Melanopsin, a tri-stable photopigment found in intrinsically-photosensitive retinal ganglion cells (ipRGCs), drives circadian rhythms and other non-image forming functions in the nervous system. Despite increased understanding of the biomolecular and spectroscopic properties of melanopsin, its multiphoton and ultrafast optical absorption properties remain underexplored. We demonstrate the effects of two-photon absorption of melanopsin using 900-1160 nm optical stimulation. Excitation in this bandwidth causes consistent increases in calcium levels in transfected HEK293T cells. Our results demonstrate the first reported nonlinear optical properties and corresponding functional responses of two-photon excitation of melanopsin in vitro, along with the effects of spectral-phase modulation on activation.
The anomalous diffusion characteristics of neuronal dynamics are analyzed by label-free, phase-sensitive optical coherence microscopy. The technique provides low-noise images, enabling cellular dynamic characteristics to be measurable. The phase variance is a conventional dynamic parameter that cannot elucidate the ballistic components of neuronal dynamics. Determining the dynamics by phase variance alone omits the ballistic information that can occur from the ion exchange across cellular membranes. The probability density function of phase displacements exerted by cellular dynamics was acquired and the shape of the power-law tail was analyzed. The development of the power-law tail provides a more sensitive dynamic feature.
We present Superfast Polarization-sensitive Off-axis Full-field Optical Coherence Microscopy (SPoOF OCM) as a novel all-optical technique for neurophysiology. Both the optical path length and birefringence induced by the millisecond-scale electrical activity of neurons are captured by SPoOF OCM at 4000 frames per second and with a field-of-view of 200×200 µm sq., 1 µm transverse resolution, 4.5 µm axial resolution, and 300 pm phase sensitivity. With an ability to capture responses spanning three orders of magnitude in both space and time, SPoOF OCM meets the exacting needs of a comprehensive neurophysiology tool and overcomes the existing limitations of traditional electrophysiology and fluorescence microscopy.
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