, S. (1989) The FASEB J. 3, 2184-2188. Edstrom, R.D., Meinke, M.H., Yang, X., Yang, R., and Evans, D.F. (1989) Biochen. 28, 4939-4942. Snellm,an, H., Pelliniemi, L.J., Penttinen, R., and Laiho, R. (1990) J. Vat. Szi. Technol. A8, 692-694. Smith, D.P.E., Bryant, A., Quate, C.F., Rabe, J.P., Gerber, Ch., and Swalen, J.D. (1987) Proc. Natl. Acad. Sci. USA 84, 969-972. Zasadzinski, J.A.N., Schneir, J., Gurley, J., Elings, V., and Hansma, P.K. (1988) Science 239, 1013-1015. Mizutani, W., Shigeno, M., Saito, K., Watanabe, M., Sugi, M., Ono, M., and Kajimura, K. (1988) Jpn. J. Appl. Phys. 27, 1803-1807. Ruppersberg, J.P., Horber, J.K.H., Gerber, Ch., and Binnig, G. (1989) FEBS Lett. 258, 460-464. Guckenberger, R., Wiegrabe, W., Hillebrand, A., Hartmann, T., Wang, Z., and Baumeister, W. (1989) Ultramicroscopy 31, 327-332. Mann, B. and Kuhn, H. (1971) J. Appl. Phys. 42, 4395-4405. Hansma, P.K., Drake, B., Marti, O., Gould, S.A.C., and Prater, C.B. (1989) Science 243, 641-643. Drake, B., Prater, C.B., Weisenhorn, A.L., Gould, S.A.C., Albrecht, T.R., Quate, C.F., Cannel, D.S., Hansma, H.G., and Hansma, P.K. (1989) Science 243,

22.

2, 1991

1586-1589.

Horber, J.K.H., Haberle, Proceedings of STM '90, Yasutake, M. and Miyata,

W., and Binnig, G., to be published in the J. Vat. Sci. Technol. C. (1990) J. Vat. Sci. Technol. A8, 350-353.

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