The importance that intracellular pH plays in cell biology has been highlighted in a new paper published by the American Chemical Society. Written by a group of researchers from Harvard Medical School led by Professor Gary Yellen, it reports the engineering of the first ratiometric, single-protein red fluorescent sensor of pH, called pHRed, and its use to image intracellular pH in live neuronal cells.
Using Andor's Revolution DSD spinning disk confocal technology, the team also established the unique capability of pHRed to be used in simultaneous measurements of intracellular ATP and pH because of its spectral compatibility with a second ratiometric ATP sensor, green fluorescent protein (GFP)-based Perceval. Further, the team showed that pHRed provides a new tool for imaging pH in thick samples and in vivo when imaged by two-photon FLIM (fluorescence lifetime imaging microscopy).
'Sensitivity to pH is a critical problem for GFP-based sensors and can cause severe artefacts if not taken into account,' said Dr Yellen. 'Our multicolour imaging experiment illustrates the value of pHRed in providing a simultaneous pH signal. This can be used to correct the pH sensitivity of a green sensor and optical sectioning with excitation ratio-imaging enables access to intracellular compartments and organelles.'
Cellular pH, though often ignored, is a key parameter that affects not only most cellular processes but also the sensing properties of most of the available genetically encoded fluorescent indicators (GEFIs). Because of its red emission colour, pHRed may be used simultaneously with any of the many green or yellow fluorescing GEFIs to correct for their pH sensitivity as well as to give intrinsically interesting information about pH changes.
'We chose the Andor Revolution DSD structured illumination system because of its speed, flexibility and price,' commented Dr Yellen. 'This spinning disk system is compatible with a range of non-laser light and filters are user-selectable to suit individual applications. Compared to expensive, maintenance-heavy, laser-based instrumentation, the DSD is ideal for individual laboratories. In eliminating the need to share with other research groups, the DSD allowed us to optimise the experimental set-up and experimental scheduling.'
DSD offers simple and flexible confocal imaging in a cost-effective add-on to existing upright or inverted microscopes. Capable of delivering high contrast, low background images of fixed and live specimens, it provides a flexible alternative to laser scanning for individual researchers and small laboratories.
