Oleg Pustovyy

Resolution of 90 nm (λ/5) in an optical transmission microscope with an annular condenser

Resolution of 90 nm was achieved with a research microscope simply by replacing the standard bright-field condenser with a homebuilt illumination system with a cardioid annular condenser. Diffraction gratings with 100 nm width lines as well as less than 100 nm size features of different-shaped objects were clearly visible on a calibrated microscope test slide. The resolution increase results from a known narrower diffraction pattern in coherent illumination for the annular aperture compared with the circular aperture. This explanation is supported by an excellent accord of calculated and measured diffraction patterns for a 50 nm radius disk.

Enhancement of Odorant-Induced Responses in Olfactory Receptor Neurons by Zinc Nanoparticles

Zinc metal nanoparticles in picomolar concentrations strongly enhance odorant responses of olfactory sensory neurons. One- to 2-nm metallic particles contain 40-300 zinc metal atoms, which are not in an ionic state. We exposed rat olfactory epithelium to metal nanoparticles and measured odorant responses by electroolfactogram and whole-cell patch clamp. A small amount of zinc nanoparticles added to an odorant or an extracellular/intracellular particle perfusion strongly increases the odorant response in a dose-dependent manner. Zinc nanoparticles alone produce no odor effects. Copper, gold, or silver nanoparticles do not produce effects similar to those of zinc. If zinc nanoparticles are replaced by Zn(+2) ions in the same concentration range, we observed a reduction of the olfactory receptor neuron odorant response. Based on these observations, we hypothesize that zinc nanoparticles are closely located to the interface between the guanine nucleotide-binding protein and the receptor proteins and are involved in transferring signals in the initial events of olfaction. Our results suggest that zinc metal nanoparticles can be used to enhance and sustain the initial olfactory events.

Real-time optical detection of methicillin-resistant Staphylococcus aureus using lytic phage probes.

Staphylococcus aureus (S. aureus)-specific bacteriophage was used as a probe for detection of methicillin-resistant S. aureus (MRSA) in aqueous solution using a novel optical method. Biorecognition phage monolayers transferred to glass substrates using Langmuir-Blodgett (LB) technique were exposed individually to MRSA in solution at logarithmic concentrations ranging from 10(6) to 10(9)cfu/ml, and observed for real-time binding using a CytoViva optical light microscope system. Results indicate that LB monolayers possessed high levels of elasticity (K), measuring 22 and 29 mN/m for 10(9) and 10(11)pfu/ml phage concentrations, respectively. Near-instantaneous MRSA-phage binding produced 33+/-5%, 10+/-1%, 1.1+/-0.1%, and 0.09+/-0.01% coverage of the substrate that directly correlated to a decrease in MRSA concentrations of 10(9), 10(8), 10(7), and 10(6)cfu/ml. The exclusive selectivity of phage monolayers was verified with Salmonella enterica subsp. enterica serovar typhimurium (S. typhimurium) and Bacillus subtilis.

Novel Metal Clusters Isolated from Blood Are Lethal to Cancer Cells

Unfolding and subsequent aggregation of proteins is a common phenomenon that is linked to many human disorders. Misfolded hemoglobin is generally manifested in various autoimmune, infectious and inherited diseases. We isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons lack nucleic acids but contain two major polypeptide populations with homology to the hemoglobin α-chain. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1- to 2-nm metallic nanoclusters containing 40–300 atoms. Each milliliter of human blood contained approximately 7 × 1013 PNCs and approximately 3 × 108 proteons. Exposure of isolated blood plasma to elevated temperatures increased the number of proteons. When an aliquot of this heated plasma was introduced into untreated plasma that was subsequently heated, the number of proteons further increased, reaching a maximum after a total of three such iterations. Small concentrations of PNCs were lethal to cultured cancer cells, whereas noncancerous cells were much less affected.

Detection and identification of methicillin resistant and sensitive strains of Staphylococcus aureus using tandem measurements.

Discrimination of methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus, was achieved by the specially selected lytic bacteriophage with a wide host range of S. aureus strains and a penicillin-binding protein (PBP 2a) specific antibody. A quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to analyze bacteria-phage interactions. The lytic phages were transformed into phage spheroids by exposure to water-chloroform interface. Phage spheroid monolayers were transferred onto QCM-D sensors by Langmuir-Blodgett (LB) technique. Biosensors were tested in the flow mode with bacterial water suspensions, while collecting frequency and energy dissipation changes. Bacteria-spheroid interactions resulted in decreased resonance frequency and an increase in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors were further exposed to a flow of the penicillin-binding protein (PBP 2a) specific antibody conjugated latex beads. Sensors tested with MRSA responded to PBP 2a antibody beads; while sensors examined with MSSA gave no response. This experimental difference establishes an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Both free and immobilized bacteriophages strongly inhibit bacterial growth on solid/air interfaces and in water suspensions. After lytic phages are transformed into spheroids, they retain their strong lytic activity and demonstrate high bacterial capture efficiency. The phage and phage spheroids can be used for screening and disinfection of antibiotic resistant bacteria. Other applications may include use on biosensors, bacteriophage therapy, and antimicrobial surfaces.

Olfactory responses to explosives associated odorants are enhanced by zinc nanoparticles

Many odorants related to manufactured explosives have low volatilities and are barely detectable as odors. We previously reported that zinc metal nanoparticles increased rat olfactory epithelium responses, measured by electroolfactogram (EOG), to several odorants. Here, we report that nanomolar concentrations of zinc metal nanoparticles strongly enhanced olfactory responses to the explosives related odorants cyclohexanone, methyl benzoate, acetophenone, and eugenol. Rat olfactory epithelium was exposed to metal nanoparticles and odorant responses were quantified by EOG. Zinc nanoparticles added to explosive odorants strongly increased the odorant response in a dose-dependent manner. The enzymatic breakdown of the second messenger cyclic adenosine monophosphate (cAMP) was prevented by adding the membrane-permeable phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This caused the olfactory cilia cAMP concentration to increase and generated EOG signals. The EOG responses generated by IBMX were not enhanced by zinc nanoparticles. Based on these observations, we conclude that zinc nanoparticles act at the receptor site and are involved in the initial events of olfaction. Our results suggest that zinc metal nanoparticles can be used to facilitate a canine detection of explosive odorants.

Enhancement of Odor-Induced Activity in the Canine Brain by Zinc Nanoparticles: A Functional MRI Study in Fully Unrestrained Conscious Dogs

Using noninvasive in vivo functional magnetic resonance imaging (fMRI), we demonstrate that the enhancement of odorant response of olfactory receptor neurons by zinc nanoparticles leads to increase in activity in olfaction-related and higher order areas of the dog brain. To study conscious dogs, we employed behavioral training and optical motion tracking for reducing head motion artifacts. We obtained brain activation maps from dogs in both anesthetized state and fully conscious and unrestrained state. The enhancement effect of zinc nanoparticles was higher in conscious dogs with more activation in higher order areas as compared with anesthetized dogs. In conscious dogs, voxels in the olfactory bulb and hippocampus showed higher activity to odorants mixed with zinc nanoparticles as compared with pure odorants, odorants mixed with gold nanoparticles as well as zinc nanoparticles alone. These regions have been implicated in odor intensity processing in other species including humans. If the enhancement effect of zinc nanoparticles observed in vivo are confirmed by future behavioral studies, zinc nanoparticles may provide a way for enhancing the olfactory sensitivity of canines for detection of target substances such as explosives and contraband substances at very low concentrations, which would otherwise go undetected.

Anterior-posterior dissociation of the default mode network in dogs.

The default mode network (DMN) in humans has been extensively studied using seed-based correlation analysis (SCA) and independent component analysis (ICA). While DMN has been observed in monkeys as well, there are conflicting reports on whether they exist in rodents. Dogs are higher mammals than rodents, but cognitively not as advanced as monkeys and humans. Therefore, they are an interesting species in the evolutionary hierarchy for probing the comparative functions of the DMN across species. In this study, we sought to know whether the DMN, and consequently its functions such as self-referential processing, are exclusive to humans/monkeys or can we also observe the DMN in animals such as dogs. To address this issue, resting state functional MRI data from the brains of lightly sedated dogs and unconstrained and fully awake dogs were acquired, and ICA and SCA were performed for identifying the DMN. Since anesthesia can alter resting state networks, confirming our results in awake dogs was essential. Awake dog imaging was accomplished by training the dogs to keep their head still using reinforcement behavioral adaptation techniques. We found that the anterior (such as anterior cingulate and medial frontal) and posterior regions (such as posterior cingulate) of the DMN were dissociated in both awake and anesthetized dogs.

High-resolution light microscopy of nanoforms

We developed a high resolution light imaging system. Diffraction gratings with 100 nm width lines as well as less than 100 nm size features of different-shaped objects are clearly visible on a calibrated microscope test slide (Vainrub et al., Optics Letters, 2006, 31, 2855). The two-point resolution increase results from a known narrowing of the central diffraction peak for the annular aperture. Better visibility and advanced contrast of the smallest features in the image are due to enhancement of high spatial frequencies in the optical transfer function. The imaging system is portable, low energy, and battery operated. It has been adapted to use in both transmitting and reflecting light. It is particularly applicable for motile nanoform systems where structure and functions can be depicted in real time. We have isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1-2nm metallic nanoclusters containing 40-300 atoms. Proteons are capable of spontaneous assembling into higher nanoform systems assuming structure of complicated topology. The arrangement of complex proteon system mimics the structure of a small biological cell. It has structures that imitate membrane and nucleolus or nuclei. Some of these nanoforms are motile. They interact and divide. Complex nanoform systems can spontaneously reduce to simple proteons. The physical properties of these nanoforms could shed some light on the properties of early life forms or forms at extreme conditions.

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species (1,2). The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique (3). The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.