Second-harmonic imaging microscopy of living cells.

Second harmonic generation (SHG) has been developed in our laboratories as a high-resolution nonlinear optical imaging microscopy for cellular membranes and intact tissues. SHG shares many of the advantageous features for microscopy of another more established nonlinear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce three-dimensional images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It can be used to image membrane probes with high membrane specificity and displays extraordinary sensitivity in reporting membrane potential; it also has the ability to image highly ordered structural proteins without any exogenous labels.

Inhibition of inducible nitric oxide synthase by acetamidine derivatives of hetero-substituted lysine and homolysine.

The synthesis and in vitro evaluation of the acetamidine derivatives of hetero-substituted lysine and homolysine analogues have identified potent inhibitors of human nitric oxide synthase enzymes, including examples with marked selectivity for the inducible isoform.

Optical nanosensors for chemical analysis inside single living cells. 1. Fabrication, characterization, and methods for intracellular delivery of PEBBLE sensors.

Spherical optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced in sizes including 20 and 200 nm in diameter. These sensors are fabricated in a microemulsion and consist of fluorescent indicators entrapped in a polyacrylamide matrix. A generalized polymerization method has been developed that permits production of sensors containing any hydrophilic dye or combination of dyes in the matrix. The PEBBLE matrix protects the fluorescent dye from interference by proteins, allowing reliable in vivo calibrations of dyes. Sensor response times are less than 1 ms. Cell viability assays indicate that the PEBBLEs are biocompatible, with negligible biological effects compared to control conditions. Several sensor delivery methods have been studied, including liposomal delivery, gene gun bombardment, and picoinjection into single living cells.

Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors.

Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of fluorescent indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB. Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.

Cause, prevalence, and response to occupational musculoskeletal injuries reported by physical therapists and physical therapist assistants.

BACKGROUND AND PURPOSE: Physical therapists (PTs) and physical therapist assistants (PTAs) are susceptible to occupational musculoskeletal injuries. The purpose of this study was to examine the reported causes and prevalence of occupational musculoskeletal injuries to PTs and PTAs during a 2-year period. SUBJECTS: A questionnaire was mailed to 500 PTs and 500 PTAs randomly selected from the American Physical Therapy Association 1996 active membership list. Six hundred sixty-seven questionnaires were returned, giving a response rate of 67%. METHOD: Based on a literature review and a pilot study, an occupational injury questionnaire was constructed and mailed. Self-reports of injuries were obtained. RESULTS: Thirty-two percent of the PTs and 35% of the PTAs reported sustaining a musculoskeletal injury. The highest prevalence of injury was to the low back (62% of injured PTs and 56% of injured PTAs). The PTs reported the upper back and the wrist and hand as having the second highest prevalence (23%). The PTAs reported the upper back as having the second highest prevalence (28%). The PTs and PTAs reported making changes in their work habits of improved body mechanics, increased use of other personnel, and frequent change of work position. The majority of PTs and PTAs reported they did not limit patient contact time or area of practice after sustaining an injury. CONCLUSION AND DISCUSSION: Although PTs and PTAs are recognized to be knowledgeable in prevention and treatment of musculoskeletal injuries, they are susceptible to sustaining occupational musculoskeletal injuries because of performing labor-intensive tasks.

Ratiometric and fluorescence-lifetime-based biosensors incorporating cytochrome c' and the detection of extra- and intracellular macrophage nitric oxide.

Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c', a hemoprotein known to bind nitric oxide selectively. The cytochrome c' is labeled with a fluorescent reporter dye, and changes in this dye's intensity or fluorescence lifetime are observed as the protein binds nitric oxide. The ratiometric sensors are composed of dye-labeled cytochrome c' attached to the optical fiber via colloidal gold, along with fluorescent microspheres as intensity standards. These ratiometric sensors exhibit linear response, have fast response times (< or = 0.25 s), and are completely reversible. The sensors are selective over numerous common interferents such as nitrite, nitrate, and oxygen species, and the limit of detection is 8 microM nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c' in solution and have a limit of detection of 30 microM nitric oxide. The use of these two techniques has allowed measurement of intra- and extracellular macrophage nitric oxide. Employing the ratiometric fiber sensors gave a multicell culture average extracellular nitric oxide concentration of 210 +/- 90 microM for activated macrophages, while an average intracellular concentration of 160 +/- 10 microM was determined from the lifetime-based measurements of dye-labeled cytochrome c' in the macrophage cytosol. Microscopic adaptation of the lifetime-based methods described here would allow direct correlation of intracellular nitric oxide levels with specific cellular activities, such as phagocytosis.

Use of ultrasound to facilitate antibiotic diffusion through Pseudomonas aeruginosa alginate.

The effect of ultrasonic treatment of two pseudomonal alginate samples was studied by investigation of their rheological characteristics and interaction with tobramycin, piperacillin and ciprofloxacin. Whilst exposure to ultrasound at an amplitude of 20 microns for 8 min caused a significant decrease in viscosity of both alginates, molecular fragmentation, as demonstrated by PAGE and gel-permeation chromatography, was minimal. Antibiotic penetration was improved substantially however with sonication, leading to a 100% increase in tobramycin and piperacillin diffusion from the alginate compared with an untreated control polymer.