FEATURE MICROSCOPY
Light labels illuminate cell biology
Jessica Rowbury looks at the development of new fluorescent proteins and label-free imaging methods, helping scientists probe deeper into tissue
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n recent years, super-resolution fluorescence microscopy has evolved at a fast pace; scientists can now image at a nanometre scale to observe molecular interactions and dynamic processes. One limitation of fluorescence microscopy, however, is that the red fluorescent probes used for labelling tissue specimens are not very bright or efficient. To solve this, researchers are developing a new group of near-infared fluorescent proteins for enabling greater tissue depths with improved sensitivity.
Meanwhile, in separate research, scientists are advancing label-free live imaging
30 ELECTRO OPTICS l OCTOBER 2016
techniques that, for certain applications, eliminate the drawbacks associated with using fluorophores, such as phototoxicity. The availability of bright fluorescent markers that can highlight molecules and structures specifically and efficiently is central to successful fluorescence microscopy. For live imaging, the green fluorescent protein (GFP) family of markers have become the gold standard, because of their ability to be genetically encoded and expressed in cells, resulting in a fusion construct with the protein of interest. This provides a powerful and versatile technique for labelling live samples. Since
its discovery in the 1960s, scientists have worked on optimising marker proteins of the GFP family, and the colour palette has been extended from the blue to the far-red side of the spectrum. It’s safe to say that the green fluorescent protein and its colour-shifted genetic derivatives have revolutionised live cell imaging. Far-red emitting fluorescent proteins (FPs)
are advantageous as markers for live specimens for multiple reasons. Not only is there less absorption, scattering and autofluorescence in the red part of the spectrum, but red light is less phototoxic, and far-red FPs offer the possibility of realising an additional detection channel for multi-colour imaging. However, despite the urgent need for far-red
FPs, researchers have not yet succeeded in engineering markers that can compete with the performance of GFP. Existing far-red FPs do not go far enough into the infrared to emit
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