Seeing Is Believing – Development of microscopy for biology

                 Understanding the ‘events’ around us through visualization and observation has always been a fundamental part of our philosophical activity and the microscope has played a central role in these processes within the biological sciences. Since the early days of the invention of the light microscopes in the 17th century, vast numbers of pioneering discoveries including ‘Cell Theory’ of Schleiden (1838) and Schwann (1839) have been made by using this key research tool. More recent modifications of the light microscope in relation to oil emission, fluorescence and phase contrast have enabled it to maintain its role as an invaluable research tool in modern biology. However, a major limitation associated with the light microscope is that its resolution is half the wavelength of light. This is the reason scientists and engineers are devoted to the development of new instruments with higher and better resolution.

                The application of AFM to biological samples dates back to the late 1980s. The significance of this microscopy is the achievement of high spatial resolutions similar to EM and of lesser requirements for sample preparation, allowing living matter to be monitored under physiological conditions. The observation of DNA strands by AFM was the first application of this technique to a biological sample. The first notable application was made in the early 1990s, for the observation of double-stranded DNA. This achievement greatly encouraged many biological researchers to jump into the nano-world in the late 1990s. When AFM was invented, scientists’ immediate thought was that it was a potential tool that could be a bridge between light microscopy and X-ray crystallography; i.e., to visualize working molecules under physiological conditions. Unfortunately, the slow imaging speed of the device at that time made it impossible to directly visualize the molecules in action. An extraordinary improvement in the device was made by Ando’s group at Kanazawa University in 2001. The increased temporal resolution of several frames per second (fps) in the newly developed ‘fast-scanning AFM’ allows the action scenes of biological molecules to be monitored more closely in the sub-second time scale.

                In addition to molecular imaging capability, AFM has another capability of force measurement to measure the elasticity of living cells. When an AFM cantilever approaches and pushes against the cell surface, a large indentation in the cell and its surface is usually observed when the probe first contacts the cell surface. This indentation can be plotted against the force of the cantilever and fitted to the Hertz model equation to estimate the Young’s modulus, which describes the elasticity of the sample. The actin network may be responsible for the elasticity of the cell. Elasticity measurements have shown that both the plasma membrane and the nuclear envelope are “flexible” enough to absorb a large deformation formed by an AFM probe. Penetration of the plasma membrane and the nuclear envelope are possible when a probe with a sharp tip (tip angle of ~25 degree) deeply indents the cell membrane, causing the membrane to come close to a hard glass surface. These types of experiments will provide useful information for the development of single-cell manipulation techniques that are applicable to the evaluation of cell properties under physiological and pathological conditions. The recent development of recognition imaging using the TREC™ mode has enabled identification of a specific molecule in the AFM image. It is possible to simultaneously obtain a topographic image and also the position of a specific interaction caused by attractive forces between the specimen and the protein- (e.g., specific antibody) coupled cantilever.