Optical excitation and detection of neuronal activity

Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can modulate behavior in live organisms. However, detecting the response to the optical stimulation requires electrophysiology with physical contact or fluorescent imaging at target locations, which is often limited by photobleaching and phototoxicity. In this paper, we show that phase imaging can report the intracellular transport induced by optogenetic stimulation. We developed a multimodal instrument that can both stimulate cells with high spatial resolution and detect optical pathlength changes with nanometer scale sensitivity. We found that optical pathlength fluctuations following stimulation are consistent with active organelle transport. Furthermore, the results indicate a broadening in the transport velocity distribution, which is significantly higher in stimulated cells compared to optogenetically inactive cells. It is likely that this label-free, contactless measurement of optogenetic response will provide an enabling approach to neuroscience. Introduction Optogenetics is a transforming tool in the field of neuroscience. By genetic engineering, a neuron can express light-sensitive proteins, and its activity can be initiated or inhibited using light. For example, delivering light stimulation on opsin family proteins causes changes in the cation level, which can trigger or prevent action potentials [1]. Unlike electrical stimulation, light stimulation manipulates specific neurons, and makes it possible to investigate the role of subpopulation neurons in a neural circuit [2]. Over the past decade, accompanied by advances in virus targeting methods (i.e., adeno-associated virus), as well as novel light delivery mechanisms (e.g., twophoton excitation combined with spatial light modulation), the optogenetics toolbox has been growing and gradually becoming a standard method for studying neural functions at both the cellular and behavioral level [2-5]. Currently, electrophysiological methods are considered to provide the highest fidelity readout of neural activity, which is essentially achieved by attaching physical electrodes to the sample [6]. Though these approaches offer high sensitivity, they require physical contact and cell impaling, while the throughput is low. Recent developments in micro-electrode arrays allows for a simultaneous recording of up to a few hundred neurons [2]. Unfortunately, the low spatial resolution, lack of control over the excitation, and photoelectric effects induced in the electrode by the process of stimulation are inevitable limitations [7]. Optical imaging is a potential solution for circumventing these limitations of electrophysiology. This is typically realized by introducing exogenous fluorescent labels or sensors that change their properties when cells are activated [8, 9]. However, the process requires careful sample preparation and is often toxic to cells [10]. Recently, quantitative phase imaging (QPI) [11] has emerged as a valuable tool for live cell imaging, especially because it is label-free and nondestructive. QPI relies on the principle of interference, whereby an image field is overlaid with a reference field. As a result, even the most transparent objects, such as unlabeled live cells, can be imaged with high contrast and sensitivity using the phase information of the field. Because the phase of the image field is measured quantitatively, it can report on both the thickness and dry mass density of the specimen. A number of methods have been proposed, especially over the past 1-2 decades, to optimize the following properties: spatial and temporal resolution, spatial and temporal sensitivity [12-16]. With the recent advances, QPI has become a significant method for studying live cells, such as red blood cell dynamics [17-20], cell growth [21, 22], cell dynamics [23-26] and cell tomography [27-29]. Using its multi-scale coverage and high sensitivity to sub-nanometer changes in optical pathlength, QPI has also found applications in neuroscience and enabled non-invasive studies of neurons at both single-cell level and network level [30-32]. In this paper, we present a new instrument that is capable of both exciting optogenetic signals and detecting the cell response using interferometry. The overall optical pathlength sensitivity is 1.1 nm. We demonstrate that the optical pathlength signals measured via QPI reports on neurite transport associated with optogenetically activated PC12-derived neurons. The new approach for measuring the neural response to stimulation, without labels or physical contact, combines a high spatial resolution optogenetic stimulation system with a high-sensitivity quantitative phase imaging instrument.