An Overview of Cell Adhesion
What is Cell Adhesion?
Introduction to Cell Adhesion
Adhesion describes the tendency of two surfaces or particles to cling to one another. The forces that cause this adhesion can differ significantly but depend on the nature of the surfaces/particles and include mechanical, chemical, and electrostatic forces, among others. Adhesion is an essential process for a variety of applications that range from material coatings to colloids to biological applications. In biology, this phenomenon is key to the formation and function of a vast range of organisms, from microorganisms like bacteria to complex multicellular organisms. In bacteria, cell adhesion contributes to microbial cell development, metabolic activity, and cell viability.  In mammalian cells, cell adhesion plays a fundamental role in formation of three-dimensional tissues during development, tissue maintenance and disease. 
More specifically, adhesion reflects how cells behave when they come into contact with other cells or surfaces. These contacts are mediated by a complex interplay of dynamic interactions at the cell-cell or cell-surface interface, which provide the platform by which signals from neighboring cells and the underlying substrate are transmitted to intracellular signaling pathways that ultimately regulate cell behavior. Understanding how these interactions contribute to biological processes thus requires the study and measurement of adhesion forces at the molecular, single-cell and population/tissue scale.
The multidimensional role of adhesion in many fields like cell biology, biophysics, and biomedical engineering, has led to the development of tools to quantify adhesion forces from the nano- to the micro-scale.
Existing methods to study cell adhesion
A broad range of techniques have been developed to measure cell adhesion, including micropipette aspiration, optical and magnetic tweezers, and atomic force microscopy (AFM).  AFM was first developed in the 1980s as a surface-imaging tool for material sciences , but it has been increasingly used in biology research. AFM can scan and probe sample surfaces non-invasively with a resolution down to the nanometer. Thanks to its compatibility with aqueous environments, the AFM allows for measurements of cell properties in near-physiological conditions with the possibility of precisely controlling the liquid medium and environmental conditions such as temperature and humidity. This instrument has proven its power/versatility in research, notably with the development of the single-cell force spectroscopy (SCFS) mode on the AFM. Several AFM-based tools and methods have been employed in the literature to perform cell adhesion measurements. In the early stage of direct force spectroscopy measurements for biological samples, the colloidal probe technique was commonly used in interface science and biomechanics. This technique is also based on AFM but instead of having a sharp AFM tip, it makes use of a colloidal particle glued to the end of the AFM cantilever.  Nevertheless, the fast development of SCFS was a game-changer. Indeed, AFM-based SCFS has become the most widely used tool to measure cell-substrate adhesion forces, as it is both versatile and highly precise. Briefly, in a SCFS setup, cells are attached to the end of an AFM cantilever and the cantilever deflects proportionally to the cell detachment force.  In addition to this straightforward working principle, SCFS has proven to be a particularly powerful approach to quantify adhesion under physiologically relevant conditions and at the single-molecule scale.  While the above approaches allow for precise quantification of adhesion forces, the use of AFM for adhesion measurements in live cells comes with a number of intrinsic limitations.
Limitations of the current state-of-the-art of cell adhesion measurement techniques
In the following section, the FluidFM technique is compared to “traditional” AFM-based techniques for SCFS. The experimental setup preparation, the workflow and performance (throughput and force range) with respect to single-cell adhesion measurements, are considered. Advantages of FluidFM technology for SCFS, with a focus on the key limitations that it overcomes, are laid out below.
Firstly, adhesion measurements with AFM-based SCFS are time consuming and low-throughput. To achieve firm adhesion of the cell to the cantilever for force measurements, the cantilever must be functionalized first. This chemical functionalization of the cantilever “glues” the cell of interest to the cantilever irreversibly,  which means that each cell requires a separate functionalized and calibrated cantilever, a process that can take 30 min.  This approach is thus low throughput, with a complex and time-consuming cell attachment/detachment methodology. Importantly, the process of immobilization could also alter cell physiology by affecting the cell surface. Secondly, the force range of measurements with current AFM-based SCFS is limited by the force with which the cell is bound to the cantilever, meaning that that only forces below the coupling strength of the cell to the force sensor can be measured. This approach is thus intrinsically limited to measurements in the early phases of cell adhesion formation, unable to provide the kinetics of adhesion forces at longer timescales (i.e. mature cell-cell contacts). [10,11] This first generation of SCFS setup allowed researchers to reach another level of single-cell analysis and manipulation. Yet, the creation and development of the FluidFM technology for SCFS went beyond the initial capabilities of the technique by overcoming several of the limitations presented by the conventional AFM. 
Single-cell adhesion enabled with the FluidFM technology
By replacing conventional chemical fixation of cells in AFM-based SCFS with under-pressure immobilization using FluidFM, this solution takes SCFS to a new level. The FluidFM picks up cells by applying negative pressure at the aperture of the hollow cantilever, which serves as a micro- or nano-pipette. This step occurs within a few seconds and thus drastically reduces the time required to obtain statistically relevant data compared to conventional SCFS.
Features and benefits of single-cell adhesion measurement
Direct and semi-automated force measurement
Through a simple and reversible cell immobilization
Broad force range
Measure forces from pN up to µN!
Measure up to 200 cells a day! Isolate single-cell, accurately and reliably.
Applications of single-cell adhesion measurement
In the following, single-cell adhesion measurement demonstrates its potential in a broad of biological applications and studies.
Data - image courtesy of Tanya Konry group, Northeastern University, Boston, USA. 
Study of tumor progression and metastasis with cell-cell adhesion forces
This work was conducted with the Nanosurf Flex-FPM by Dr. Noa Cohen, group of Prof. Tanya Konry, Northeastern University in Boston, on cell-cell adhesion forces to gain more insights into tumor progression and metastasis. 
On the left-hand side, three optical images illustrate:
(A) a single cell to be picked up by a FluidFM Probe
(B) a single cell aspired to the cantilever
(C) the FluidFM Probe with aspired cell during a cell-cell adhesion measurement.
Below, two typical force curves were obtained to illustrate the interaction between a MCF7 cell aspired to the cantilever and non-cancerous, fibroblast (HS5) on the substrate at different contact times. Over time, the cell-cell interaction evolved as depicted in the curve (B). 
Data - image courtesy of Tanya Konry group, Northeastern University, Boston, USA. (A) Typical force curves between a MCF7 cell aspired to the cantilever and non-cancerous, fibroblast (HS5) on the substrate at different contact times. (B) Development of the force with contact time between the cells. 
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