Environmental contaminants are continuously being released into our ecosystems with limited knowledge of their health implications. The development of 2D 1H-13C in-vivo NMR spectroscopy has allowed for real-time monitoring of living organisms, such as adult Daphnia magna [1], providing a better understanding of toxic mechanisms at the biochemical level. However, studies have found that dormant eggs, which are essential for a population’s survival under stress, can respond very differently to contaminants at the biochemical level compared to adults, making research at this life stage crucial [2]. Current in-vivo methods use a 5 mm cryogenically cooled probe for NMR studies, however, due to the size of the eggs (~350 um in diameter) this would require hundreds of eggs per sample, making culturing of 13C enriched organisms difficult and costly. Alternatively, the increase of mass sensitivity from microcoils allows for single egg or single organism analysis, eliminating natural variability within groups. Unfortunately, this forfeits the sensitivity enhancements offered by cryogenically cooled probes and often restricts the analysis to a singerly tuned microcoil for maximum sensitivity. The low sensitivity of NMR is especially problematic with in-vivo samples due to their inhomogeneous nature, high water compositions, and extremely low metabolite concentrations. Here, paramagnetic lenses are used to miniaturize NMR while maintaining cryoprobe sensitivity. The broadband lenses enhance the signal to noise ratio (SNR) by conforming to the sample as closely as possible and refocusing the B1 field on a much smaller area (Fig. 1) [3].
The lenses act as a micro “amplifier” where the 1H 90 degree pulse occurs at a much shorter pulse width (~3.5 us) relative to the same sample without the lens (~9 us) (Fig 2). By reciprocity, the enhanced excitation field translates to increased detection sensitivity. Given the broadband nature, we can acquire data of multiple nuclei without dramatic detuning of the cryoprobe. To illustrate the performance of the lenses, Fig. 3 and 4 show the 1D 13C and 1H spectra of 540 nL of 13C enriched glucose with and without the lens. The lens show SNR improvements of ~3.5x and ~2x for 13C and 1H respectively (cryoprobe with inner 1H coil). The integration of paramagnetic lenses and cryoprobe technology creates a detection system that should be sensitive enough for environmental monitoring of single eggs and organisms.