Observing living cells - up close and personal

(Nanowerk Spotlight) Cells are the smallest 'brick' in life's building structures. Every living organism is made of cells. Individual cells carry their own DNA and have their own life cycle. Considering that larger organisms, such as humans, are basically huge, organized cell cooperatives, the study of individual live cells is a hugely important scientific task. Among the most significant technical challenges for performing successful live-cell imaging experiments is to maintain the cells in a healthy state and functioning normally on the microscope stage while being illuminated. Especially if scientists want to look into cellular processes that occur in cells in their natural state and that cannot be observed by traditional cytological methods. It is well known that cells move, grow, duplicate, and move from point A to point B. Up to now people studied these mechanical properties with optical microscopes because it is the most common and simple method, very efficient, a very well developed and advanced technology. However, with optical microscopes detection is limited to objects no smaller than the wavelengths of the visible region of light, roughly between 400 and 700 nanometers. Distances or movement smaller than this range cannot be seen with these instruments. Researchers in Kyoto, Japan have applied a near-field optical approach to measure cell mechanics and were able to show intriguing data of nanoscale cell membrane dynamics associated with different phenomena of the cell’s life, such as cell cycle and cell death.
Special devices and methods have to be used to perform high-resolution investigations of cell structures. Usually the cell has to be killed and then SEM, TEM, or other imaging techniques will be used. The only available technique to measure mechanical movement of a live cell, with high resolution, probably is the Atomic Force Microscope. Here, the cell's natural life activity is disturbed by the presence of a cantilever, a metal sensor that is practically in contact with the cell membrane, and could lead to pressure on the cell or other perturbations.
"We have successfully used Scanning Near-field Optical Microscopy (SNOM) to measure the natural beating motion of heart cells and we demonstrated its ability to identify a specific signal associated to different crucial cell phenomena of the cell’s life, such as cell cycle and cell death" Dr. Ruggero Micheletto explains to Nanowerk. "A near-field microscope uses a very sharp optical fiber, called a SNOM probe, which we placed near the cell, about half a micron away from the cell membrane. The cell is kept undisturbed in its culture media. Any movement that occurs is detected by this nano-probe because of the extreme sensitivity of the optical field to sample-to-probe distance. We demonstrated that vertical displacement of about 0.1 nanometers can be detected. This is a purely optical approach, so the cell receives no mechanical disturbances and actually it 'does not know' that it is under scrutiny. So it is a non-invasive, non-intrusive super-high sensitivity detection method."
Micheletto, a post doctoral researcher in professor Yoichi Kawakami's Optoelectronic Materials Science and Engineering lab at Kyoto University, co-authored a recent paper ("Acoustical nanometer-scale vibrations of live cells detected by a near-field optical setup"), together with Dr. Kawakami and Dr. Rosaria Piga, a researcher at the Human Stress Signal Research Center at AIST in Osaka.
In this SNOM approach the scientists measure the optical coupling between cell membrane and the optical probe. If the membrane vibrates, the coupling changes and one can observe a signal variation with an extremely high sensitivity to small displacements.
The fundamentals of the optical process involved are not completely known. Nano-apertured SNOM probes – like the one the team in Japan uses – are very sensitive to sample probe separation. However, the signal that is measured is also influenced by other processes, like absorption, refraction index changes, or any phenomenon that may perturb the optical propagation. In other words, if there is a change in some inner part of the cell a researcher may observe optical variations that could be confused with a mechanical vibration.
Micheletto explains that nobody knows how the nano-movement they observed are important to the cell life-cycle. "Even if we did demonstrate that the vibrations do actually convey information about the physiological status of the cell, we do not know how this is important to the observed cell, or to the surrounding cells. Since cells naturally are in contact with one another, it may be possible that cells are sensitive and actually 'feel' vibrations of adjacent cells, and maybe in fact communicate each other by these small movements. This would be a great discovery. However we may be wrong, maybe these movements are natural vibrations, more related to spontaneous Brownian motions than something regulated internally by the cell."
"A detailed analytical three dimensional model of the optical process is required to discern and distinguish processes, but this has not yet been developed and it is a very difficult task" notes Kawakami.
"Our findings could well imply a wider use of SNOM as a scientific as well as medical tool to reveal new aspects of cell biology, for instance for the discovery of dynamic nanomechanical activity still unknown and eventually associated to a physiological, morphological, or pathological state of the cell" says Piga. "It is already known that some pathologies are correlated with a number of abnormal activities such as the emission of specific substances from cells, or their inglobation into cells; an increased cellular division; a different kind of cell death; or a rearrangement of organelles, molecules or cytoplasmic material inside the cells. Any of these aspects could potentially be detected with SNOM observations and associated with a specific pathology, thus increasing the possibility of a successful diagnosis."
The researchers used P12 cell (a rat pheochromocytoma cell line) samples that are not known to have evident membrane movements, to demonstrate for the first time that cells membranes do show natural nano-sized movement. They showed that these vibrations do actually convey information about the status of the cell, using different drugs and analyzing how vibration Fourier spectrum changes depending on the drug.
"We believe this field is rather new" says Kawakami. "Cell membranes are known to vibrate or move only in special kinds of cells – for example cardiomyocytes where cells 'beat' naturally. Our new technique will allow studying these cells with higher sensitivity. However, the technique is also opening a new door to the study of much finer membrane movements as the one we observed in P12 cells. So far, the significance of these nanoscale movements is unknown and we hope that other scientists will follow our approach and be able to investigate and discover more about the nature of these mechanical processes."
The researchers are now planning to verify if cells are sensitive to nano-mechanical movements. They are planning to do that using their technique, coupled with nano-actuators to reproduce artificially similar movements, and observe if cells react to these mechanical stimuli or not.
Micheletto cautions that there are quite a number of technical difficulties and fundamental biophysical problems that need to be clarified. For example: if cells are actually sensible to nano-mechanical stimuli, what is the information conveyed by them? Is there a sort of language involved? "In other words: can we induce a particular cell behavior by a specific mechanic stimulus? These are open questions that arise from our work."
By Michael is author of three books by the Royal Society of Chemistry:
Nano-Society: Pushing the Boundaries of Technology,
Nanotechnology: The Future is Tiny, and
Nanoengineering: The Skills and Tools Making Technology Invisible
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