High-sensitivity Q-band electron spin resonance imaging system with submicron resolution.

A pulsed electron spin resonance (ESR) microimaging system operating at the Q-band frequency range is presented. The system includes a pulsed ESR spectrometer, gradient drivers, and a unique high-sensitivity imaging probe. The pulsed gradient drivers are capable of producing peak currents ranging from ∼9 A for short 150 ns pulses up to more than 94 A for long 1400 ns gradient pulses. Under optimal conditions, the imaging probe provides spin sensitivity of ∼1.6 × 10(8) spins∕√Hz or ∼2.7 × 10(6) spins for 1 h of acquisition. This combination of high gradients and high spin sensitivity enables the acquisition of ESR images with a resolution down to ∼440 nm for a high spin concentration solid sample (∼10(8) spins∕µm(3)) and ∼6.7 µm for a low spin concentration liquid sample (∼6 × 10(5) spins/µm(3)). Potential applications of this system range from the imaging of point defects in crystals and semiconductors to measurements of oxygen concentration in biological samples.

Electron spin resonance micro-imaging of live species for oxygen mapping.

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution(1). Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature(2). The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 µm is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

Microimaging of oxygen concentration near live photosynthetic cells by electron spin resonance.

We present what is, to our knowledge, a new methodology for high-resolution three-dimensional imaging of oxygen concentration near live cells. The cells are placed in the buffer solution of a stable paramagnetic probe, and electron spin-resonance microimaging is employed to map out the probe's spin-spin relaxation time (T(2)). This information is directly linked to the concentration of the oxygen molecule. The method is demonstrated with a test sample and with a small amount of live photosynthetic cells (cyanobacteria), under conditions of darkness and light. Spatial resolution of approximately 30 x 30 x 100 microm is demonstrated, with approximately microM oxygen concentration sensitivity and sub-fmol absolute oxygen sensitivity per voxel. The use of electron spin-resonance microimaging for oxygen mapping near cells complements the currently available techniques based on microelectrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen microimaging, a capability which other methods find very difficult to achieve.

ESR micro-imaging of LiNc-BuO crystals in PDMS: spatial and spectral grain distribution.

Microcrystals of lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) in a bio-compatible and oxygen-permeable polymer matrix of poly-dimethyl-siloxane (PDMS) can be used for repetitive non-invasive imaging of oxygen in live specimens by means of mm-scale electron spin resonance (ESR) imaging. This probe denoted as "oxychip" was characterized by high-resolution mum-scale ESR microcopy to reveal the fine details of its spatial and spectral properties. The ESR micro-images of a typical oxychip device showed that while the spatial distribution of the microcrystals in the polymer is fairly homogenous (as revealed by optical microscopy), the ESR signal originates only from a very few dominant crystals. Furthermore, spectral-spatial analysis in a microcrystal and a sub-microcrystal spatial resolution reveals that each crystal has a slightly different g-factor and also exhibits variations in linewidth, possibly due to the slightly different individual crystallization process.

ESR imaging in solid phase down to sub-micron resolution: methodology and applications.

Electron spin resonance microcopy (ESRM) is an imaging method aimed at the observation of paramagnetic species in small samples with micron-scale spatial resolution. At present, this technique is pursued mainly for biological applications at room temperature and in relatively low static magnetic fields. This work is focused on the use of ESRM for the measurement of solid samples. First, a brief comparison of various electron spin resonance (ESR) detection techniques is provided, with an emphasis on conventional "induction detection". Following that, some methodological details are provided along with experimental examples carried out at room temperature and in a static field of approximately 0.5 T. These examples show for the first time the imaging of solid samples measured by "induction detection" ESR with a resolution better than 1 mum. Based on these experimental examples and capabilities, an outlook for the future prospects of this methodology in terms of spin sensitivity and resolution is provided. It is estimated that single-spin sensitivity could be achieved for some samples at liquid-helium temperatures and static fields of approximately 2 T. Furthermore, under these conditions, spatial resolution could reach the nanometer scale. Finally, a description of possible applications of this new methodology is provided.

Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay.

Membrane binding and relative penetration of indolicidin analogs were studied using lipid/polydiacetylene (PDA) chromatic biomimetic membranes. Colorimetric and fluorescence analyses determined that an indolicidin analog with a proline and tryptophan residue substituted with lysines showed more pronounced bilayer surface interactions, while indolicidin and particularly an indolicidin analog in which all prolines were replaced with alanine residues exhibited deeper insertion into the lipid bilayer. The colorimetric data demonstrated that more pronounced blue-red transitions were observed when the chromatic vesicles incorporated lipopolysaccharide (LPS) within the lipid bilayer, indicating that LPS promoted preferred binding and incorporation of the peptides at the lipid/water interface. The fluorescence quenching experiments further confirmed this outcome. The results indicate that the antibacterial activity of indolicidin most likely requires initial binding to the LPS moieties within bacterial membranes, as well as disruption of the bilayer interface. The degree of hemolysis induced by the analogs, on the other hand, correlated to the extent of penetration into the hydrophobic core of the lipid assembly.