Wide-field fluorescence lifetime imaging of neuron spiking and subthreshold activity in vivo.

The development of voltage-sensitive fluorescent probes suggests fluorescence lifetime as a promising readout for electrical activity in biological systems. Existing approaches fail to achieve the speed and sensitivity required for voltage imaging in neuroscience applications. We demonstrated that wide-field electro-optic fluorescence lifetime imaging microscopy (EO-FLIM) allows lifetime imaging at kilohertz frame-acquisition rates, spatially resolving action potential propagation and subthreshold neural activity in live adult Drosophila. Lifetime resolutions of <5 picoseconds at 1 kilohertz were achieved for single-cell voltage recordings. Lifetime readout is limited by photon shot noise, and the method provides strong rejection of motion artifacts and technical noise sources. Recordings revealed local transmembrane depolarizations, two types of spikes with distinct fluorescence lifetimes, and phase locking of spikes to an external mechanical stimulus.

Semi-field evaluation of a novel controlled release device using transfluthrin as spatial repellent to prevent entry of mosquitoes into military tents.

Mosquitoes can impact military operational readiness by transmission of disease-causing pathogens or through secondary effects, e.g., annoyance and bites. The focus of this research was to determine if an array of novel controlled release passive devices (CRPD) utilizing the spatial repellent, transfluthrin (TF), as the active ingredient could prevent entry of mosquitoes into military tents for up to 4 weeks. The TF-charged CRPDs were spaced along six strands of monofilament and hung across the tent entrance. Efficacy was evaluated with caged Aedes aegypti to indicate knockdown/mortality effects, and four species of free-flying mosquitoes, Ae. aegypti, Aedes taeniorhynchus, Anopheles quadrimaculatus and Culex quinquefasciatus, to indicate repellent effects. Bioassay cages containing Ae. aegypti were hung vertically at 0.5, 1.0 and 1.5 â€‹m above ground level at designated locations inside of the tents. Knockdown/mortality counts were made every 15 min for the first hour, then at 2, 4 and 24 h post-exposure. Free fliers were recaptured in BG traps operated from 4 to 24 h post-exposure. Knockdown/mortality was gradual until 4 h post-exposure. This increased to near 100% by 24 h in the treated tent but was < 2% in the control tent. There was a significant reduction in the recapture rates of all free-flying species in the treated tent compared with the control tent. Results indicate that TF-charged CRPDs can significantly reduce the numbers of mosquitoes entering military tents and that the four species were affected similarly by the TF. The needs for additional research are discussed.

Resonant Electro-Optic Imaging for Microscopy at Nanosecond Resolution.

We demonstrate an electro-optic wide-field method to enable fluorescence lifetime microscopy (FLIM) with high throughput and single-molecule sensitivity. Resonantly driven Pockels cells are used to efficiently gate images at 39 MHz, allowing fluorescence lifetime to be captured on standard camera sensors. Lifetime imaging of single molecules is enabled in wide field with exposure times of less than 100 ms. This capability allows combination of wide-field FLIM with single-molecule super-resolution localization microscopy. Fast single-molecule dynamics such as FRET and molecular binding events are captured from wide-field images without prior spatial knowledge. A lifetime sensitivity of 1.9 times the photon shot-noise limit is achieved, and high throughput is shown by acquiring wide-field FLIM images with millisecond exposure and >108 photons per frame. Resonant electro-optic FLIM allows lifetime contrast in any wide-field microscopy method.

Fast pulse shaping for a novel gated electron mirror.

We present the design and prototype of a switchable electron mirror, along with a technique for driving it with an arbitrary pulse shape. We employ a general technique for electronic pulse-shaping, where high fidelity of the pulse shape is required, but the characteristics of the system, which are possibly nonlinear, are not known. This driving technique uses an arbitrary waveform generator to pre-compensate the pulse, with a simple iterative algorithm used to generate the input waveform. This is a broadly applicable, general method for arbitrary pulse shaping. Driving our switchable electron mirror with a flat-top pulse, we demonstrate an improvement in rms error of roughly two orders of magnitude compared to an uncompensated waveform. Our results demonstrate the feasibility of high fidelity waveform reproduction in the presence of nonidealities, with immediate applications in the realization of novel electron optical components.

Electro-optic imaging enables efficient wide-field fluorescence lifetime microscopy.

Nanosecond temporal resolution enables new methods for wide-field imaging like time-of-flight, gated detection, and fluorescence lifetime. The optical efficiency of existing approaches, however, presents challenges for low-light applications common to fluorescence microscopy and single-molecule imaging. We demonstrate the use of Pockels cells for wide-field image gating with nanosecond temporal resolution and high photon collection efficiency. Two temporal frames are obtained by combining a Pockels cell with a pair of polarizing beam-splitters. We show multi-label fluorescence lifetime imaging microscopy (FLIM), single-molecule lifetime spectroscopy, and fast single-frame FLIM at the camera frame rate with 103-105 times higher throughput than single photon counting. Finally, we demonstrate a space-to-time image multiplexer using a re-imaging optical cavity with a tilted mirror to extend the Pockels cell technique to multiple temporal frames. These methods enable nanosecond imaging with standard optical systems and sensors, opening a new temporal dimension for wide-field low-light microscopy.

Design for a 10 keV multi-pass transmission electron microscope.

Multi-pass transmission electron microscopy (MPTEM) has been proposed as a way to reduce damage to radiation-sensitive materials. For the field of cryo-electron microscopy (cryo-EM), this would significantly reduce the number of projections needed to create a 3D model and would allow the imaging of lower-contrast, more heterogeneous samples. We have designed a 10 keV proof-of-concept MPTEM. The column features fast-switching gated electron mirrors which cause each electron to interrogate the sample multiple times. A linear approximation for the multi-pass contrast transfer function (CTF) is developed to explain how the resolution depends on the number of passes through the sample.

Note: A single-chamber tool for plasma activation and surface functionalization in microfabrication.

We present a simple apparatus for improved surface modification of polydimethylsiloxane (PDMS) microfluidic devices. A single treatment chamber for plasma activation and chemical/physical vapor deposition steps minimizes the time-dependent degradation of surface activation that is inherent in multi-chamber techniques. Contamination and deposition irregularities are also minimized by conducting plasma activation and treatment phases in the same vacuum environment. An inductively coupled plasma driver allows for interchangeable treatment chambers. Atomic force microscopy confirms that silane deposition on PDMS gives much better surface quality than standard deposition methods, which yield a higher local roughness and pronounced irregularities in the surface.

The impact of surgical specialty on outcomes for carotid endarterectomy.

BACKGROUND: Carotid endarterectomy (CEA) is one of the most frequently performed surgical procedures in the United States. Traditionally, this procedure has been performed by surgeons in at least four specialties. The purpose of this study was to examine the effect of surgeon specialty on the long-term outcomes of CEA among patients receiving the procedure in Pennsylvania. MATERIALS AND METHODS: Data included 17,635 patient admissions for CEA performed between 1995 and 1997, and patient readmission data for the 5-y follow-up period ending in 2002. Five-y outcomes for these patients were compared between vascular, cardiothoracic, general, and neurosurgeons. The primary outcome measures were mortality, stroke, combined stroke and mortality, transient ischemic attack (TIA), and re-occlusion of the ipsilateral artery. Secondary outcomes measured were length of stay and total charges. RESULTS: Using general surgeon as the reference group, and controlling for age, race, severity, and admission type, we found no significant difference across surgical specialties in overall mortality at 5 y post-CEA. Patients treated by vascular surgeons were found to have significantly fewer (P=0.012) strokes and significantly lower re-occlusion rate (P=0.021) at 5 y compared with patients of general surgeons. Patients treated by vascular surgeons also had significantly shorter hospital stay (P<0.0001) but significantly higher charges (P<0.0001) relative to general surgeons. CONCLUSIONS: These results suggest that there are significant differences in outcomes following carotid endarterectomy according to surgeon training. Additional research is needed to explore differences across specialties that may be driving outcomes and to explore the role of surgeon volume at the profession level and cross-volume effects on CEA outcomes.

Bilateral lacrimal system involvement by sclerosing extramedullary hematopoietic tumor.

A 60-year-old man with myelofibrosis and extramedullary hematopoiesis was referred for evaluation of "swollen lacrimal glands." Examination disclosed bilateral firm, immobile, nontender masses in the lacrimal sac fossa with no associated signs of inflammation. MRI of the orbits disclosed lobulated enhancing mass lesions involving the lacrimal sacs and nasolacrimal ducts bilaterally, with bony scalloping of the lacrimal sac fossa and expansion of the bony walls of the nasolacrimal ducts. Incisional biopsy and subsequent debulking of the tumors confirmed sclerosing extramedullary hematopoiesis lesions. To the authors' knowledge, involvement of the lacrimal drainage system with such tumors has not been previously described.

Comprehensive geriatric assessment and home-based rehabilitation for elderly people with a history of recurrent non-elective hospital admissions.

OBJECTIVE: To determine whether a co-ordinated programme of geriatric assessment and multidisciplinary home-based rehabilitation reduces disability and prevents non-elective hospital readmission in high-risk elderly patients. DESIGN: Nested case-control study comparing usual post-discharge care versus usual care plus a comprehensive geriatric assessment and home-based rehabilitation service, comprising nursing, occupational therapy and physiotherapy with geriatric medical review. Patients were >or=65 years with >or=2 non-elective hospital admissions within the previous 12 months. Disability was assessed using the 100-point Barthel index and Nottingham extended activities of daily living (EADL) score. Non-elective hospital admissions were recorded over 1-year follow-up. RESULTS: We studied 84 patients; 56 receiving the new service were matched to 28 controls. Intervention subjects received a median of 19 h [interquartile range (IQR) (7,35)] rehabilitation over 19 [IQR (6,42)] domiciliary visits. At 3 months, there was improvement in median Barthel and Nottingham EADL scores in the intervention group of 3 and 2 points, respectively, compared with reductions in controls of 3 and 6 points (both P<0.001, changes in intervention group versus controls); similar differences persisted in survivors at 12 months. There was a non-significant trend for reduction in the proportion of patients with further non-elective hospital admission in the intervention group (36/56, 64%) compared with controls (21/28, 75%; OR 0.70, 95% CI 0.34, 1.46). CONCLUSIONS: A co-ordinated programme of geriatric assessment and multidisciplinary home-based rehabilitation reduced disability in elderly patients at high risk for non-elective hospital admission. Further research is required to determine whether this approach can reduce the need for hospital admission.