Browsing by Author "Saggau, Peter"

Now showing 1 - 7 of 7
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    A dual acousto-optic laser scanning microscope system for the study of dendritic integration: Design, construction, and preliminary results
    (2003) Iyer, Vijay; Saggau, Peter
    Recent research has highlighted the vital role played by dendrites in effecting the computational properties of single neurons in the central nervous system (CNS). An ultraviolet (UV) acousto-optic laser scanning microscope system was developed that enables UV laser pulses to be delivered to multiple user-selected sites in the microscope's specimen plane with high spatial (<10mum) and temporal (<20mus) resolution. By employing "caged" neurotransmitters, the system can effect physiologically realistic spatio-temporal patterns of "synaptic" stimulation to the dendrites of a single cultured neuron. This system was combined with a previously developed acousto-optic laser scanning system for fast, multi-site optical recording of electrical activity (Bullen et al. 1999). This combination---the "Dual Scanner"---allows the study of important dendritic questions such as the underlying mechanisms of spatial and temporal summation. This thesis describes several current outstanding questions of dendritic integration, the design and construction of the system, and some promising preliminary results.
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    A multi-photon microscope for three dimensional functional recording of fast neuronal activity
    (2007) Reddy, Gaddum Duemani; Saggau, Peter
    A thorough understanding of how neurons work is one of the greatest scientific goals in the field of experimental neuroscience. However, four fundamental technical limitations complicate any attempt to study neuronal function with sub-cellular resolution: First, neurons and neuronal processes are small, second, in realistic experimental situations they can be located deep within optically scattering tissue, third, the chemical and electrical signaling that characterizes neuronal behavior happens quickly, and fourth, neurons and neuronal processes have very three dimensional (3D) shapes. Here we develop a tool that overcomes all four listed limitations by combining the technique of multi-photon microscopy with a unique method for 3D laser beam steering. The result is an instrument capable of monitoring physiological signals at multiple locations in the volume of space occupied by a neuron, a task that is unachievable with any other available instrument.
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    Acousto-optic multiphoton laser scanning microscopy and multiphoton photon counting spectroscopy: Applications and implications for optical neurobiology
    (2005) Iyer, Vijay; Saggau, Peter
    Multiphoton excitation of molecular probes has become an important tool in experimental neurobiology owing to the intrinsic optical sectioning and low light scattering it affords. Using molecular functional indicators, multiphoton excitation allows physiological signals within single neurons to be observed from within living brain tissue. Ideally, it would be possible to record from multiple sites located throughout the elaborately branching dendritic arbors, in order to study the correlations of structure and function both within and across experiments. However, existing multiphoton microscope systems based on scanning mirrors do not allow optical recordings to be obtained from more than a handful of sites simultaneously at the high rates required to capture the fast physiological signals of interest (>100Hz for Ca2+ signals, >1kHz for membrane potential transients). In order to overcome this limitation, two-dimensional acousto-optic deflection was employed, to allow an ultrafast laser beam suited for multiphoton excitation to be rapidly repositioned with low latency (∼15mus). This supports a random-access scanning mode in which the beam can repeatedly visit a succession of user-selected sites of interest within the microscope's field-of-view at high rates, with minimal sacrifice of pixel dwell time. This technique of acousto-optic multiphoton laser scanning microscope (AO-MPLSM) was demonstrated to allow the spatial profile of signals arising in response to physiological stimulation to be rapidly mapped. Means to compensate or avoid problems of dispersion which have hampered AO-MPLSM in the past are presented, with the latter being implemented. Separately, the combination of photon counting detection with multiphoton excitation, termed generally multiphoton photon counting spectroscopy (MP-PCS), was also considered, with particular emphasis on the technique of fluorescence correlation spectroscopy (FCS). MP-PCS was shown to allow information about molecular numbers and mobility, as well as the focal volume itself, to be obtained. This capability may in the future be employed to study the number and transport of native neuronal signaling molecules. MP-PCS was also found to be a promising off-line tool which can allow the performance of AO-MPLSM to be optimized, with respect to both the instrument and the indicators employed.
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    An addressable confocal microscope for functional imaging of neuronal activity
    (2005) Bansal, Vivek; Saggau, Peter
    The study of computation occurring in single neurons and small networks of interconnected neurons is often limited by (1) the number of sites that can be simultaneously probed with electrophysiology tools such as patch pipettes and (2) the recording speed of fluorescence imaging tools such as confocal or multiphoton microscopy. Even in the line scan mode of galvanometer-based scanners, where one scan dimension is sacrificed to gain overall speed, the effective frame rate is limited to less than 1 kHz with no flexibility in site selection. To overcome these limitations and allow the study of many sites throughout the dendritic arbor, we have developed an addressable confocal laser-scanning microscope that permits recording from user-selected sites-of-interest at high frame rates, in addition to conventional full frame imaging. Our system utilizes acousto-optic deflectors (AODs) in the illumination pathway to allow for rapid user-defined positioning of a focused laser spot. However, since AODs rely on diffraction to steer a laser beam, they cannot effectively descan the fluorescence emission spectrum as done in mirror-based systems which utilize reflection; this prevents the use of a stationary pinhole as a spatial filter. Instead, we implement an addressable spatial filter using a digital micromirror device (DMD) in conjunction with the AODs to achieve confocality. A registration algorithm synchronizes the AODs and DMD such that point illumination and point detection are always colocalized in conjugate image planes. The current version of the confocal system has a spatial resolution of ∼1 mum. Furthermore, by letting the user tailor which sites are visited, we have shown that recordings can be made at an aggregate frame rate of ∼40 kHz. We have successfully demonstrated that the system is capable of optical sectioning and thus exhibits the main advantage of a confocal microscope for light-scattering biological tissue. This property was used to create three-dimensional reconstructions of fluorescently labeled test specimens. Additionally, we have used the system to record intracellular calcium transients using the fluorescent calcium indicator Oregon Green BAPTA-1. The transients were a result of back-propagating action potentials elicited via 1 nA current injections in cultured hippocampal neurons from wild-type mice.
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    Imaging and vibrometry of the mouse cochlear apex using spectral domain optical coherence tomography
    (2014-03-13) Gao, Simon; Raphael, Robert M.; Oghalai, John S; Baraniuk, Richard G.; Brownell, William; Saggau, Peter; Tkaczyk, Tomasz S.
    Hearing loss affects millions of Americans and is of increasing concern to an aging population. It can occur as a result of congenital malformations or damage to the functional soft tissues within the hearing organ, the cochlea. Current clinical imaging modalities such as magnetic resonance imaging and computed tomography do not have the necessary resolution to detect such changes and, furthermore, provide no functional information. As well, research into how the intracochlear tissues vibrate and thus transduce sound pressure waves into neural signals has stagnated because of the limitations inherent to currently available technologies. To address these challenges, we developed a spectral domain optical coherence tomography system to visualize and measure nanoscale vibrations of intracochlear structures. Using this system and mouse models, we first imaged excised cochlea from a transgenic mouse model of human hearing loss with an altered tectorial membrane. The soft tissue structures and expected anatomical variations were visible using OCT, and quantitative measurements confirmed the ability to detect critical changes relevant to hearing. We then compared the vibratory patterns of the intracochlear structures of live and dead normal hearing mice and found that active force generation by outer hair cells produced larger displacements of the tectorial membrane than any other structure, including the basilar membrane. As well, there was a traveling wave that emanated from the point of outer hair cell attachment and moved radially. This presumably propels fluid and drives the stimulation of inner hair cell stereociliary bundles. Because inner hair cells provide the majority of the afferent auditory input, these nanoscale movements thus describe how the forces produced by outer hair cells improve the auditory sensitivity and frequency selectivity of mammalian hearing.
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    Method for high speed microscopy with three-dimensional laser beam scanning
    (2008-02-19) Saggau, Peter; Reddy, Duemani; Iyer, Vijay; Baylor College of Medicine; Rice University; United States Patent and Trademark Office
    A system and method for independently controlling the collimation and lateral positioning of a light beam comprises at least one acousto-optic deflector and a pair of counter propagating acoustic waves with offset frequencies. While the frequency offset controls the lateral positioning of the light beam, a frequency gradient across the acousto-optic deflectors controls the collimation of the light beam.
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    Random access high-speed confocal microscope
    (2010-04-27) Saggau, Peter; Bansal, Vivek; Patel, Saumil; Baylor College of Medicine; Rice University; United States Patent and Trademark Office
    Disclosed herein is a confocal imaging system for imaging a specimen. The system comprises a light source, a light deflector capable of positioning a beam of light produced by the light source at one of a series of predetermined points on the specimen, an addressable spatial filter capable of selectively filtering light from the specimen, and a central processing unit capable of providing selective position control to the light deflector and the addressable spatial filter.