Final Report Summary - NMCEL (A modular micro nuclear magnetic resonance in vivo platform for the nematode Caenorhabditis elegans)
The nematode C. elegans possesses several attributes that make this organism attractive as a model system. Biologically, its genome has been completely sequenced, it consists of a small number of cells, its nervous system has been mapped, its lifecycle is relatively short, and it is highly prolific. From a practical perspective, the nematode is non-toxic, non-pathogenic, and easy to handle requiring minimal specialized laboratory equipment. While certainly not immediately obvious, C. elegans is an important organism in the study of human disease, with an estimated 40% of its protein-coding genes possessing a human homolog. Importantly, it has been estimated that 85-90% of the ca. 20100 genes do not produce a visible phenotype when deactivated, and therefore a window to the metabolic responses that result from such deactivation is highly desirable.
Nuclear magnetic resonance (NMR) is a technique capable of providing insight at the molecular level in a non-invasive manner. Capable of determining the chemical environment at a nucleus, NMR can probe multiple molecular properties (e.g. identification, localization within the organism or cell, participation in metabolic pathways, metabolic flux, diffusion, etc.). A current limitation of standard NMR techniques is the throughput, where significant operator input is required on a per sample basis. However, this limitation is alleviated by using micro-fabrication techniques which hold potential to yield intelligent probe and fluidic systems in combination with other detection modes, highly parallel signal acquisition from multiple measurement cells, which together could deliver unprecedented structural and quantitative information from complex samples. A key feature is the miniaturization of the NMR detector, with micro-coil technology coupled with microfluidics promising to propel NMR into the high throughput arena.
Within this context, the NMCEL team aimed to integrate organism culturing, transport to and from the NMR detection region, and high resolution spectroscopy and imaging measurements into an automated, modular system. Such a system would permit high throughput measurements on a per-organism basis, yielding molecular information at the level of the individual that is otherwise averaged over a population in standard NMR techniques. In the development of this system, C. elegans was chosen as the model organism, whose dimensions (1 mm length, 0.1 mm diameter) are highly compatible with the team’s micro-NMR detector and fluidic technology. This choice does not preclude the application of this technology to other organisms; it is anticipated that the modular system will be transferable to a multitude of model organisms, organism embryos, and cell clusters, providing a highly important tool to the molecular and systems biologists.
Towards this goal, significant progress was made in NMR detector design and fabrication, C. elegans position sensing, understanding electronic function in the presence of high magnetic fields, and NMR signal enhancement strategies. Through our computational optimization and fabrication experience, we developed a new generation of NMR micro-coils capable of high resolution NMR spectroscopy (most designs approaching and one design achieving less than 1 Hz line width) with high sensitivity (comparable to micro-coil sensor data found in the literature). This new generation includes microfluidic interconnections as proof-of-concept that the design is functional under MR conditions. Imaging applications require magnetic field gradients, and our second generation micro-gradient coils were shown to produce up to 50 times the field gradient compared to their commercial counterparts. In order to track the position of the nematode while being transported, a CMOS based camera was validated to operate while immersed in a high magnetic field. Additionally, a new conductive photoresist material was used pattern sub-micron sensing structures capable of distinguishing not only the presence of a single C. elegans, but also its lifestage. Two routes to NMR signal enhancement on sub-microliter sample volumes were developed, including para-hydrogen induced polarization and dynamic nuclear polarization.
Nuclear magnetic resonance (NMR) is a technique capable of providing insight at the molecular level in a non-invasive manner. Capable of determining the chemical environment at a nucleus, NMR can probe multiple molecular properties (e.g. identification, localization within the organism or cell, participation in metabolic pathways, metabolic flux, diffusion, etc.). A current limitation of standard NMR techniques is the throughput, where significant operator input is required on a per sample basis. However, this limitation is alleviated by using micro-fabrication techniques which hold potential to yield intelligent probe and fluidic systems in combination with other detection modes, highly parallel signal acquisition from multiple measurement cells, which together could deliver unprecedented structural and quantitative information from complex samples. A key feature is the miniaturization of the NMR detector, with micro-coil technology coupled with microfluidics promising to propel NMR into the high throughput arena.
Within this context, the NMCEL team aimed to integrate organism culturing, transport to and from the NMR detection region, and high resolution spectroscopy and imaging measurements into an automated, modular system. Such a system would permit high throughput measurements on a per-organism basis, yielding molecular information at the level of the individual that is otherwise averaged over a population in standard NMR techniques. In the development of this system, C. elegans was chosen as the model organism, whose dimensions (1 mm length, 0.1 mm diameter) are highly compatible with the team’s micro-NMR detector and fluidic technology. This choice does not preclude the application of this technology to other organisms; it is anticipated that the modular system will be transferable to a multitude of model organisms, organism embryos, and cell clusters, providing a highly important tool to the molecular and systems biologists.
Towards this goal, significant progress was made in NMR detector design and fabrication, C. elegans position sensing, understanding electronic function in the presence of high magnetic fields, and NMR signal enhancement strategies. Through our computational optimization and fabrication experience, we developed a new generation of NMR micro-coils capable of high resolution NMR spectroscopy (most designs approaching and one design achieving less than 1 Hz line width) with high sensitivity (comparable to micro-coil sensor data found in the literature). This new generation includes microfluidic interconnections as proof-of-concept that the design is functional under MR conditions. Imaging applications require magnetic field gradients, and our second generation micro-gradient coils were shown to produce up to 50 times the field gradient compared to their commercial counterparts. In order to track the position of the nematode while being transported, a CMOS based camera was validated to operate while immersed in a high magnetic field. Additionally, a new conductive photoresist material was used pattern sub-micron sensing structures capable of distinguishing not only the presence of a single C. elegans, but also its lifestage. Two routes to NMR signal enhancement on sub-microliter sample volumes were developed, including para-hydrogen induced polarization and dynamic nuclear polarization.