In RBCs, FRET can occur, e.g., between the dye Fura-red and haemoglobin (unpublished results). It must be noted that FRET can also be used in a beneficial way, as nicely shown by Esposito et al.89 for imaging the haemoglobin concentrations in malaria-infected RBCs. Yet another factor that influences the fluorescence intensity is RBC

volume changes because a change in volume results in a change in the dye concentration and hence an altered fluorescence signal. Fortunately, most of the above mentioned sources of artefacts are rather small and Forskolin might be neglected when the observed signals are robust. However, if minute signals are expected or observed, the artefacts are likely to become relevant. An almost unavoidable artificial situation in live cell imaging is the fact that the RBCs are attached to a (coated or uncoated) coverslip. The only way to exclude artificial conclusions is the comparison/combination check details with complementary methods. Last but not least, live cell imaging is often used to detect hormonal or pharmacological stimulation of RBCs. To have a proper control of the solution surrounding the cell, a local perfusion (a micro-manipulator-associated cannula placed close to the RBCs to apply a laminar flow) is preferred over an exchange of the bulk solution of the entire dish that almost certainly would lead to slow gradients of the exchanged

solutions and a loss of control concerning the timing of the drug or hormonal stimulation.

Because RBCs contain a number of mechanically sensitive proteins,38 one has to make sure that the flow does not change with the application, and therefore, the flow must be kept constant (also under control conditions) and just the solution composition needs to be switched from the battery of solutions. Adhesion is traditionally measured by either microscopic investigation, quantifying a microscopic aggregation index90 or by indirect methods based on the Glutathione peroxidase properties of RBC suspensions. Such techniques include sedimentation-associated procedures, transmission light or ultrasound scattering, impedance measurements, determination of viscosity or other rheometric methods.91 The classical methods to measure RBC aggregation have been recently reviewed.92 However, with regard to adhesion force measurements, a focus was set to rheometric techniques.[93] and [94] These methods are all indirect and suffer from a limited amount of information on the number of cells involved or the impact of RBC morphological and deformability changes. Recently, two quantitative RBC intercellular adhesion measurements were introduced at the single-cell level and compared to each other.[95] and [96] The two techniques are holographic optical tweezers (HOT) and atomic force microscope-based single cell force spectroscopy (SCFS). To exert forces on cells with optical tweezers, a limited force regime is available due to cell damage with increasing laser power, i.e.