Shedding light on life

[PointRadix]

physicsworld.com – May 30, 2008

Visualizing and understanding complex biological systems at the cellular level is an endeavour that requires the best efforts of both biologists and physicists.

Since the discovery of X-rays in 1895, curiosity as well as clinical need has produced huge advances in our ability to visualize structures inside the human body. X-ray radiographic imaging led the way, followed in 1957 by the use of gamma-ray cameras, and then ultrasound imaging, magnetic resonance imaging (MRI), and positron emission tomography (PET). Together, these tools, in which physics plays a key part, underpin modern clinical-imaging practice. The length scales that these techniques interrogate, however, leave much to be desired if one is interested in the very small. Clinical MRI, for example, can only resolve structures down to 100 µm. While some living cells are more than 80 µm across, interesting and important cellular processes — such as signalling between cells — may take place over length scales of much less than 1 µm.

Living cells are essentially defined by their complex spatial structures — for example the doughnut shape of red blood cells and the elongated projections (known as axons) of neuronal cells are key to their functions. Underlying these broad morphological characteristics, however, are much finer-scale molecular assemblies, such as the cytoskeleton (a protein “scaffolding” that stabilizes the larger-scale intracellular structures) and micro-domains within cell membranes, which are the locii of many molecular signalling events. Any technique to study the properties of biological molecules and their many interactions should ideally provide spatial information, because researchers increasingly need to integrate information about the interactions that underlie a biological effect with data on where in cells these interactions take place. Short wavelength X-rays can be used to provide information on very small length scales and can even produce images of individual molecules — as in X-ray crystallography. The downside is that such radiation can severely damage biological materials. X-rays with slightly longer wavelengths and lower energy — which can be produced by synchrotrons — are, however, much less destructive to tissue. Indeed, these “soft” X-rays can be used to generate 3D images of living cells with a resolution of up to 10 nm, as has been shown by research at the Advanced Light Source synchrotron at the Lawrence Berkeley National Laboratory in the US. Unfortunately, even soft X-rays are destructive and difficult to handle, and they require access to a synchrotron.

Most techniques used for cellular imaging, therefore, tend to be optical, exploiting mainly the ultraviolet and visible parts of the electromagnetic spectrum. Of these techniques, fluorescence microscopy has become the most important because it is very sensitive, enormously versatile and relatively easy to implement. Fluorescence is the phenomenon by which certain molecular structures, known as fluorophores, emit photons when excited via irradiation with light of a specific wavelength. This emission typically occurs over a timescale of 1–10 ns, which is suitable for measuring the movements and re-orientations of molecules within cells, thus allowing many biological processes to be followed. In rare cases, the biological molecule of interest is inherently fluorescent. But usually fluorescence has to be “built into” the molecules that researchers wish to study by tagging them with a fluorophore. The tag can even be a whole protein in its own right, such as the green fluorescent protein (GFP), which is attached to and expressed at the same time as the protein of interest and only fluoresces when both are actually manufactured by the cell.

The energy, momentum, polarization state and emission time of photons emitted by fluorophores can all provide vital information about biological processes at the microscopic and nanoscopic scales. The polarization of fluorescence photons, for instance, is affected by the orientation of the fluorophore — and hence of any protein to which it is attached — and can therefore provide information about the molecular dynamics of the molecule of interest. Much work is being done by physicists and biologists that takes advantage of each of the attributes of fluorescence.

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