High-speed laser microsurgery of alert fruit flies for fluorescence imaging of neural activity.

Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-µm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12- to 350-µm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca(2+) signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5-20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation.

Sorting droplets into many outlets.

Droplet microfluidics is a commercially successful technology, widely used in single cell sequencing and droplet PCR. Combining droplet making with droplet sorting has also been demonstrated, but so far found limited use, partly due to difficulties in scaling manufacture with injection molded plastics. We introduce a droplet sorting system with several new elements, including: 1) an electrode design combining metallic and ionic liquid parts, 2) a modular, multi-sorting fluidic design with features for keeping inter-droplet distances constant, 3) using timing parameters calculated from fluorescence or scatter signal triggers to precisely actuate dozens of sorting electrodes, 4) droplet collection techniques, including ability to collect a single droplet, and 5) a new emulsion breaking method to collect aqueous samples for downstream analysis. We use these technologies to build a fluorescence based cell sorter that can sort with high (>90%) purity. We also show that these microfluidic designs can be translated into injection molded thermoplastic, suitable for industrial production. Finally, we tally the advantages and limitations of these devices.

Long-term optical brain imaging in live adult fruit flies.

Time-lapse in vivo microscopy studies of cellular morphology and physiology are crucial toward understanding brain function but have been infeasible in the fruit fly, a key model species. Here we use laser microsurgery to create a chronic fly preparation for repeated imaging of neural architecture and dynamics for up to 50 days. In fly mushroom body neurons, we track axonal boutons for 10 days and record odor-evoked calcium transients over 7 weeks. Further, by using voltage imaging to resolve individual action potentials, we monitor spiking plasticity in dopamine neurons of flies undergoing mechanical stress. After 24 h of stress, PPL1-α'3 but not PPL1-α'2α2 dopamine neurons have elevated spike rates. Overall, our chronic preparation is compatible with a broad range of optical techniques and enables longitudinal studies of many biological questions that could not be addressed before in live flies.

Investigation of the suitability of silicate bonding for facet termination in active fiber devices.

We demonstrate that silicate bonding an optical flat to the output facet of an active fiber device can increase the reliability of high-peak power systems and subsantially reduce the effective feedback at the termination of a double-clad fiber. We determine the bonding parameters and conditions that maximize the optical damage threshold of the bond and minimize the Fresnel reflection from the bond. At 1-mum wavelength, damage thresholds greater than 70 J/cm(2) are demonstrated for 25-ns pulses. We also measured Fresnel reflections less than -63 dB off the bond. Finally, we determined that the strength of the bond is sufficient for most operating environments.

In Vivo Imaging of Human Sarcomere Twitch Dynamics in Individual Motor Units.

Motor units comprise a pre-synaptic motor neuron and multiple post-synaptic muscle fibers. Many movement disorders disrupt motor unit contractile dynamics and the structure of sarcomeres, skeletal muscle's contractile units. Despite the motor unit's centrality to neuromuscular physiology, no extant technology can image sarcomere twitch dynamics in live humans. We created a wearable microscope equipped with a microendoscope for minimally invasive observation of sarcomere lengths and contractile dynamics in any major skeletal muscle. By electrically stimulating twitches via the microendoscope and visualizing the sarcomere displacements, we monitored single motor unit contractions in soleus and vastus lateralis muscles of healthy individuals. Control experiments verified that these evoked twitches involved neuromuscular transmission and faithfully reported muscle force generation. In post-stroke patients with spasticity of the biceps brachii, we found involuntary microscopic contractions and sarcomere length abnormalities. The wearable microscope facilitates exploration of many basic and disease-related neuromuscular phenomena never visualized before in live humans.

Linearly polarized, 3.35 W narrow-linewidth, 1150 nm fiber master oscillator power amplifier for frequency doubling to the yellow.

A high-power linearly polarized Yb-doped silica fiber master oscillator power amplifier at 1150 nm is reported. It produced 3.35 W cw and 2.33 W of average power in 1 micros pulses at a 100 kHz repetition rate, both with 8 pm linewidth. This is the first report, to the best of our knowledge, of a high-power Yb-doped fiber amplifier at a wavelength longer than 1135 nm. The pulsed output was frequency doubled in a bulk periodically poled near-stoichiometric LiTaO(3) chip to generate 976 mW of average power at 575 nm with an overall system optical-to-optical efficiency of 9.8% with respect to launched pump power.

Zigzag slabs for solid-state laser amplifiers: batch fabrication and parasitic oscillation suppression.

We have developed a 100 W class Nd:YAG master oscillator power amplifier system based in part on an end-pumped zigzag slab power amplifier. This amplifier incorporates parasitic oscillation suppression by using roughened edges and achieves a small-signal gain coefficient (g(0)l) of 8.06. We describe a novel technique for suppression of parasitic oscillations using claddings on slab edges that significantly increases g(0)l to 11.63 and increases the single-pass extracted power in a power amplifier by 50%. Commercial use of these zigzag slab amplifiers has been limited by the time and cost of production. We describe a new batch fabrication technique that improves the quality and significantly reduces the cost of zigzag slabs.

Efficient yellow-light generation by frequency doubling a narrow-linewidth 1150 nm ytterbium fiber oscillator.

A linearly polarized, narrow-linewidth, diode-pumped, Yb-doped silica-fiber oscillator operating at 1150 nm was frequency doubled to produce 40 mW of 575 nm radiation. The oscillator generated 89 mW of cw linearly polarized output power and was tunable over 0.80 nm. The laser output was coupled to a periodically poled LiNbO3 waveguide that converted 67% of the coupled power to the yellow. The system was fully integrated, with no free-space optics, and had an overall optical-to-optical efficiency of 7.0% with respect to the incident diode-laser pump power.