The overall aim in this area is to develop and implement high resolution ESR microscopy for biological and medical applications. Instrumental development efforts are directed to enable the routine imaging of a variety of tissues, cells and other bio-samples for clinical evaluation, biophysical and biomedical research. Several classes of biological applications will be demonstrated with the new experimental tools.
NMR microscopy is routinely employed to observe magnetic parameters and transport phenomena in small scale structures such as biomaterials. Despite extensive efforts, the spatial resolution of this method is limited (>10µm for short acquisition times). In contrast to NMR, ESR microscopy is just being developed. Most efforts in ESR imaging have been directed towards low resolution imaging of large biological objects to identify the radical and the oxygen concentration. Recently, we have shown that ESR microscopy can improve upon the resolution limits of NMR, and successfully undertake the 1µm resolution challenge. We are now able to obtain 3D images with a resolution ranging from ~10×10×30 microns (9 GHz CW ESR) up to ~3×2×8 microns (16 GHz pulsed ESR), in a few minutes of acquisition. The detection sensitivity of the pulse system at room temperature enables the measurement of ~2×107spins in each voxel after ~15 minutes of acquisition. The resolution and detection sensitivity are the best obtained so far for ESR at ambient conditions of temperature and pressure, which can be applied to biological systems. These capabilities rely upon the development of an advanced miniature resonator, fed via microstrip line, optimized gradient coils capable of achieving gradient of more than 25 T/(m·A), and an advanced control and imaging software. The modular design of the CW imaging system enables one to achieve a micro-imaging capability in conjunction with any commercial CW ESR spectrometer, while the pulse system is entirely home made (spectrometer and imaging accessories).
ACERT Specific Aims are:
Continuous wave ESR microscope: We plan to develop a CW ESR microscopy system operating at 35 GHz, which is expected to provide a superior magnetic resonance image resolution of about 2-5 µm in 10-60 min of acquisition, and capable of imaging a range of solid or fluid biosamples. Imaging is achieved through the introduction of stable organic free radicals into these samples, similar to the contrast agents of NMR or dyes in optics.
Pulsed ESR microscopes: Pulse ESR systems are more complex than the CW ESR microscopes and cannot be used in conjunction with short relaxation time radicals. Nevertheless pulse ESRM provides better image quality, faster image acquisition, and more importantly, is able to provide several images of the same sample with different contrast parameters (i.e. spatially resolved relaxations times, diffusion coefficients, lineshape, or spin concentraion). We plan to develop a pulsed ESRM system operating at 35 GHz. This higher frequency system should enable the acauisition of 3D images of a variety of bio-samples with a resolution of ~2×2×5 µm in a few mintues of acquisition.
Sample preparation methods: We prefer to use flat (up to ~0.5 mm) ”optical microscopy-like” samples. This is, we believe, the most appropriate way to conduct most ESR microscopy experiments. We are developing appropriate sample preparation methods.
Test samples for micro-imaging:The development of CW and pulsed systems with high quality performances in terms of signal to noise and resolution, requires well-defined model samples. Such samples include solid radicals placed at certain points in the sample, samples with two types of radicals, liquid samples in various states of oxygenation, and simple live model samples. Special techniques such as photolithography, or micro machining, are required to produce these samples with high precision.
Advanced applications of ESR microscopy: Projects include studies of the slow release of drugs from microscopic biodegradable polymeric spheres by acquiring 3D images with spin labeled drugs; imaging the O2 concentration in live cells with sub-cellular spatial resolution through the use of free radicals with high linewidth sensitivity to O2; and imaging the free radicals internalized in cancerous tissues to verify their location at the cellular level.
These technologies will be available at ACERT to the scientific community.
Highlights of recent ESR microscopy & imaging developments.