Frequency shift feedback imaging in liquid forbiological molecules

Hiroshi Sekiguchia, Takaharu Okajimaa, Hideo Arakawaa,Sumihiro Maedab, Akihiko Takashimab, Atsushi Ikaia,*

aGraduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho,

Midori-ku, Yokohama 226-8501, JapanbLaboratory for Alzheimer’s disease, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan

Abstract

A commercially available atomic force microscope (AFM) equipped with a hand made simple self-oscillation circuit was

used in imaging biomolecular samples in liquid environments, i.e. under physiological conditions. Assembled tau proteins,

which are the major component of the neurofibrillary deposits in Alzheimer’s disease, was taken as a trial sample. In order to

image its native structure, the protein was physically absorbed on a cleaved mica surface without fixation. Using the frequency

feedback imaging with a self-oscillation technique, the structure of protein fibers was clearly imaged even in a wide scanning

range (3.75 mm) with a contact force less than 100 pN. Furthermore, no damage of the proteins was observed in successive

imagings. This indicates that the deformation of proteins was negligible in our method. In contrast, the proteins were destroyed

when the vertical applied force of above 300 pN was applied using the amplitude feedback imaging with the self-oscillation

technique.

# 2002 Elsevier Science B.V. All rights reserved.

PACS: 07.79.Lh; 87.15.By

Keywords: Atomic force microscope; Self-oscillation; FM detection; Tau protein; Neurofibrill

1. Introduction

The atomic force microscope (AFM) is a suitable

instrument for the investigation of biological samples

for two reasons. First, structure and mechanical prop-

erties of the samples can be analysed simultaneously

using the imaging and force curve mode of the AFM,

respectively. Second, the sample can be kept under

physiological condition in AFM experiments, which

makes it possible for us to study the dynamic beha-

viour of living biological samples. Therefore, AFM is

expected to be a useful tool to elucidate the relation-

ship between the structure and function of biological

molecules.

So far, the contact mode as well as the tapping

mode has been widely used for imaging biological

samples in liquid, but it is not easy to routinely obtain

high-resolution images of them. Main reasons of this

difficulty is that, in the contact mode, an inherent

strong lateral force often peels off the samples

Applied Surface Science 210 (2003) 61–67

Abbreviations: AFM, atomic force microscope; FM, frequency

modulation; PHF, paired helical filaments* Corresponding author. Tel.: þ81-45-924-5828;

fax: þ81-45-924-5806.

E-mail address: aikai@bio.titech.ac.jp (A. Ikai).

0169-4332/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0169-4332(02)01480-0

physically absorbed on the substrate as has been

discussed previously [1] except for limited cases

of proteins assembled into 2D crystals [2], while

in the tapping mode, a high vertical force that is needed

to overcome the damping effect in liquid tends to

deform samples. One way to avoid these effects are

to fix the sample using glutaraldehyde or other reagents

[3]. The fixation process, however, often causes arti-

facts in the sample images, and native mechanical

properties of the sample cannot be studied. Thus, in

order to obtain reliable images of native biomolecules

using an AFM in liquid environments, it is crucial to

image them with as low loading force as possible.

Recently, the Q-control method, which can enhance

the effective quality factor of an oscillating cantilever

by an additional feedback loop [4] was utilized for

imaging in liquid with an improved force sensitivity,

achieving imaging with a low applied force [5]. How-

ever, the effectivity of Q-control method for imaging

biological material in liquid has recently been brought

into question. Jaggi et al. [6] reported that the tapping

mode and the Q-control method are comparable if

scan parameters are optimized. Additionally, increas-

ing the Q-value requires a longer relaxation time for

the perturbation of amplitude.

We found that frequency modulation (FM) detec-

tion method with a self-oscillation technique [7] could

be more useful for imaging in liquid. This method can

be more sensitive compared to the detection of

decrease in amplitude. Self-oscillation at 8–10 kHz

which is commonly used in tapping mode was avail-

able, and we could image by detecting a few hertz shift

using a soft cantilever of 0.15 N/m corresponding to

below 100 pN in applied force.

This method was applied to image the assembled

tau proteins that is the major component of the neu-

rofibrillary deposits, which constitute a defining neu-

ropathological characteristic of Alzheimer’s disease

[8]. As a result, we could obtain stable images of a soft

biological sample in liquid and were convinced the

effectiveness of this method.

2. Materials and methods

2.1. Atomic force microscopy

A Multimode NanoScope III (Digital Instruments,

Santa Barbara, CA) equipped with a tapping mode

fluid cell was used. Fig. 1 shows a schematic diagram

Fig. 1. Schematic diagram of the FM feedback method with self-oscillation. Inside the dotted rectangle is the system of a commercially

available AFM, NanoScope III. The other electronics were home made and introduced to the AFM for the present purpose.

62 H. Sekiguchi et al. / Applied Surface Science 210 (2003) 61–67

of the FM feedback method with self-oscillation.

Mechanical–acoustic excitation was performed using

a positive feedback loop composed of a deflection

sensor, a phase shifter, a gain amplifier, and a built-in

actuator for cantilever oscillation. In the positive feed-

back loop, first a sinusoidal-like deflection signal was

filtered to remove extremely low-frequency compo-

nents and converted into square wave on a logic level

Fig. 2. (a) Frequency spectra of the system driven by mechanical–acoustic excitation in liquid condition. A cantilever whose resonance

frequency was about 8 kHz in liquid was mounted. (b) The vibration amplitude, the frequency shift and the deflection of the cantilever signal

as functions of tip–surface distance. The reference point (0 nm) on the abscissa was set to the point where the frequency began to increase.

H. Sekiguchi et al. / Applied Surface Science 210 (2003) 61–67 63

by a zero-cross comparator. Next, the phase of the

square wave was shifted by a variable phase shifter

(CD-951V4, NF Co., Japan), and the amplitude of the

square wave was regulated by a gain amplifier. Finally,

the square wave was filtered to reduce high frequency

components, and then resulting signal was used as the

excitation signal of the cantilever. The amplitude of

the signal was adjusted to less than 10 nm by a gain

amplifier. The frequency shift of the cantilever oscil-

lation was detected using analog FM demodulation

and the frequency signal was inputted to the Nano-

Scope controller through a Signal Access Module

(Digital Instruments, Santa Barbara, CA). The canti-

lever used was OMCL-TR800PSA (Olympus Co.,

Japan) with a nominal spring constant of 0.15 N/m

and a resonance frequency of about 8 kHz in liquid,

respectively. All measurements were performed in

phosphate buffered saline (pH ¼ 7:5). The vibration

spectrum of a cantilever driven by mechanical–acous-

tic excitation was obtained by the ‘‘cantilever tune’’

command of NanoScope. Force curve measurements

were performed on a cleaved mica surface at 1 Hz scan

rate and 100 nm in scan width. Imaging was per-

formed at 512 � 256 pixels, at 1 Hz scan rate and

with a low loading force.

An applied force during imaging was directly esti-

mated from averaged cantilever deflection signals

from force curve which was measured immediately

after imaging, according to F ¼ k � d, where F is the

applied force, k the cantilever constant and d the

averaged cantilever deflection. The performance of

the system was described in [9].

3. Samples

Wild type tau proteins were expressed and purified

in a similar protocol reported in [10]. Purified tau

proteins (0.5 mg/ml) were assembled by incubating

them with heparin (10 mM), which acts as a polymer-

ization accelerator for tau [11], in HEPES buffer

(10 mM HEPES, 100 mM NaCl, pH ¼ 7:4) at 37 8Cfor 100 h. Assembling reaction was stopped by freez-

ing the sample solution at �30 8C. After removing

heparin and salts by ultracentrifugation (10 k G,

20 min, 4 8C, 6 times), the assembled protein (about

0.05 mg/ml, 10 ml) was deposited on a freshly cleaved

mica surface and imaged in physiological condition.

4. Results and discussion

In Fig. 2(a), the response of a cantilever driven by a

conventional mechanical–acoustic excitation in liquid

is shown. The shape of the spectra was affected by

subtle change of cantilever setting, however, large

peaks were commonly observed around 8–10 kHz

even different cantilever was mounted. So the spec-

trum is due to a convolution of resonances of the fluid

cell, cantilever base, liquid and cantilever itself. Tap-

ping mode images of good quality obtained in liquid

and found in published papers were obtained in this

frequency range [12,13]. We tuned the self-oscillation

frequency to be in the range 8–10 kHz and adjusted the

phase of excitation signal so that the amplitude of

cantilever deflection became stable and a maximum

value at a fixed excitation amplitude. In Fig. 2(b), the

vibration amplitude, the frequency shift and the can-

tilever deflection of the self-oscillation are given as

functions of the tip–surface distance. We set the point

where the frequency signal began to increase (Fig. 2(b)

center) as the reference point (0 nm) of tip surface

Fig. 3. Images of assembled tau proteins at different scan sizes: (a)

3:75 mm � 2:5 mm and (b) 1:5 mm � 1:2 mm.

64 H. Sekiguchi et al. / Applied Surface Science 210 (2003) 61–67

distance. It is clear that a positive shift of the frequency

was more sensitive than the decrease in the amplitude.

Therefore, we concluded that it would be better to

apply the frequency signal as a feedback signal for

imaging. Since the deflection of cantilever increased

below 0 nm (Fig. 2(b) bottom), the positive frequency

shift of the oscillation must have been caused by a

repulsive force between the tip and the sample. Using

a cantilever whose resonance frequency was far away

from 8 to 10 kHz, we could not observe such a sharp

Fig. 4. Sequential amplitude feedback images of assembled tau protein by self-oscillation method. Top, center and bottom images were

obtained at 1st, 3rd and 4th scanning from the time when amplitude feedback imaging was started.

H. Sekiguchi et al. / Applied Surface Science 210 (2003) 61–67 65

increase of frequency signal and could not get fre-

quency feedback imaging probably because the fluid

cell worked as a narrow band-pass filter.

Fig. 3(a) and (b) include frequency feedback images

of assembled tau proteins with different scan sizes. We

used reconstructed fibers as a model of paired helical

filaments (PHFs) in the brain of Alzheimer patients.

We could obtain stable height traces with a scan rate as

fast as 1 Hz with 3:75 mm � 2:5 mm in scan size as

shown in Fig. 3(a). This showed that the response of

the frequency shift in our self-oscillating system was

sensitive enough for imaging biological samples in

liquid. The stability of oscillation, another practical

requirement for the oscillating method in AFM, was

also demonstrated.

The observed fiber had a striped feature with highs

(18:8 � 1:9 nm, n ¼ 15) and lows (9:5 � 1:4 nm,

n ¼ 14) in Fig. 3(b). Both the highs and lows were

significantly higher, respectively, than those reported

for dried samples with AFM, which were 13:68�1:46 nm and 6:66 � 0:78 nm, respectively [14]. This

discrepancy suggests that the dried fibers were flat-

tened, while the present sample was kept in more

native condition during our AFM imaging. When we

compared the observed height values with reported

diameters obtained by electron microscopy (18–20

and 8–10 nm in wider and narrower regions, respec-

tively [15]), deformation of the sample caused by the

applied force during imaging seemed to be extremely

small. Applied force during imaging was estimated to

be below 100 pN.

This conclusion was supported by the results of

imaging using an amplitude feedback also as shown in

Fig. 4. In this method, we switched to take the feed-

back signal from the frequency shift to the amplitude

of oscillation with the same self-oscillating method.

Good images were also obtained as shown in Fig. 4

(top image) but the background seemed to be more

flat, which may indicate a lower resolution in the

amplitude feedback method. An interesting difference

between the images obtained by the feedback methods

was the height of the sample. In the amplitude feed-

back method, the averaged height of highs and lows of

the striped fiber (Fig. 4, top) was decreased to

12:4 � 2:6 nm (n ¼ 15) and 7:0 � 2:4 nm (n ¼ 14),

respectively. This was thought to have resulted by a

larger loading force of about 300 pN in the amplitude

feedback method. Additionally, since as the force was

too large for this soft fibrous sample, it was destroyed

during imaging as shown in Fig. 4 (middle and bot-

tom). These results confirmed that when the frequency

feedback was employed the AFM probe was touching

fragile samples quite gently.

We observed a periodicity of 70–100 nm in each

fiber, which corresponded to the reported pitch of PHF

of 81 nm [14]. Although we could see some helical

images, we could not yet conclude about the helicity

of this fiber, because it is known that tip convolution

effect is severe for helical molecules [16], which can

give right-handed helical images for left-handed helix

in some cases. Further details of the structure of the

assembled tau are currently under investigation.

5. Conclusion

Using an FM feedback in liquid with a self-oscilla-

tion technique, we could obtain clear images of soft

fibrillary proteins under physiological conditions with

a low applied force. Since the present AFM method

with FM is simple, it will be useful for imaging various

soft materials in liquid.

Acknowledgements

This work was supported in part by grant-in-aid to AI

from the Japan Society for the Promotion of Science

(Research for the Future Program #99R167019) and

from the Japanese Ministry of Education, Science,

Culture and Sports (Scientific Research on Priority

Areas (B) #11226202).

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