Kilohertz 3D voltage imaging using near-infrared confocal squeezed light field microscopy

University Of California Los Angeles, Los Angeles CA

Investigators

Abstract

Project Summary: High-speed volumetric imaging of dynamic neuronal activity over extended periods is a challenging yet crucial objective in neuroscience. Conventional optical measurements of neuronal activity primarily rely on calcium signals, which provide limited information about natural signal processing in the nervous system and offer minimal data on the continuous inhibitory and excitatory signals in most neurons. In contrast, voltage imaging directly measures neuronal electrical activity, potentially overcoming the limitations inherent to calcium imaging. Recent advances in NIR voltage-sensitive dyes have significantly expanded the use of voltage imaging in brain research, driving the development of new optical instrumentation optimized for this purpose. Capturing neural action potentials in 3D poses a significant challenge for imaging instrumentation, requiring millisecond temporal resolution for precise recording. Traditional 3D optical imaging techniques, such as confocal or multiphoton microscopy, rely on extensive point scanning to create volumetric images. However, these methods often result in lengthy acquisition times, making them unsuitable for capturing rapid changes in neural activity. Recent advancements in light field microscopy have significantly boosted the frame rate of 3D microscopy, making it an ideal strategy for imaging neuronal networks. However, because light field imaging records both spatial and angular information, it typically requires a large-format image sensor, which has a low frame rate due to limited electronic bandwidth. The temporal resolution achieved thus far (tens of milliseconds) is insufficient to resolve individual neuron firing events (~one millisecond). Moreover, despite its digital refocusing capabilities at specific depths, light-field microscopy lacks inherent optical sectioning abilities. This limitation becomes pronounced in environments with high background light, reducing image contrast and diminishing its efficacy for in-vivo imaging. To overcome the above limitations, we propose to develop a NIR confocal Squeezed Light-field Microscopy (SLIM) method for kilohertz volumetric imaging of neuronal action potentials. This method has recently become feasible due to the emergence of SLIM, which is highly efficient in acquiring light field data for 3D imaging. Instead of measuring the entire light field data cube, SLIM captures a compressed light field representation, significantly reducing the data load and enabling a kilohertz volumetric frame rate. Additionally, by incorporating a confocal slit, we will be able to suppress background light and significantly increase image contrast, which is crucial for in-vivo imaging. When combined with NIR voltage-sensitive dyes, the resulting system will provide a comprehensive solution for high-speed voltage imaging of 3D neuronal networks in deep tissues. Furthermore, the proposed system will be compatible with experiments in behaving animals, allowing researchers to link neuronal activity to behavior. The insights gained from this research will be instrumental in interpreting complex animal behaviors based on electrical activities at the single-cell level.