FLUOLAPS · A two-photon electrochemical and fluorescence microscope for imaging of cell-surface interactions

A two-photon electrochemical and fluorescence microscope for imaging of cell-surface interactions

owadays, interest in the study of structure, function and characteristics of living cells, which are concerned with our understanding nature and ourselves, has increased. Cells are equipped with a host of receptors that can transduce chemical signals into electrical ones. If efficiently coupled to an electronic readout device, cells could thus function as versatile biosensors in a variety of applications [1]. Concentration changes of extracellular metabolism products are induced by the transformation of intracellular physiological status. Thus, investigations concerned with the extracellular environment or extracellular action potentials interest many researchers deeply. Light-addressable potentiometric sensors (LAPS) can be used to measure changes in the surface potential, thus determining a particular analyte of interest with spatial resolution [10,11,12,13,14]. LAPS are based on an electrolyte/insulator/silicon (EIS) field-effect structure that can be addressed by scanning a focused light beam across the sample, thereby exciting local photocurrents [9]. The potential changes are measured as a shift of the depletion region of the photocurrent voltage curve along the voltage axis. Scanning photo-induced impedance microscope (SPIM), which was developed by Krause et.al [15], works in a similar way to LAPS. It is also based on a field effect metal/electrolyte-insulator-semiconductor structure except that the applied voltage results in an inversion layer, thus it measures the impedance change in the system. One of the most challenging parts of this project was to obtain a perfect insulator which permits high-resolution LAPS measurements. In order to develop the insulator we chose organic monolayers, which have the advantages that they are easy to produce using self-assembly and allow further synthetic modifications. Two different linera hydrocarbons, one a C14 alkene and the second a C16 alkyne, have been attached to the sensor surface to produce high quality monolayers. Further chemical modifications were successfully carried out providing a hydrophilic surface sensor, modified with a specific peptide suitable for cell growth. As a model of neuronal cells, organic microcapsules were attached to the surface. High resolution and good sensitivity were achieved using a two-photon effect for charge carrier excitation and organic monolayer modified silicon on sapphire (SOS) as the SPIM substrate. SPIM allowed impedance imaging of collapsed microcapsules with unprecedented detail. SPIM images of capsules labelled with gold nanoparticles (AuNPs) showed a good agreement with the corresponding optical images, including the creases resulting from the collapse of the hollow shells. The significant increase in impedance caused by the impregnation with AuNPs was also verified by conductive Atomic Force Microscopy (C-AFM) measurements in the dry state. These improvements in the monolayer modification provide good quality substrates for recording 2D images of a single neuron and the measurement of the changes in the photocurrents where the neuron is placed. This technique should in the future be capable of producing electrical images of the surface attachment area of one single cell. The obtained results have an important socioeconomic impact due to the fact that the study of the neuron response provides a better understanding of the neuronal communication which can be use in the future to investigate disease mechanisms of degenerative diseases such as Alzheimer’s. So, the obtained results in this project will have a high impact in the society. Further information about the project and the results can be found on the group webpage: http://webspace.qmul.ac.uk/skrause/SPIM.html

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