The human brain consists of approximately a hundred billion of neurons that communicate through a unique series of biochemical and electrical processes. When neuronal networks are solicited, electrochemical processes happen involving millions of cells within a complex network. The morphology of the neuronal cytoskeleton is fundamental in the establishment of these complex neuronal networks and crucial for the functional integrity of electrical signaling. Traditionally, the neuronal morphology and electrical signals are measured with invasive optical probes, exogenous dyes, or by exogenous electrical recordings. To fully understand the underlying mechanisms involved in neuronal activity and for eventual clinical applications a direct label-free non-invasive optical probe is of great significance. In this thesis we demonstrated the possibility of label-free imaging of neuronal structures and electrical neuronal activity employing the unique intrinsic sensitivity of nonlinear optical techniques.
We developed a 3D wide-field second harmonic (SH) imaging system that increases the SH imaging throughput for label-free elastic SH imaging by several orders of magnitude. The increase in throughput was achieved with a wide-field geometry and medium repetition rate laser source in combination with gated detection. In addition dynamic and ultrafast measurements can be performed readily with different possible polarization configurations.
First, we performed label-free SH and two photon excitation fluorescence (2PEF) imaging of living cultured neurons with short acquisition time and at very low fluences. We demonstrated the use of wide-field high throughput SH microscopy for investigating dynamic changes in cytoskeletal morphology on the single cell level. The method allows real-time in vitro label-free measurements of cytoskeletal changes that can, under certain conditions, be quantified by orientational distribution or changes in the number of microtubules.
Then, we investigated the changes in neuronal morphology and metabolic activity by performing label-free SH polarimetry. We calculated the coefficient of polarization, which reports on orientational irregularities in the microtubule cytoskeleton, and used endogenous 2PEF as a metabolic marker in cultured neurons throughout the stages of their morphological development. Being able to observe morphological changes in the cytoskeleton in a label-free way with clear markers of organization in combination with indicators for metabolic activity, allows us to follow neuronal differentiation in detail, and in a non-invasive way.
Finally, we employed SH imaging to label-freely capture direct information of neuronal membrane potentials. We performed SH imaging of cultured neurons undergoing a depolarization by a temporary extracellular excess of K+ ions. To demonstrate the concept, we performed a patch-clamp and SH imaging comparison and showed that whole neuron membrane potential changes correlated linearly with the square root of the SH intensity. Finally, we used the nonlinear optical response of the membrane bound water to create membrane potential and ion flux maps of living cultured neurons in real time.
With these results obtained with the 3D wide-field high throughput SH microscope, we demonstrated the possibilities to image in time, label-free and with low fluence, neuronal structural changes and electrical neuronal activity employing the unique intrinsic sensitivity of nonlinear phenomena.