Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo.

Recording of the concerted activity of neuronal assemblies and the dendritic and axonal signal integration of downstream neurons pose different challenges, preferably a single recording system should perform both operations. We present a three-dimensional (3D), high-resolution, fast, acousto-optic two-photon microscope with random-access and continuous trajectory scanning modes reaching a cubic millimeter scan range (now over 950 × 950 × 3000 μm3) which can be adapted to imaging different spatial scales. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (>290 × 290 × 200 μm3) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSFx < 1.9 μm, PSFz < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. The system contains new design concepts: it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors, and it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures. We demonstrate the use of the microscope at different spatial scales by first showing 3D optical recordings of action potential back propagation and dendritic Ca2+ spike forward propagation in long dendritic segments in vitro, at near-microsecond temporal resolution. Second, using the same microscope we show volumetric random-access Ca2+ imaging of spontaneous and visual stimulation-evoked activity from hundreds of cortical neurons in the visual cortex in vivo. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest

Monte Carlo Simulations of Single-Molecule Fluorescence Detection Experiments

Several Monte Carlo simulations of single-molecule fluorescence systems are developed to help evaluate and improve ongoing experiments. In the first simulation, trapping of a single molecule in a nanochannel is studied. Molecules move along the nanochannel by diffusion and electrokinetic flow. Single-molecule fluorescence signals excited by two spatially offset laser beams are detected and the direction of the flow is adjusted to try to equalize the signals and center the molecule between the beams. An algorithm is evaluated for trapping individual molecules in succession by rapidly reloading the trap after a molecule photobleaches or escapes. This is shown to be effective for trapping fast-diffusing single-chromophore molecules in succession within a micron-sized confocal region while accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware. In the second simulation, trapping of a molecule in a two-dimensional fluidic device consisting of sub-micron-separated glass plates is studied. Two different illumination schemes for sensing the molecule\u27s position are compared: (i) a single continuous laser spot circularly scanned at 40 KHz or 240 KHz in the plane of the device; and (ii) four pulsed laser spots arranged in a square and temporally alternated at 304 MHz In either case, the times of detected photons are used by algorithms to control the electrokinetic flow in two dimensions to compensate diffusion and achieve single-molecule trapping. However each scheme is found to have limitations, as circular scanning produces a modulation in the fluorescence signal and in the autocorrelation function, whereas the four-pulse scheme becomes ineffective if the fluorescence lifetime of the molecule is greater than the time between laser pulses, The third simulation investigates appropriate conditions for detection of single molecules flowing through an array of fluidic channels for an application to high-throughput screening for pharmaceutical drug discovery. For parallelized single-molecule detection, illumination is provided by a continuous laser focused to a line intersecting all channels and fluorescence is imaged to a single row of pixels of an electron-multiplying CCD with sufficient gain for single-photon detection. The simulation separately models each channel to determine laser, flow, and camera operating conditions suitable for efficient detection

Optimization of two-photon excited fluorescence for volumetric imaging

Two-photon microscopy is often used in biological imaging due to its optical sectioning and depth penetration capabilities. These characteristics have made two-photon microscopy especially useful for neurobiological studies where imaging a volume at single cell resolution is typically required. This dissertation focuses on the optimization of two-photon excited fluorescence for volumetric imaging of biological samples, with special attention to imaging the mouse brain. Chapter 2 studies wavefront manipulation as a way of optimizing two-photon excited fluorescence. We show, through numerical simulations and experiments, that the magnitude of the two-photon fluorescence signal originating from cell-sized objects can be used as a metric of beam quality. We also show that the cranial window used in mouse experiment is a major source of aberrations, which can readily be represented in the Zernike basis. Finally, we implement a modal wavefront optimization scheme that optimizes the wavefront based entirely on the magnitude of the fluorescence. Along with this scheme, Zernike functions are found to be a useful basis for correcting aberrations encountered in mouse brain imaging while the Hadamard basis is found to be useful for scattering compensation. Corrections performed in mouse brain using Zernike functions are found to be valid over hundreds of microns, allowing a single correction to be applied to a whole volume. Finally, we show that the wavefront correction system can double as a wavefront encoding system for experiments that require custom point-spread-functions. Chapter 3 aims to significantly improve the volume imaging rate of two-photon microscopy. The imaging speed is improved by combining two-photon excitation with scanning confocally-aligned planar excitation microscopy (SCAPE). Numerical simulations, analytical arguments, and experiments reveal that the standard method of combining nano-joule pulses with 80 MHz repetition rates is inadequate for two-photon light-sheet excitation. We use numerical simulations and experiments to explore the possibility of achieving fast volumetric imaging using line and sheet excitation and find that the sheet excitation scheme is more promising. Given that two-photon excitation requires high photon-flux-densities near the focus, achieving high enough fluorescence has to be balanced with restrictions placed by saturation, photodamage, photobleaching and sample heating effects. Finally, we experimentally study light sheet excitation at various pulse repetition rates with femtosecond pulses and find that repetition rates near 100 kHz allow imaging of nonbiological samples of ~200x300x300 μm^3 volume at 20 volumes per second while balancing the above constraints. This work paves the way for achieving fast, volumetric two-photon imaging of the mouse brain

Doppler Encoded Excitation Patterning (DEEP) Microscopy

Traditional optical imaging systems rely on lenses and spatially-resolved detection to probe distinct locations on the object. We develop a novel computational approach to 2D and 3D imaging that instead measures the object\u27s spatial Fourier transform using a single-element detector and without requiring precision optics. This wide-field technique can be used to image biological and synthetic structures in fluoresced or scattered light using coherent or broadband illumination. It employs dynamic structured illumination, acousto-optics, RF electronics, and tomographic algorithms to circumvent several trade-offs in conventional imaging, such as the dependence of the optical transfer function on the imaging lenses and the coupling of resolution and depth of field. We use Fourier optics concepts to derive the dynamic optical transfer function, evaluate different Fourier sampling strategies, and investigate and compare tomographic algorithms for 2D and 3D image synthesis. We also develop conceptual and analytical models to describe imaging of fluorescent as well as amplitude and phase scattering objects, the effects of broadband and spatially-incoherent illumination, and nonlinear wide-field super-resolution imaging. We consider sources of noise, analyze and simulate SNR behavior for several types of noise and Fourier sampling strategies, and compare the sensitivity of the technique to conventional imaging. We describe several experimental proof-of-concept systems and present two-dimensional high-resolution tomographic image reconstructions in both scattered and fluoresced light demonstrating a thousandfold improvement in the depth of field compared to conventional lens-based microscopy. Finally, we explore approaches for high-speed Fourier sampling and propose several related sensing techniques, including wide-field fluorescence imaging in scattering media

DEVELOPMENT OF A VERSATILE HIGH SPEED NANOMETER LEVEL SCANNING MULTI-PROBE MICROSCOPE

The motivation for development of a multi-probe scanning microscope, presented in this dissertation, is to provide a versatile measurement tool mainly targeted for biological studies, especially on the mechanical and structural properties of an intracellular system. This instrument provides a real-time, three-dimensional (3D) scanning capability. It is capable of operating on feedback from multiple probes, and has an interface for confocal photo-detection of fluorescence-based and single molecule imaging sensitivity. The instrument platform is called a Scanning Multi-Probe Microscope (SMPM) and enables 45 microm by 45 microm by 10 microm navigation of specimen with simultaneous optical and mechanical probing with each probe location being adjustable for collocation or for probing with known probe separations. The 3D positioning stage where the specimen locates was designed to have nanometer resolution and repeatability at 10 Hz scan speed with either open loop or closed loop operating modes. The fine motion of the stage is comprises three orthogonal flexures driven by piezoelectric actuators via a lever linkage. The flexures design is able to scan in larger range especially in z axis and serial connection of the stages helps to minimize the coupling between x, y and z axes. Closed-loop control was realized by the capacitance gauges attached to a rectangular block mounted to the underside of the fine stage upon which the specimen is mounted. The stage's performance was studied theoretically and verified by experimental test. In a step response test and using a simple proportional and integral (PI) controller, standard deviations of 1.9 nm 1.8 nm and 0.41 nm in the x, y and z axes were observed after settling times of 5 ms and 20 ms for the x and y axes. Scanning and imaging of biological specimen and artifact grating are presented to demonstrate the system operation. For faster, short range scanning, novel ultra-fast fiber scanning system was integrated into the xyz fine stage to achieve a super precision dual scanning system. The initial design enables nanometer positioning resolution and runs at 100 Hz scan speed. Both scanning systems are capable of characterization using dimensional metrology tools. Additionally, because the high-bandwidth, ultra-fast scanning system operates through a novel optical attenuating lever, it is physically separate from the longer range scanner and thereby does not introduce additional positioning noise. The dual scanner provides a fine scanning mechanism at relatively low speed and large imaging area using the xyz stage, and focus on a smaller area of interested in a high speed by the ultra-fast scanner easily. Such functionality is beneficial for researchers to study intracellular dynamic motion which requires high speed imaging. Finally, two high end displacement sensor systems, a knife edge sensor and fiber interferometer, were demonstrated as sensing solutions for potential feedback tools to boost the precision and resolution performance of the SMPM

Optical imaging of dendritic spikes in apical oblique dendrites of layer 5 pyramidal neurons

Dendrites have active properties capable of generating dendritic spikes that could boost the impact of distal synaptic inputs. The strong passive filtering of the membrane and generation of local dendritic spikes enable different dendritic regions to function as independent computational compartments. While there is a wealth of information about cortical processing in apical tuft and basal dendrites, the functional role of apical oblique dendrites of layer 5 pyramidal neurons (L5PNs) are less understood. In this thesis, I aim to understand to functional role of thin apical oblique dendrites of L5PNs in the cortex. Using a previously published multi-compartment model of a L5PN, I first investigated the excitability of apical oblique dendrites and the extent of action potential (AP) back-propagation. In the model, I found that a 2-AP train at f > 35 Hz elicited an oblique branch spike in certain dendrites. The spike is mediated by activation of voltage-gated sodium and voltage-gated calcium channels. In addition, oblique branch spikes manifest as after-depolarizing potentials (ADP) at the soma. I then experimentally verified the generation of spikes by imaging the dendritic activity of oblique branches of L5PNs from the somatosensory cortex in vitro. I used our custom-built two-photon (2P) holographic microscope to perform functional calcium imaging on thin oblique branches of L5PNs in vitro. Oblique branch spikes are evoked at a critical frequency of fc = 57+/-5 Hz (from calcium imaging) and fc = 72+/-4 Hz (from ADP measurements) of a 2-AP train. Generation of spikes in oblique dendrites could establish their role as independent computational compartments that could boost coincident synaptic inputs. To further improve optical recording along dendrites, I proposed novel optical recording techniques to enhance the signal-to-noise ratio (SNRs) of the detected signals. A 4-fold improvement in the SNR was obtained with temporal gating for multi-site holographic calcium imaging, while a 6-fold increase in SNR was obtained for voltage imaging when using scattered photons to excite voltage indicators. These two novel SNR enhancement techniques can facilitate experiments that require imaging of activity in thin dendrites