Research activities are increasingly focusing on the identification and the spatial and temporal distribution of e.g. local “hot spots” for dynamic changes in ion concentration, voltage or pH in a cell or a cellular network. Such “hot spots” are often localized in specialized parts of a cell or in certain cells in a cellular network. Additionally, these areas often have different properties compared to the rest of the specimen in terms of cell metabolism or structure. Conventional fluorophores used to investigate dynamic physiological states change their emission intensity upon ion binding, pH change or voltage change (e.g. fluo-4 has increased emission upon calcium binding). However, these markers do not take into account that differences in structure, diameter or marker uptake/expression can cause changes in the quantity of emitted light that are not in correlation with the actual ion concentration, voltage or pH. To quantitatively and comparably detect the changes in cellular structures or different cells, a method insensitive to structure diameter and fluorophore concentration is needed. In contrast to non-ratiometric imaging methods, ratiometric imaging offers the opportunity to reproducibly measure absolute intracellular ion, voltage and pH levels/changes with respect to cell diameter, fluorophore concentration and optical properties of the imaging setup. However, ratiometric imaging depends on a fast change of excitation wavelength or the detected wavelength, a strong light source, excellent transmission of optical components and fast signal detection. The recent development of ultrafast filter wheels, UV-light optimized objectives, highly sensitive fluorophores and new
Analyzing Ion Concentrations by Measuring Fluorophore Shifts
Many fundamental functions of a cell strongly depend on delicate, but nevertheless dynamic balances of ions (e.g. calcium, magnesium), voltage potentials and pH between the cell’s cytosol and the surrounding extracellular space. Changes in these balances significantly alter a cell’s behavior and function. Therefore, measurements of intracellular ion, voltage and pH dynamics in real time are of tremendous interest for researchers in neuroscience, cell biology and cell physiology in general. In many cases, however, exact estimations of actual ion concentrations or relative changes in different locations in a cell or a cellular network are difficult with conventional fluorescence methods. The reason is that these methods do not take account of the fact that differences in cell morphology within different parts of a single cell or between cell types in cellular networks might influence the quality and quantity of emitted light. This can lead to substantial misinterpretations when dynamic changes of ion concentrations, voltage or pH are investigated. Ratiometric imaging techniques bypasses these issues by observing emission wavelength shifts of fluorophores or by comparing the emission intensity of a fluorophore combination instead of measuring mere intensity changes.