Biophysical Reviews (2021) 13:455–458
https://doi.org/10.1007/s12551-021-00820-x

LETTER

Biophysical reviews top five: atomic force microscopy in biophysics
Toshio Ando 1
Accepted: 1 July 2021 / Published online: 10 July 2021
# The Author(s) 2021

Abstract
Since its invention in the late 1980s, atomic force microscopy (AFM), in which a nanometer-sized tip is used to physicallyinterrogate the properties of a surface at high resolution, has brought about scientific revolutions in both surface science andbiological physics. In response to a request from the journal, I have prepared a top-five list of scientific papers that I feel represent
truly landmark developments in the use of AFM in the biophysics field. This selection is necessarily limited by number (just five)and subjective (my opinion) and I offer my apologies to those not appearing in this list.

The technique of atomic force microscopy (AFM) constitutes
a multifunctional toolbox in modern biophysics (see reviews:
Müller and Dufrêne 2008; Ando et al. 2014; Dufrêne et al.
2017; Valotteau et al. 2019). Using AFM, we can perform
various measurements under physiological conditions thatare infeasible with other techniques: e.g., dynamic highresolution imaging of biomolecules during their functional
activity, recognition and localization of specific molecules,
force measurements to estimate the strength of intra- and intermolecular bonds at the single-molecule level, and the sitespecific recording of the elasticity of biological surfaces. This
current metrological prosperity can, of course, be traced backto its point of origin (Binnig et al. 1986) but it also owes agreat deal to many technical developments and creative studies carried out since then.
In terms of technical advances, although all AFM experiments utilize similar instrumental components, for biological/
biophysical applications AFM devices must be operated in aparticular manner best suited to the peculiarities of the sample(e.g., typically soft, hydrated/in solution, and highly dynamic). In this regard, AFM instruments that are geared to biophysical measurements are typically operated in the AC mode
where the cantilever is oscillated at its resonant frequency insolution. Each of these technical components, necessary toachieve this specialization, has its own origins: e.g., Binnig
et al. 1987 for cantilevers, Meyer and Amer 1988 for the* Toshio Ando
tando@staff.kanazawa-u.ac.jp
1

Nano Life Science Institute (WPI-NanoLSI), Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan

optical beam deflection technique able to measure cantileverdeflection, Zhong et al. 1993 for the AC mode, and Marti et al.
1987 for in-liquid imaging. Such technical and instrumental
advances made possible a number of groundbreaking biophysical studies (Drake et al. 1989; Weisenhorn et al. 1993;
Butt et al. 1993; Florin et al. 1994; Hinterdorfer et al. 1996) inwhich core principles and techniques were established andwhich have gone on to form the basis of much of modern
AFM-based biophysical research. It is from these pioneeringstudies that I have made my top five selection (Table 1).
Although the actual results produced from these pioneeringstudies may not be necessarily particularly refined, they haveall opened new avenues for studying different aspects of biological samples with AFM. In the next section, I offer a short
description of these studies along with a brief justification of
their selection in this list.

Top five list justification
Very soon after the advent of AFM, Paul Hansma and hiscolleagues observed, at ~1-min intervals, the clotting process
by fibrin molecules following the digestion of fibrinogen withthrombin (Drake et al. 1989). This study inspired exploration
into the potential of AFM for the observation of dynamic
biological processes with a few such examples being
antibody-antigen binding (Ohnesorge et al. 1992), live cells
infected by viruses (Häberle et al. 1992), DNA bending uponbinding to λ Cro protein (Erie et al. 1994), DNA digestion
with DNase (Bezanilla et al. 1994), DNA–RNA polymerase
binding process (Guthold et al. 1994), RNA replication reaction (Kasas et al. 1997), and the diffusion of RNA polymerase

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Table 1 Five selected studies

Biophys Rev (2021) 13:455–458

Drake et al. 1989
Weisenhorn et al. 1993
Butt et al. 1993
Florin et al. 1994
Hinterdorfer et al. 1996

The first attempt to visualize dynamic biomolecular processes
The first quantification of elasticity of living cells
The first consideration of scan speed limit in AFM
The first attempt to measure adhesion forces between individual ligand-receptor pairs
The first attempt of recognition imaging

along a DNA strand (Guthold et al. 1999). One of the inventors of AFM, Gerd Binnig, said, “In biology, use of the force
microscope will probably become quite common because of
its ability to deliver films of processes” (Binnig 1992).
However, at the time it took at least 30 s to capture an AFM
image. In this regard, I point out that high-speed AFM capableof filming at ~10 frames per second was established 16 years
later (Ando et al. 2008).
AFM-based mechanobiological measurements are now widely performed, in particular for living normal and cancerous cells(see reviews: Krieg et al. 2019; Runel et al. 2021). The elasticity(Young’s modulus) of living cells was first quantified to be
0.013–0.15 MPa from the force-versus-indentation curve measurements on a lung carcinoma cell (Weisenhorn et al. 1993). In
that study, the measured value of Young’s modulus for the cancer cell was significantly smaller than that recorded for normal
cells, a finding which is now well accepted. Moreover, metastatic
tumor cells are more than 70% “softer” compared with benign
tumor cells (Xu et al. 2012). Thus, this initial study by
Weisenhorn et al. (1993) established the basis of using AFM asa diagnostic tool.
The study by Butt et al. (1993) was the first to provide atheoretical consideration for a scan speed limit in AFM imposedby the cantilever’s dynamic response. This study did not intend
to propose or develop faster AFM but just quantified the scan
speed limit in the constant height DC mode AFM based on
available (at that time) cantilever properties. Nonetheless, beyond
the authors’ intention, this study likely influenced the development of high-speed AFM that was initiated in 1993. A few years
later, prototypic HS-AFM systems and acquired molecularmovies were reported (Vianni et al. 2000; Ando et al. 2001).
Gaub and colleagues were the first to quantify the adhesive
force of a single protein-ligand bond using AFM for thebiotin-avidin pair (Florin et al. 1994). A “bait molecule”(avidin) was attached to the biotin-coated cantilever tip, while
the sample (an agarose bead) was biotinylated. Then, unbinding forces were measured from force-versus-distance (FD)curves, in a pulling process somewhat like fishing. Later, thisstudy was refined by changing the rate of force application,
allowing the quantification of the energy landscape of thebiotin-avidin interaction (Yuan et al. 2000), based on the
Bell-Evans model (Bell 1978; Evans and Ritchie 1997). This
force spectroscopy method has been widely applied to various

biomolecular interactions and unfolding of proteins (seeReview: Puchner and Gaub 2009). Moreover, the rate of force
application has now been enhanced to the level comparable tomolecular dynamics simulations (Rico et al. 2013).
Biological surfaces, such as membrane surfaces, often containa diverse range of proteins and other biomolecules. In such acase, it is difficult to specify and localize individual species froma topography image alone. In the first attempt to detect andlocalize a specific type of species, the just described “fishinglike” experiment (Florin et al. 1994) was extended to twodimensional measurements (Hinterdorfer et al. 1996), where bait
molecules were covalently attached to the cantilever tip via a
flexible linker. To overcome the time-consuming FD curve measurements, a fast method called “TREC imaging” was later
invented (Stroh et al. 2004) in which the cantilever’s upwardand downward swings are separately detected for simultaneous
capturing of recognition and topography images, respectively.
This type of recognition imaging has been used to localize receptors and ion channels on cell surfaces (see Review:
Chtcheglova and Hinterdorfer 2018) and specific binding sites
in protein and DNA molecules (Zhu et al. 2010).

Concluding remarks
It was very difficult to limit my selection to only five papers
among the numerous great works within the AFM field but
this was the brief given with the invitation to write this topfive list. Rather than scientific quality, I have set a high value
on pioneering studies that have influenced or inspired subsequent studies. I apologize to all whose works have not been
selected in the top-five format. There are many great
pioneering studies showing beautiful high-resolution AFM
images of membrane proteins but it was impossible to selectone because of multiple papers published in the same year
(1990). Moreover, I could have included, but did not, many
excellent AFM studies on mapping of material properties suchas flexibility, electrostatic potential, and adhesiveness.
It is hoped that this list may be useful to those just startingout in the bio-AFM field wishing to have some advice on
important historical papers from the perspective of a colleague
who has been working in this area for a long time.

Biophys Rev (2021) 13:455–458
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