Nuclear Magnetic Resonance (NMR) spectroscopy is a non-invasive analytical technique which allows for the study of intact samples. Comprehensive Multiphase NMR (CMP-NMR) combines techniques and hardware from solution state and solid state NMR to allow for the holistic analysis of all phases (i.e. solutions, gels and solids) in unaltered samples. This study is the first to apply CMP-NMR to deceased, intact organisms and uses 13 C enriched Daphnia magna (water fleas) as an example. D. magna are commonly used model organisms for environmental toxicology studies. As primary consumers, they are responsible for the transfer of nutrients across trophic levels, and a decline in their population can potentially impact the entire freshwater aquatic ecosystem. Though in vivo research is the ultimate tool to understand an organism’s most biologically relevant state, studies are limited by conditions (i.e. oxygen requirements, limited experiment time and reduced spinning speed) required to keep the organisms alive, which can negatively impact the quality of the data collected. In comparison, ex vivo CMP-NMR is beneficial in that; organisms do not need oxygen (eliminating air holes in rotor caps and subsequent evaporation); samples can be spun faster, leading to improved spectral resolution; more biomass per sample can be analyzed; and experiments can be run for longer. In turn, higher quality ex vivo NMR, can provide more comprehensive NMR assignments, which in many cases could be transferred to better understand less resolved in vivo signals. This manuscript is divided into three sections: 1) multiphase spectral editing techniques, 2) detailed metabolic assignments of 2D NMR of 13 C enriched D. magna and 3) multiphase biological changes over different life stages, ages and generations of D. magna . In summary, ex vivo CMP-NMR proves to be a very powerful approach to study whole organisms in a comprehensive manner and should provide very complementary information to in vivo based research.

Planar microcoils with diameter ranging from 20 to 1000 μm I.D. (130-1130 μm O.D.) are evaluated for their applications in NMR spectroscopy. The coils are first overfilled with a standard sucrose solution and compared against each other. Coils with smaller I.D. (≤100 μm) perform extremely well. One hypothesis is that as the coils get smaller the volume occupied by the copper turns increases relative to the open I.D.; as such a large proportion of the sample is brought in close proximity to the coil turns and likely gives rise to strong sample-coil magnetic coupling, which increases the signal. The applications of the planar microcoils are demonstrated on Cypselurus poecilopterus (fish) and Daphnia magna (water flea) eggs. A single D. magna egg on a 50 μm coil yielded at least 3000 times the mass sensitivity (∼9,000,000 time saving) when compared to a 5 mm probe. This value could be at least 4 times higher if the B homogeneity of the coils could be improved. With the current design, 80% of the signal is lost in multiple pulse experiments that rely on phase inversion and signal cancellation between scans. The data were extrapolated to predict that biological samples as small as ∼4 μm may become accessible via planar microcoil designs. To fulfill their potential for in situ metabolic screening, specialized magnetic susceptibility matched sample holders that restrict the sample to the homogeneous B field region (i.e. within the 90% RF field) of the coil and advanced experiments that narrow spectral lines, suppress lipids and disperse signals into multiple dimensions will be required.

Nuclear magnetic resonance microscopy at an isotropic resolution of 3.0 lm was realized by using dedicated hardware such as RF surface microcoils, a planar triple-axis gradient with 6,500 G/cm, and a static magnetic field of 18.8 T. Purely phaseencoded constant time imaging was used to allow increasing the gradient strength for the suppression of diffusion effects without reducing the signal-to-noise ratio. For this method the relationship between gradient strength and true spatial resolution was investigated, and an empirical formula is provided that is useful for practical applications. The characteristics of the different hardware components were investigated experimentally. Furthermore, microscopic phantom images were acquired and evaluated for their true resolution. It is demonstrated that the use of sufficiently large gradients enables suppressing diffusion-related loss of spatial resolution.

Magnetic resonance imaging (MRI) is now a leading diagnostic technique. As technology has improved, so has the spatial resolution achievable. In 1986 MR microscopy (MRM) was demonstrated with resolutions in the tens of microns, and is now an established subset of MRI with broad utility in biological and non-biological applications. To date, only large cells from plants or aquatic animals have been imaged with MRM limiting its applicability. Using newly developed microsurface coils and an improved slice preparation technique for correlative histology, we report here for the first time direct visualization of single neurons in the mammalian central nervous system (CNS) using native MR signal at a resolution of 4–8µm. Thus MRM has matured into a viable complementary cellular imaging technique in mammalian tissues.

We describe a two-plate digital microfluidic method for interfacing with nuclear magnetic resonance spectroscopy (DMF-NMR) for microscale chemical analysis.