Depth-multiplexing spectral domain OCT for full eye length imaging with a single modulation unit

Measuring the axial length of the eye is emerging as a crucial approach to measure progression and monitor management of myopia. The high cost of current swept-source OCT devices, the preferred method for such measurements, limits their broad use, especially in lower-income communities.While spectral domain (SD) OCT is a more affordable option, its limited imaging range falls short for full eye length measurement. Existing depth-multiplexing (DM) techniques for SD-OCT provide a workaround by capturing images at multiple depths within the eye. However, these methods typically require multiple light modulation units or detectors for simultaneous imaging across depths, adding complexity and cost. In response, we propose a novel DM-SD-OCT approach that utilizes a single light modulation unit for depth encoding. This innovative method facilitates the capture of images at multiple depths within the eye using a single line scan camera, with subsequent computational demixing. Our implementation of this system successfully enabled simultaneous acquisition and demixing of signals from three distinct depths within the eye. The system's effectiveness was demonstrated using a model eye, confirming its potential as a cost-effective solution for comprehensive eye length measurement in clinical myopia research

In vivo measurement of afferent activity with axon-specific calcium imaging.

In vivo calcium imaging from axons provides direct interrogation of afferent neural activity, informing the neural representations that a local circuit receives. Unlike in somata and dendrites, axonal recording of neural activity-both electrically and optically-has been difficult to achieve, thus preventing comprehensive understanding of neuronal circuit function. Here we developed an active transportation strategy to enrich GCaMP6, a genetically encoded calcium indicator, uniformly in axons with sufficient brightness, signal-to-noise ratio, and photostability to allow robust, structure-specific imaging of presynaptic activity in awake mice. Axon-targeted GCaMP6 enables frame-to-frame correlation for motion correction in axons and permits subcellular-resolution recording of axonal activity in previously inaccessible deep-brain areas. We used axon-targeted GCaMP6 to record layer-specific local afferents without contamination from somata or from intermingled dendrites in the cortex. We expect that axon-targeted GCaMP6 will facilitate new applications in investigating afferent signals relayed by genetically defined neuronal populations within and across specific brain regions

High-throughput volumetric imaging of neural dynamics in vivo

Brain is composed of complex neural networks that work in concert to underlie the animal’s cognition and behavior. Optical microscopy has become an indispensable tool to study brain due to its non-invasiveness and high spatial resolution. Two-photon fluorescence microscopy is the method of choice to image the optically opaque mammalian brain. However, conventional two-photon fluorescence microscopy has to perform serial 3D point scanning for volumetric imaging, which renders it extremely difficult to study neural circuits in 3D at subcellular resolution with sufficient imaging speed.Taking advantage of the fact that neurons remain largely stationary during in vivo imaging, when their temporal activities are the subject of interest, one can acquire an axially projected view of the objects without constantly tracking their 3D location. Incorporating a Bessel-like beam into a conventional two-photon fluorescence microscope extends the system’s depth-of-focus, which turns the 2D frame rate into an axially projected volume rate. As a result, Bessel focus scanning technology enables high-speed volumetric imaging without sacrificing the lateral resolution and reduces data size by an order of magnitude when compared with conventional serial 3D scanning. This thesis first explores the application of Bessel focus scanning technology in two-photon fluorescence microendoscopy to achieve high-throughput neural circuit imaging in deeply-buried nuclei of the mouse brain. The thesis then presents efficient data analysis approaches for high-throughput calcium neural imaging data. Finally, the thesis depicts a custom-designed and easy-to-operate two-photon fluorescence microscope combining several state-of-the-art technologies together, i.e., adaptive optics, Bessel focus scanning technology, and remote focusing, which will open up possibilities for neurobiology questions that could not be well addressed before

Ultrafast two-photon fluorescence imaging of cerebral blood circulation in the mouse brain in vivo

Characterizing blood flow dynamics in vivo is critical to understand the function of vascular network under physiological and pathological conditions. Existing methods for hemodynamic imaging have insufficient spatial and temporal resolution to monitor blood flow at cellular level in large blood vessels. By employing an ultrafast line-scanning module based on free-space angular chirped enhanced delay (FACED), we achieved two-photon fluorescence imaging of cortical blood flow at 1,000 2D frames and 1,000,000 1D line scans per second in the awake mouse. This orders-of-magnitude increase in temporal resolution allowed us to measure cerebral blood flow up to 49 mm/s and observe pulsatile blood flow at harmonics of heart rate. Directly visualizing red blood cell (RBC) flow through vessels down to >800 µm in depth, we characterized cortical-layer-dependent flow velocity distributions of capillaries, obtained radial velocity profiles and kilohertz 2D velocity mapping of multi-file blood flow, and carried out RBC flux measurements from penetrating blood vessels

Rapid mesoscale volumetric imaging of neural activity with synaptic resolution.

Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over the mesoscale with synaptic resolution. We applied the technology to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain

Rapid mesoscale volumetric imaging of neural activity with synaptic resolution.

Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over the mesoscale with synaptic resolution. We applied the technology to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain

Ultrafast two-photon fluorescence imaging of cerebral blood circulation in the mouse brain in vivo.

Characterizing blood flow dynamics in vivo is critical to understanding the function of the vascular network under physiological and pathological conditions. Existing methods for hemodynamic imaging have insufficient spatial and temporal resolution to monitor blood flow at the cellular level in large blood vessels. By using an ultrafast line-scanning module based on free-space angular chirped enhanced delay, we achieved two-photon fluorescence imaging of cortical blood flow at 1,000 two-dimensional (2D) frames and 1,000,000 one-dimensional line scans per second in the awake mouse. This orders-of-magnitude increase in temporal resolution allowed us to measure cerebral blood flow at up to 49 mm/s and observe pulsatile blood flow at harmonics of heart rate. Directly visualizing red blood cell (RBC) flow through vessels down to >800 µm in depth, we characterized cortical layer–dependent flow velocity distributions of capillaries, obtained radial velocity profiles and kilohertz 2D velocity mapping of multifile blood flow, and performed RBC flux measurements from penetrating blood vessels

High-throughput synapse-resolving two-photon fluorescence microendoscopy for deep-brain volumetric imaging in vivo.

Optical imaging has become a powerful tool for studying brains in vivo. The opacity of adult brains makes microendoscopy, with an optical probe such as a gradient index (GRIN) lens embedded into brain tissue to provide optical relay, the method of choice for imaging neurons and neural activity in deeply buried brain structures. Incorporating a Bessel focus scanning module into two-photon fluorescence microendoscopy, we extended the excitation focus axially and improved its lateral resolution. Scanning the Bessel focus in 2D, we imaged volumes of neurons at high-throughput while resolving fine structures such as synaptic terminals. We applied this approach to the volumetric anatomical imaging of dendritic spines and axonal boutons in the mouse hippocampus, and functional imaging of GABAergic neurons in the mouse lateral hypothalamus in vivo