Representative plots of different linear chirps on peak currents from patch-clamp electrophysiology.
In addition to imaging, the Biophotonics Imaging Laboratory has made optical developments to facilitate activation of neural tissue using optogenetics. Ultrafast lasers have been used to activate neural excitation using optogenetic probes, but modulation of the spectral phase to more finely drive this process has not been leveraged in the field. By spectral-temporal modulation of incident optical pulses, we have demonstrated that we can control the peak amplitude response of single neurons, in addition to altering their natural firing patterns. Due to the ultrafast nature of the optical pulses and of many of these opsins, we are continuing to explore these mechanisms in other opsins to see how different pulse shapes affect the firing patterns and communication of single neurons at numerous timescales.
Representative electrophysiological recordings of fast-spiking interneurons after illuminating with ultrafast light.
Optogenetics has been utilized widely in the past two decades for activation of collections of neurons to understand brain function. Recent advancements in optogenetics have leveraged ultrafast optics for multiphoton activation of opsins, which promotes increased optical depth penetration, reduced out-of-focus activation, and single-cell precision. The spectral-temporal properties of the incident light’s effects on opsins, however, have seldom been investigated. Initial results on this phenomena, known as coherent control, in ChR2 shows that the phase between multiple wavelengths of light in a broadband laser pulse promotes notable differences in single-neuron firing properties. As this technology is furthered, its implications in other opsins and with combinations of different chromophores of interest in neuroscience will continue to be investigated.
Peak-currents acquired from single-neurons after ultrafast optogenetic activation. Note the asymmetric peak amplitude from this activation (C) compared to the symmetric, normalized intensity SHG intensity (d).
Multiphoton optogenetics and calcium imaging have found increasing utilities in the neuroscience field for a variety of applications. The use of infrared light sources promotes increased penetration depth, reduced out-of-focus excitation, and thus facilitates single-cell precision for activation and detection of neural activity. Many commercial and academic systems require the use of multiple lasers to minimize cross-talk between these molecules, and promote chromatically-distinct excitation of these molecules. Furthermore, additional dispersion-compensation optics need to be added for each laser path to ensure near transform-limited pulses arrive at the sample to optimize image quality. In this work, we leveraged the supercontinuum technology developed in the Biophotonics Imaging Laboratory to create separate optical paths for imaging calcium activity using GCaMP6s, and single-cell stimulation using C1V1-mCherry—both from a single supercontinuum source. Our results demonstrate that by using a photonic crystal fiber (PCF) to generate a supercontinuum of light, single-source calcium imaging and stimulation can be realized, reducing the need for multiple bulky lasers for the neural sciences.
Left: System diagram for single-source stimulation and activation of cellular activity. Right: Representative optogenetic activation results for continuous-wave (CW) stimulation, (B), and two-photon stimulation (D).
- Liu Y-Z, Renteria C, Courtney CD, Ibrahim B, You S, Chaney EJ, Barkalifa R, Iyer RR, Zurauskas M, Tu H, Llano DA, Chistian-Hinman CA, Boppart SA. Simultaneous two-photon activation and imaging of neural activity based on spectral-temporal modulation of supercontinuum light. Neurophotonics, 7:045007, 2020.