Targeted pruning of a neuron's dendritic tree via femtosecond laser dendrotomy.

Neurons are classified according to action potential firing in response to current injection. While such firing patterns are shaped by the composition and distribution of ion channels, modelling studies suggest that the geometry of dendritic branches also influences temporal firing patterns. Verifying this link is crucial to understanding how neurons transform their inputs to output but has so far been technically challenging. Here, we investigate branching-dependent firing by pruning the dendritic tree of pyramidal neurons. We use a focused ultrafast laser to achieve highly localized and minimally invasive cutting of dendrites, thus keeping the rest of the dendritic tree intact and the neuron functional. We verify successful dendrotomy via two-photon uncaging of neurotransmitters before and after dendrotomy at sites around the cut region and via biocytin staining. Our results show that significantly altering the dendritic arborisation, such as by severing the apical trunk, enhances excitability in layer V cortical pyramidal neurons as predicted by simulations. This method may be applied to the analysis of specific relationships between dendritic structure and neuronal function. The capacity to dynamically manipulate dendritic topology or isolate inputs from various dendritic domains can provide a fresh perspective on the roles they play in shaping neuronal output.

Simultaneous multi-site two-photon photostimulation in three dimensions.

We demonstrate simultaneous multi-site two-photon photolysis of caged neurotransmitters with close to diffraction-limited resolution in all three dimensions (3D). We use holographic projection of multiple focal spots, which allows full control over the 3D positions of uncaging sites with a high degree of localized excitation. Our system incorporates a two-photon imaging setup to visualize the 3D morphology of the neurons in order to accurately determine the photostimulation sites. We show its application to studies of synaptic integration by performing simultaneous and controlled glutamate delivery at multiple locations on dendritic trees.

Optimal complex field holographic projection.

We describe a technique that uses complex field holograms to project three-dimensional light patterns. Holographic projection commonly uses phase-only encoding since accurately representing complex holograms using both amplitude and phase spatial light modulators reduces the optical throughput significantly. Here, we use a lossless projection via the generalized phase contrast method to produce the necessary amplitude pattern required for complex field holographic projection. We numerically evaluate the technique and demonstrate high optical throughput with reduced undesired high diffraction orders.

Optical trapping and surgery of living yeast cells using a single laser.

We present optical trapping and surgery of living yeast cells using two operational modes of a single laser. We used a focused laser beam operating in continuous-wave mode for noninvasive optical trapping and manipulation of single yeast cell. We verified that such operational mode of the laser does not cause any destructive effect on yeast cell wall. By changing the operation of the laser to femtosecond-pulsed mode, we show that a tightly focused beam dissects the yeast cell walls via nonlinear absorption. Lastly, using the combined technique of optical microsurgery and trapping, we demonstrate intracellular organelle extraction and manipulation from a yeast cell. The technique established here will be useful as an efficient method for both surgery and manipulation of living cells using a single laser beam.

An efficient visualization tool for the analysis of protein mutation matrices.

BACKGROUND: It is useful to develop a tool that would effectively describe protein mutation matrices specifically geared towards the identification of mutations that produce either wanted or unwanted effects, such as an increase or decrease in affinity, or a predisposition towards misfolding. Here, we describe a tool where such mutations are efficiently identified, categorized and visualized. To categorize the mutations, amino acids in a mutation matrix are arranged according to one of three sets of physicochemical characteristics, namely hydrophilicity, size and polarizability, and charge and polarity. The magnitude and frequencies of mutations for an alignment are subsequently described using color information and scaling factors. RESULTS: To illustrate the capabilities of our approach, the technique is used to visualize and to compare mutation patterns in evolving sequences with diametrically opposite characteristics. Results show the emergence of distinct patterns not immediately discernible from the raw matrices. CONCLUSION: Our technique enables effective categorization and visualization of mutations by using specifically-arranged mutation matrices. This tool has a number of possible applications in protein engineering, notably in simplifying the identification of mutations and/or mutation trends that are associated with specific engineered protein characteristics and behavior.

Holographic projection of arbitrary light patterns with a suppressed zero-order beam.

We present what is to our knowledge a novel technique for efficient suppression of the zero-order beam inherent in light patterns projected via phase-only computer-generated holograms (CGHs). Encoding a CGH on a spatial light modulator (SLM) with a limited fill factor produces a disturbing zero-order beam at the optical axis. Here, we propose to derive a CGH, which includes holographic information to project a corrective beam that destructively interferes with the zero-order beam. The CGH for projecting arbitrary light patterns plus a corrective beam are derived using the Gerchberg-Saxton algorithm where the iterations impose both amplitude and phase constraints for the target field pattern at the Fourier plane. As proof of principle, we analyze the viability of the technique by simulating the performance when applied on a practical SLM with a limited fill factor, fixed number of phase-shifting pixels, and wavefront distortion associated with the surface roughness of the SLM.

Effect of spurious diffraction orders in arbitrary multifoci patterns produced via phase-only holograms.

We analyze the effect of spurious diffraction orders when generating functional multifoci patterns produced by illuminating a phase-only hologram with a single Gaussian beam. Using a practical device for encoding a hologram generates an undesirable zero order and high-diffraction orders at the Fourier plane. This translates to the fact that a significant fraction of the incident light does not necessarily convert to functional multifoci patterns. In most applications, the zero order can be avoided by generating foci patterns shifted off the optical axis, which further increases the amount of light distributed to spurious high-diffraction orders owing to the reduction of light directed to the desired foci pattern. We analyze the amount of light dispersed to spurious orders and show that these unwanted orders can be a major limiting factor for most applications based on arbitrary multifoci patterns.

Real-time three-dimensional optical micromanipulation of multiple particles and living cells.

Counterpropagating light fields provide a stationary optical potential well for a Brownian particle. Introducing variability in the relative strengths of the counterpropagating beams allows us to create a more general configuration-the optical elevator. An optical elevator dynamically controls the axial location of the potential minimum where the particle finds a stable equilibrium position. We describe the implementation of multiple real-time reconfigurable optical elevators with the generalized phase contrast method for dynamic manipulation of polystyrene spheres and yeast cells S. cerevisiae in three dimensions.

Reply to comment on "Excitation with a focused, pulsed optical beam in scattering media: diffraction effects.".

We address the issues that were raised by Tycho and Jørgensen [Appl. Opt. 41, 4709 (2002)] concerning our strategy [Appl. Opt. 39, 5244 (2000)] for incorporating the wave properties of light in the description of a propagating focused excitation beam in a highly scattering medium. We explain that the strategy is consistent with the Huygens-Fresnel principle and does not violate the energy conservation principle.