Nuclear magnetic resonance in plant science research

Summary

In vivo nuclear magnetic resonance (NMR) spectroscopy and NMR imaging are noninvasive techniques with the potential to investigate a wide range of biochemical and physiological problems in living systems. The extent to which this potential has been realized in plant tissues is discussed with reference to recent applications to a number of systems, including root tissues and plant cell suspensions. It is concluded that while the potential of NMR imaging has yet to be fully realized in plants, in vivo NMR spectroscopy has matured into a problem-solving technique that can be used to test metabolic hypotheses directly.     

Functionalization and peptide-based delivery of magnetic nanoparticles as an intracellular MRI contrast agent

We report the development of functionalized superparamagnetic iron oxide nanoparticles with a PEG-modified, phospholipid micelle coating, and their delivery into living cells. The size of the coated particles, as determined by dynamic light scattering and electron microscopy, was found to be between 12 and 14 nm. The PEG-phospholipid coating resulted in high water solubility and stability, and the functional groups of modified PEG allowed for bioconjugation of various moieties, including a fluorescent dye and the Tat peptide. Efficient delivery of the functionalized nanoparticles into living cells was confirmed by fluorescence microscopy, relaxation time measurements, and magnetic resonance imaging (MRI). This demonstrates the feasibility of using functionalized magnetic nanoparticles with uniform (~10 nm) sizes as an MRI contrast agent for intracellular molecular imaging in deep tissue. These micelle-coated iron oxide nanoparticles offer a versatile platform for conjugation of a variety of moieties, and their small size confers advantages for intracellular molecular imaging with minimal perturbation.Abbreviations CPP cell penetrating peptide - CPMG Carr–Purcell–Meiboom–Gill spin-echo method - CTAB cetyltrimethylammonium bromide - DLS dynamic light scattering - DMEM Dulbeccos modified Eagles medium - DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine - FCS fetal calf serum - FGM-2 fibroblast growth medium 2 - HDF human dermal fibroblast - HS horse serum - MDBK Madin–Darby bovine kidney - MIONs superparamagnetic iron oxide nanoparticles - mMIONs micelle-coated MIONs - MRI magnetic resonance imaging - PBS phosphate-buffered saline - PEG poly(ethylene glycol) - SPDP N-succinimidyl 3-(2-pyridyldithio)propionate - TCEP tris(2-carboxyethyl)phosphine hydrochloride - TEM transmission electron microscopy     

A new approach for the biolistic method: Bombardment of living nitrogen-fixing bacteria into plant tissues

Summary A new utilization of the biolistic gun was developed for the direct introduction of nitrogen-fixing bacteria (Azotobacter vinelandii) into strawberry (Fragaria x ananassa) tissues. This was the first case of using living bacteria as microprojectiles for the bombardment of plant tissues. Bacterial cells, adhered to tungsten particles, were accelerated by a nitrogen-powered device, and delivered into the target leaves and regenerating shoot meristems. The presence of bacteria in the developing strawberry callus tissues and regenerating plants was detected by microscopy, acetylene reduction assay, and selective polymerase chain reaction. Practically, the elaborated method proved to be suitable for the establishment of artificial intereellular, associations between nitrogen-fixing bacteria and higher plants.     

Absorption and translocation to the aerial part of magnetic carbon-coated nanoparticles through the root of different crop plants

The development of nanodevices for agriculture and plant research will allow several new applications, ranging from treatments with agrochemicals to delivery of nucleic acids for genetic transformation. But a long way for research is still in front of us until such nanodevices could be widely used. Their behaviour inside the plants is not yet well known and the putative toxic effects for both, the plants directly exposed and/or the animals and humans, if the nanodevices reach the food chain, remain uncertain. In this work we show that magnetic carbon-coated nanoparticles forming a biocompatible magnetic fluid (bioferrofluid) can easily penetrate through the root in four different crop plants (pea, sunflower, tomato and wheat). They reach the vascular cylinder, move using the transpiration stream in the xylem vessels and spread through the aerial part of the plants in less than 24 hours. Accumulation of nanoparticles was detected in wheat leaf trichomes, suggesting a way for excretion/detoxification. This kind of studies is of great interest in order to unveil the movement and accumulation of nanoparticles in plant tissues for assessing further applications in the field or laboratory.     

Interaction of a flavone loaded on surface-modified dextran-spooled superparamagnetic nanoparticles with β-cyclodextrin and DNA

Flavones are biologically active compounds obtained mainly from plant sources. Pharmaceutically important compounds can be delivered to the physiological target by loading them in carriers like cyclodextrins and magnetic nanoparticles. Herein, the binding of 6-methoxyflavone to β-cyclodextrin and DNA is studied using UV–visible absorption and fluorescence spectroscopy. The loading of 6-methoxyflavone onto a magnetic nanoparticles is employed. β-cyclodextrin encapsulates the 6-methoxyflavone to form an inclusion complex. β-cyclodextrin also used to draw forth 6-methoxyflavone loaded onto a magnetic nanoparticles. The morphology, magnetic property and the crystallite size of the nanoparticles are studied using scanning electron microscopy, vibrating sample magnetometry and X-ray diffraction techniques, respectively. The binding of the drug-loaded magnetic nanoparticles to DNA shows that the compound is accessible to DNA and available mostly on the surface of the nanoparticles despite a modified dextan polymer supposedly encapsulates the flavone.     

Carbon-iron magnetic nanoparticles for agronomic use in plants: Promising but still a long way to go

In the recent years, multiple ways of interaction between the fields of nanotechnology and biology have been opened, mainly in the biomedical research, with the development of tools for diagnosis and controlled delivery of substances.^1,2 On the other hand, in the field of plant biology, the interaction between both disciplines has been less frequent. Most of the published work on this field has focus in the environmental impact of nanoparticles on crop growth and development;^3,4 and also on the bio production of nanoparticles using plant extracts (reviewed in refs. ^5–8). Much less attention has taken other possible aspects of the interrelationship between nanotechnology and plant biology, such as the development of nanodevices for controlled delivery of drugs or different kind of substances,^9,10 in a similar way to that already developed in the medical research.Key words: plant nanobiotechnology, phytosanitaries, nanoparticle uptake, electron microscopy, nanoparticle subcellular localizationRecently, our group has developed an approach for the application of carbon-coated iron nanoparticles to pumpkin plants. The goal of this project was the development of tools for the treatment of pathologies affecting specific areas or organs of the plants. To achieve that, nanoparticles carrying the phyto-remedy will be applied to the affected plants, and magnetic fields will be applied to the organs of the plant affected by the pathology. In this way, the nanoparticles will be retained in the affected area, and the effect of the active compound linked to the nanoparticle will be concentrated in a restricted area.In a previous report, we described the capability of different microscopic methodologies to identify and locate nanoparticles in plant tissue samples, including both electron microscopy and light microscopy approaches.¹¹ Using this knowledge, we used a correlative microscopy approach, identifying first the presence of nanoparticles in sections of resin-embedded tissue by light microscopy (bright field, phase contrast and dark field), followed by a further analysis of consecutive sections from the same block, this time by transmission electron microscopy. This approach allowed us to unveil many different aspects of the behavior of the nanoparticles in the living tissue. First of all, movement of the nanoparticles was detected at different levels: chains of nanoparticle-aggregates carrying cells were apparent close to the application point, when such application was made by ‘injection’ of the nanoparticle suspension into the pith cavity of the stem, suggesting the flux of nanoparticles from one cell to another. Also, after the same kind of application, nanoparticles were detected in an area close to the vascular core, but appearing as isolated particles in the cytoplasm of the cell. Furthermore, after application of the nanoparticle by ‘spray’ (that is, application of a drop of the solution over the surface of the leaf, close to the petiole insertion point), isolated nanoparticles were also detected close to the application point (Fig. 1). This last data is particularly relevant, as this application method attempted to emulate that of breeders and coordinators of phytosanitary control. The fact that the nanoparticles are capable of penetrating through the leaf cuticule and into the cell cytoplasm opens the possibility for the use of this approach in phytosanitary applications.Open in a separate windowFigure 1Isolated nanoparticles localized close to the epidermis after spray application. (A) Low amplification image of the area were the nanoparticle is (squared area): SEC, sub estomatal cavity; GC, guard cell; E, exterior; LP, lagunar parenchyma; EP, epidermis. (B) Detail of the region squared in (A), showing an isolated nanoparticle (arrow). Bars in (A): 5 µm, (B): 200 nm.A second question of special interest in our analysis was the differential response to the presence of the nanoparticles shown by the cell cytoplasm when they appear in the form of aggregates when compared with cells carrying non-clustered nanoparticles. In fact, a dense cytoplasm with starch-containing organelles was observed concomitantly with nanoparticle aggregates in the cytosol (Fig. 2), suggesting that plant cells could respond to the presence of a high density of nanoparticles by changing their subcellular organization. On the other hand, no response was observed in those cells in which only isolated nanoparticles were detected (Fig. 1). The change on the cytoplasm of the cells was accompanied by the fact that the cell-to-cell movement of the particles in regions with a high density of aggregates seems to direct them to the exterior of the organism, what points to a physiological response from the plant to the intracellular presence of nanoparticle aggregates.Open in a separate windowFigure 2Different behavior between cell cytoplasm depending of the content in nanoparticle aggregates. (A) low amplification image showing unmodified cells (UMC) without nanoparticles, next to a nanoparticle carrying cell (NCC), whose cytoplasm is full of organelles. (B) Detail of the area squared in (A), indicating the presence of nanoparticle aggregates (asterisks). (C) Detail of a nanoparticle aggregate. Bars in (A): 10 µm, (B): 2 µm, (C): 200 nm.Despite the obvious advance that supposes the possibility of the application of nanoparticles on agronomical applications, it is also clear that there are many aspects in the protocols for detection of the nanoparticles and for their infiltration into the plant than can be improved. As shown in our work, the detection of isolated nanoparticles is almost impossible to achieve just by the resolution of the conventional optical microscopy, and their detection by direct scanning of ultrathin sections by electron microscopy is a tedious and time consuming task, especially if there is no clear evidence of the presence or not of nanoparticles in a certain part of the plant. Therefore, it is convenient to develop protocols with auxiliary methodologies to increase the sensibility of the microscopy techniques, that could allow directing the analysis by TEM to samples in which presence of nanoparticles has been previously assessed with certainty. Several methodologies have been employed for the detection of metallic nanoparticles in large tissue samples or in living tissues, with a special development in the field of therapy and diagnosis of diseases in the central nervous system. In this area magnetic resonance imaging,^12,13 phototermal interference contrast¹⁴ and conventional iron staining¹³ approaches have been successfully applied, although the resolution level is still low, with ranges between 1 µm and the cell size. A suitable solution could be the optimization of current methods for iron staining (reviewed in ref. ¹⁵), provided that lesser equipment requirements are needed than for the other approaches. In this line of action, a protocol should be optimized for their use to detect our particles in plant cells using as a starting point previous reports of iron detection in plant tissues, as the one used by Green¹⁶ for detection of inorganic ferric iron in Arabidopsis thaliana. Also, as a suitable system for the detection of global uptake of magnetic nanoparticles into plant organs, a vibrating sample magnetometer has been used successfully to measure the amount of nanoparticles taken by different organs of Cucurbita maxima plants,¹⁷ what could be a good option for the selection of samples of interest to be analyzed with microscopy in future experiments. Last, but not least, an even more straight forward approach is the attachment of colored, fluorescent or chemically detectable compounds to the nanoparticles, allowing an enhancement of the capacity of optical microscopy to detect those.¹⁸Our approach has been shown to allow the internalization of nanoparticles into plant cells in vivo, but the system as established initially is far from being functional, and several questions require improvement. First, the distribution of nanoparticles is still very limited, and most of the intracellular translocation of nanoparticles takes place near the application point, and much more efficiently when it is effectuated by injection. The most interesting way of application, the pulverization, has given only very limited results, with presence of intracellular nanoparticles but just in the first cellular layer, the epidermis. Also, long-range transport has still a low efficiency. Observations in samples equivalent to the ones that we have analyzed of fresh vibratome section, in which nanoparticles seemed to be present¹¹ may point to the possibility of loosing some NPs during the processing for EM analysis. Again, this possibility reinforces the need of a pre-scan of the samples to identify the presence of nanoparticles before a further analysis.Interestingly, it has been described recently the uptake of magnetite nanoparticles through the root system in Cucurbita maxima plants, as well as when applied by spraying, but the first method was tested only in plants growing in liquid media, and in the second only plants growing again in liquid media showed a significant uptake of particles.¹⁷ Also, in Arabidopsis and Phalaenopsis plants, it has been possible to perform live imaging of the uptake of other kind of nanoparticles (NaYF4:Yb,Er).¹⁹As stated previously, these experiments support the applicability of magnetic nanoparticles for use in agronomic purposes. But despite of the promising results, there is still a long way to go until they are suited for their use. This commentary has attempted to focus in some of the aspects to improve in the methodology employed.     

Nuclear magnetic resonance micro-imaging in the investigation of plant cell metabolism

Micro-imaging based on nuclear magnetic resonance offers the possibility to map metabolites in plant tissues non-invasively. Major metabolites such as sucrose and amino acids can be observed with high spatial resolution. Stable isotope tracers, such as (13)C-labelled metabolites can be used to measure the in vivo conversion rates in a metabolic network. This review summarizes the different nuclear magnetic resonance micro-imaging techniques that are available to obtain spatially resolved information on metabolites in plants. A short general introduction into NMR imaging techniques is provided. Particular emphasis is given to the difficulties encountered when NMR micro-imaging is applied to plant systems.     

Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model

The magnetic properties and the ultrastructure, with special emphasis on the nanometric range, of liver tissues in an iron overload rat model have been investigated. The tissues of the animals, sacrificed at different times after a single iron dextran injection, have been characterised by magnetic AC susceptibility measurements together with transmission electron microscopy (TEM) and selected area electron diffraction (SAED) as helping techniques. It has been observed that few days after the iron administration the liver contains at least two iron species: (i) akaganéite nanoparticles, coming from iron dextran and (ii) ferrihydrite nanoparticles corresponding to ferritin. The magnetic susceptibility of the tissues depends not only on the elemental iron content but also on its distribution among chemical species, and varies in a remarkable regular manner as a function of the elapsed time since the iron administration. The results are of relevance with respect to non-invasive techniques for liver iron determination, directly or indirectly based on the magnetic susceptibility of the tissues, as biomagnetic liver susceptometry (BLS) and magnetic resonance (MRI) image treatment.     

Uptake and cellular distribution,in four plant species,of fluorescently labeled mesoporous silica nanoparticles

Key message

We report the uptake of MSNs into the roots and their movement to the aerial parts of four plant species and their quantification using fluorescence, TEM and proton-induced x - ray emission (micro - PIXE) elemental analysis.

Abstract

Monodispersed mesoporous silica nanoparticles (MSNs) of optimal size and configuration were synthesized for uptake by plant organs, tissues and cells. These monodispersed nanoparticles have a size of 20 nm with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time, a combination of confocal laser scanning microscopy, transmission electron microscopy and proton-induced X-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. The translocation and widescale distribution of MSNs in plants will enable them to be used as a new delivery means for the transport of different sized biomolecules into plants.     

Sensing of silver nanoparticles on/in endothelial cells using atomic force spectroscopy

Endothelial cells, due to their location, are interesting objects for atomic force spectroscopy study. They constitute a barrier between blood and vessel tissues located deeper, and therefore they are the first line of contact with various substances present in blood, eg, drugs or nanoparticles. This work intends to verify whether the mechanical response of immortalized human umbilical vein endothelial cells (EA.hy926), when exposed to silver nanoparticles, as measured using force spectroscopy, could be effectively used as a bio‐indicator of the physiological state of the cells. Silver nanoparticles were characterized with transmission electron microscopy and dynamic light scattering techniques. Tetrazolium salt reduction test was used to determine cell viability after treatment with silver nanoparticles. An elasticity of native cells was examined in the Hanks' buffer whereas fixed cells were softly fixed with formaldehyde. Additional aspect of the work is the comparative force spectroscopy utilizing AFM probes of ball‐shape and conical geometries, in order to understand what changes in cell elasticity, caused by SNPs, were detectable with each probe. As a supplement to elasticity studies, cell morphology observation by atomic force microscopy and detection of silver nanoparticles inside cells using transmission electron microscopy were also performed. Cells exposed to silver nanoparticles at the highest selected concentrations (3.6 μg/mL, 16 μg/mL) are less elastic. It may be associated with the reorganization of the cellular cytoskeleton and the “strengthening” of the cell cortex caused by presence of silver nanoparticles. This observation does not depend on cell fixation. Agglomerates of silver nanoparticles were observed on the cell membrane as well as inside the cells.     

Nuclear magnetic resonance studies of the location and function of plant nutrients in vivo

The cytoplasmic and vacuolar pools of ammonium, inorganic phosphate and potassium can be studied non-invasively in plant tissues using high resolution nuclear magnetic resonance spectroscopy. The techniques that allow these pools to be discriminated in vivo are described and their application to plants is reviewed with reference to the phosphorus, nitrogen and potassium nutrition of root tissues.     

Detection of the third and fourth heart sounds using Hilbert-Huang transform

Background

Magnetic nanoparticles are gaining great roles in biomedical applications as targeted drug delivery agents or targeted imaging contrast agents. In the magnetic nanoparticle applications, quantification of the nanoparticle density deposited in a specified region is of great importance for evaluating the delivery of the drugs or the contrast agents to the targeted tissues. We introduce a method for estimating the nanoparticle density from the displacement of tissues caused by the external magnetic field.

Methods

We can exert magnetic force to the magnetic nanoparticles residing in a living subject by applying magnetic gradient field to them. The nanoparticles under the external magnetic field then exert force to the nearby tissues causing displacement of the tissues. The displacement field induced by the nanoparticles under the external magnetic field is governed by the Navier's equation. We use an approximation method to get the inverse solution of the Navier's equation which represents the magnetic nanoparticle density map when the magnetic nanoparticles are mechanically coupled with the surrounding tissues. To produce the external magnetic field inside a living subject, we propose a coil configuration, the Helmholtz and Maxwell coil pair, that is capable of generating uniform magnetic gradient field. We have estimated the coil currents that can induce measurable displacement in soft tissues through finite element method (FEM) analysis.

Results

From the displacement data obtained from FEM analysis of a soft-tissue-mimicking phantom, we have calculated nanoparticle density maps. We obtained the magnetic nanoparticle density maps by approximating the Navier's equation to the Laplacian of the displacement field. The calculated density maps match well to the original density maps, but with some halo artifacts around the high density area. To induce measurable displacement in the living tissues with the proposed coil configuration, we need to apply the coil currents as big as 10⁴A.

Conclusions

We can obtain magnetic nanoparticle maps from the magnetically induced displacement data by approximating the Navier's equation under the assumption of uniform-gradient of the external magnetic field. However, developing a coil driving system with the capacity of up to 10⁴A should be a great technical challenge.     

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.     

Incorporation of biodegradable nanoparticles into human airway epithelium cells-in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases

PURPOSE: Nanoparticles are able to enhance drug or DNA stability for purposes of optimised deposition to targeted tissues. Surface modifications can mediate drug targeting. The suitability of nanoparticles synthesised out of porcine gelatin, human serum albumin, and polyalkylcyanoacrylate as drug and gene carriers for pulmonary application was investigated in vitro on primary airway epithelium cells and the cell line 16HBE14o-. METHODS: The uptake of nanoparticles into these cells was examined by confocal laser scan microscopy (CLSM) and flow cytometry (FACS). Further the cytotoxicity of nanoparticles was evaluated by an LDH-release-test and the inflammatory potential of the nanoparticles was assessed by measuring IL-8 release. RESULTS: CLSM and FACS experiments showed that the nanoparticles were incorporated into bronchial epithelial cells provoking little or no cytotoxicity and no inflammation as measured by IL-8 release. CONCLUSIONS: Based on their low cytotoxicity and the missing inflammatory potential in combination with an efficient uptake in human bronchial epithelial cells, protein-based nanoparticles are suitable drug and gene carriers for pulmonary application.     

Drug delivery and nanoparticles:applications and hazards

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatine and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery. Besides the potential beneficial use also attention is drawn to the questions how we should proceed with the safety evaluation of the nanoparticle formulations for drug delivery. For such testing the lessons learned from particle toxicity as applied in inhalation toxicology may be of use. Although for pharmaceutical use the current requirements seem to be adequate to detect most of the adverse effects of nanoparticle formulations, it can not be expected that all aspects of nanoparticle toxicology will be detected. So, probably additional more specific testing would be needed.     

Systematic Determination of Various Forms of Choline in Plants

A rapid and systematic method is described for the determination of lipid-choline, free choline and two water-soluble bound cholines in plant tissues. These substances are extracted with isopropanol and methanol, avoiding the enzymatic degradation. The extract is fractionated into lipid-choline and water-soluble choline by applying the solvent-partitioning method of Folch et al. The latter fraction is passed through Amberlite IRC-50 column, and separated into free choline and bound-choline fractions. Two forms of acid-labile choline are determined after hydrolyzing the bound-choline fraction with hydrochloric acid. The method was applied to the determination of various forms of choline in several plants, and lipid-choline and free choline were found to be major components.     

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.

     

Color transformation and fluorescence of Prussian blue-positive cells: implications for histologic verification of cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide (SPIO) nanoparticles, either modified or in combination with other macromolecules, are being used for magnetic labeling of stem cells and other cells to monitor cell trafficking by magnetic resonance imaging (MRI) in experimental models. The correlation of histology to MRI depends on the ability to detect SPIO-labeled cells using Prussian blue (PB) stain and fluorescent tags to cell surface markers. Exposure of PB-positive sections to ultraviolet light at a wavelength of 365 nm commonly used fluorescence microscopy can result in color transformation of PB-positive material from blue to brown. Although the PB color transformation is primarily an artifact that may occur during fluorescence microscopy, the transformation can be manipulated using imaging process software for the detection of low levels of iron labeled cells in tissues samples.     

Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula

The disclosure of magnetic nanoparticles in five plant species growing in Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and halfwidth of the signal of 32 mT at room temperature to g = 2.71 and 50-55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170-220 mkR per h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.     

光学透明技术在植物多尺度成像中的应用

光学透明技术是一种通过各种化学试剂,将原本不透明的生物样本实现透明化,并在光学显微镜下深度成像的技术。结合多种光学显微成像新技术,光学透明技术可对整个组织进行成像和三维重建,深度剖析生物体内部空间特征与形成机制。近年来,多种植物光学透明技术和多尺度成像技术被陆续研发,并取得了丰硕的研究成果。该文综述了生物体光学透明技术的基本原理和一些新技术,重点介绍基于光学透明技术开发的新型成像方法及其在植物成像与细胞生物学中的应用,为后续植物整体、组织或器官的透明、成像与三维重构及功能研究提供理论依据和技术支持。