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ABSTRACT: During rhythmic movements, central pattern generators (CPGs) trigger bursts of motor activity with precise timing. However, the number of neurons that must be activated within CPGs to generate motor output is unknown. In the mammalian breathing rhythm, a fundamentally important motor behavior, the preBötzinger Complex (preBötC) produces synchronous population-wide bursts of activity to control inspiratory movements. We probed mechanisms underlying inspiratory burst generation in the preBötC using holographic photolysis of caged glutamate in medullary slices from neonatal mice. With stimulation parameters determined to confine photoactivation to targeted neurons, simultaneous excitation of 4-9 targeted neurons could initiate ectopic, endogenous-like bursts with delays averaging 255 ms, placing a critical and novel boundary condition on the microcircuit underlying respiratory rhythmogenesis.
Journal of Neuroscience 02/2013; 33(8):3332-8. · 7.11 Impact Factor
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ABSTRACT: Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na(+) channels.
Proceedings of the National Academy of Sciences 11/2011; 108(49):19504-9. · 9.68 Impact Factor
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ABSTRACT: We report a theoretical study that elaborates the influence of the polarization state of both the pump and the probe pulse in ultrafast coherent vibrational ladder climbing experiments in the mid-infrared. Whereas a subensemble in a randomly oriented sample of molecules is excited by the pump pulse in this multiphoton process, further inhomogeneities such as the spatial profile of the laser beams, the longitudinal attenuation in the sample, and the probe beam polarization have to be taken into account. Analytical expressions for a density function describing the number of molecules that are exposed to an effective pump intensity are introduced, and the variation of the population distribution and the actual transient absorption signal in dependence on the polarization-state combinations for pump and probe pulse are discussed in detail. In simulations on the model system carboxy-hemoglobin, it is demonstrated that the polarization states play important roles both for exciting a certain population distribution and for actually observing it. In particular, it will be discussed under which conditions experimental data indicates a population inversion.
The Journal of Physical Chemistry B 03/2011; 115(18):5554-63. · 3.70 Impact Factor
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ABSTRACT: Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells.
The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca(2+) imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells.
We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes.
PLoS ONE 01/2010; 5(2):e9431. · 4.09 Impact Factor
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ABSTRACT: We present an endomicroscope apparatus that utilizes structured illumination to produce high resolution (approximately 2.6 microm) optically sectioned fluorescence images over a field of view of about 240 microm. The endomicroscope is based on the use of a flexible imaging fiber bundle with a miniaturized objective. We also present a strategy to largely suppress structured illumination artifacts that arise when imaging in thick tissue that exhibits significant out-of-focus background. To establish the potential of our endomicroscope for preclinical or clinical applications, we provide images of BCECF-AM labeled rat colonic mucosa.
Optics Express 06/2008; 16(11):8016-25. · 3.59 Impact Factor
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ABSTRACT: Dynamic speckle illumination (DSI) provides a simple and robust technique to obtain fluorescence depth sectioning with a widefield microscope. We report a significant improvement to DSI microscopy based on a statistical image-processing algorithm that incorporates spatial wavelet prefiltering. The resultant gain in sectioning strength leads to a fundamentally improved scaling law for the out-of-focus background rejection.
Optics Letters 07/2007; 32(11):1417-9. · 3.40 Impact Factor
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ABSTRACT: Dynamic speckle illumination (DSI) microscopy is a widefield fluorescence imaging technique that provides depth discrimination. The technique relies on the illumination of a sample with a sequence of speckle patterns. We consider an image processing algorithm based on a differential intensity variance between consecutive images, and demonstrate that DSI sectioning strength depends on the dynamics of the speckle pattern. Translated speckle patterns confer greater sectioning strength than randomized speckle patterns because they retain out-of-focus correlations that lead to better background rejection. We present a theory valid for arbitrary point-spread-functions, which we corroborate with experimental results.
Optics Express 09/2006; 14(16):7198-209. · 3.59 Impact Factor
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ABSTRACT: We propose a scheme to generate intense, ultrafast mid-infrared pulses with conversion efficiencies exceeding the upper bound for single-stage difference-frequency mixing as predicted by the Manley-Rowe relations. Finite-element fast Fourier transform simulations of the mixing process show that the parametric cascade downconverter generates 1.7 times more photons (at 10 microm) than in the initial pump pulse (center wavelength of 1.48 microm, duration of 130 fs, and pulse energy of 50 microJ), with negligible pulse spatial and temporal distortion.
Applied Optics 07/2006; 45(17):4109-13. · 1.41 Impact Factor
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ABSTRACT: We present a simple modification to a conventional wide-field fluorescence microscope that provides depth discrimination in thick tissues. The technique consists of illuminating a sample with a sequence of independent speckle patterns and displaying the rms of the resultant sequence of fluorescence images. The advantage of speckle illumination is that it provides diffraction-limited illumination granularity that is highly contrasted even in scattering media. We demonstrate quasi-confocal imaging in a mouse olfactory bulb labeled with green fluorescent protein.
Optics Letters 01/2006; 30(24):3350-2. · 3.40 Impact Factor
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ABSTRACT: We demonstrate vibrational climbing in the CO stretch of carboxyhemoglobin pumped by midinfrared chirped ultrashort pulses. By use of spectrally resolved pump-probe measurements, we directly observed the induced absorption lines caused by excited vibrational populations up to v = 6. In some cases, we also observed stimulated emission, providing direct evidence of vibrational population inversion. This study provides important spectroscopic parameters on the CO stretch in the strong-field regime, such as transition frequencies and dephasing times up to the v = 6 to v = 7 vibrational transition. We measured equally spaced vibrational transitions, in agreement with the energy levels of a Morse potential up to v = 6. It is interesting that the integral of the differential absorption spectra was observed to deviate far from zero, in contrast to what one would expect from a simple one-dimensional Morse model assuming a linear dependence of dipole moment with bond length.
Proceedings of the National Academy of Sciences 10/2004; 101(36):13216-20. · 9.68 Impact Factor
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ABSTRACT: Mid-infrared ultrashort pulses of 9.2-microm center wavelength are characterized in both amplitude and phase. This is achieved by use of a variant of spectral phase interferometry for direct electric field reconstruction in which spectral interferometry has been replaced with time-domain interferometry, a technique that is well suited for infrared pulses. The setup permits simultaneous recording of the second-order interferometric autocorrelation, thus providing an independent check on the retrieved spectral phase.
Optics Letters 11/2003; 28(19):1826-8. · 3.40 Impact Factor
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ABSTRACT: Intense mid-infrared pulses tunable between 5 and 14 �m with pulse energies of several microjoules were generated by difference-frequency mixing (DFM) in a GaSe crystal. Longer wavelengths (up to 18 �m) were achieved by DFM in a CdSe crystal. The infrared pulses were then characterized using various techniques: The spectrum was measured using a Fourier-transform spectrometer, which was then modified to determine the interferometric second-order autocorrelation. The electric field spectral phase was measured using the same setup, thus leading to a full characterization of the mid-infrared pulses. The spectral phase was measured using the time-domain homodyne optical technique for spectral phase interferometry for direct electric field reconstruction, where spectral interferometry was replaced with time-domain interferometry. The measured pulse duration was 100 fs, nearly transform limited.
