Functional mapping of the connectivity of the brain is an essential procedure for neurosurgeons aiming for maximal resection of a brain tumor, while minimizing damage to the eloquent cortex. Being able to delineate the location of critical cortical areas in individual patients allows the surgeon to preserve sensorimotor and cognitive functions. Several methods exist for functional mapping, however, none are capable of delivering fast, label free mapping of the cortex with millimeter precision. In this work, we investigate the potential use of infrared neural stimulation (λ=1470nm) as a means of focal stimulation of the mouse cortex in vivo. Here, we show that the application of infrared neural stimulation induces in vivo cortical intracellular calcium signals in Layer II/III mouse neurons, using intravital calcium imaging with the genetically encoded calcium indicator GCaMP6f.
Infrared laser light radiation can be used to depolarize neurons and to stimulate neural activity. The absorption of
infrared radiation and heating of biological tissue is thought to be the underlying mechanism of this phenomenon
whereby local temperature increases in the plasma membrane of cells either directly influence membrane properties or
act via temperature sensitive ion channels. Action potentials are typically measured electrically in neurons with
microelectrodes, but they can also be observed using fluorescence microscopy techniques that use synthetic or
genetically encoded calcium indicators. In this work, we studied the impact of infrared laser light on neuronal calcium
signals to address the mechanism of these thermal effects. Cultured primary mouse hippocampal neurons expressing the
genetically encoded calcium indicator GCaMP6s were used in combination with the temperature sensitive fluorophore
Rhodamine B to measure calcium signals and temperature changes at the cellular level. Here we present our all-optical
strategy for studying the influence of infrared laser light on neuronal activity.
Interest in the interaction between laser light and biological samples has gained momentum in recent years, particularly in neurobiology, where there is significant potential to stimulate neurons with infrared laser light. Despite recent reports showing the application of infrared light for neurostimulation, the underlying mechanism is still unknown. The two main hypotheses are based on thermal or electrostatic mechanisms. Here, a novel optical method is presented to make temperature measurements in human neural cells under infrared laser excitation (λ=800nm) using the dye Rhodamine B (RhB). The measurement of temperature is based on the property of RhB, a fluorescent dye whose fluorescence intensity decreases linearly with increases in temperature. We present and detail the setup and measurement procedure that has temporal resolution of few milliseconds, based around a fluorescent live-cell imaging microscope used for cellular microfluorimetry experiments.