Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens.

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump-probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.

Measuring the signs of the methyl 1H chemical shift differences between major and 'invisible' minor protein conformational states using methyl 1H multi-quantum spectroscopy.

Carr-Purcell-Meiboom-Gill (CPMG) type relaxation dispersion experiments are now routinely used to characterise protein conformational dynamics that occurs on the µs to millisecond (ms) timescale between a visible major state and 'invisible' minor states. The exchange rate(s) ([Formula: see text]), population(s) of the minor state(s) and the absolute value of the chemical shift difference [Formula: see text] (ppm) between different exchanging states can be extracted from the CPMG data. However the sign of [Formula: see text] that is required to reconstruct the spectrum of the 'invisible' minor state(s) cannot be obtained from CPMG data alone. Building upon the recently developed triple quantum (TQ) methyl [Formula: see text] CPMG experiment (Yuwen in Angew Chem 55:11490-11494, 2016) we have developed pulse sequences that use carbon detection to generate and evolve single quantum (SQ), double quantum (DQ) and TQ coherences from methyl protons in the indirect dimension to measure the chemical exchange-induced shifts of the SQ, DQ and TQ coherences from which the sign of [Formula: see text] is readily obtained for two state exchange. Further a combined analysis of the CPMG data and the difference in exchange induced shifts between the SQ and DQ resonances and between the SQ and TQ resonances improves the estimates of exchange parameters like the population of the minor state. We demonstrate the use of these experiments on two proteins undergoing exchange: (1) the ~ 18 kDa cavity mutant of T4 Lysozyme ([Formula: see text]) and (2) the [Formula: see text] kDa Peripheral Sub-unit Binding Domain (PSBD) from the acetyl transferase of Bacillus stearothermophilus ([Formula: see text]).

A methyl 1H double quantum CPMG experiment to study protein conformational exchange.

Protein conformational changes play crucial roles in enabling function. The Carr-Purcell-Meiboom-Gill (CPMG) experiment forms the basis for studying such dynamics when they involve the interconversion between highly populated and sparsely formed states, the latter having lifetimes ranging from ~ 0.5 to ~ 5 ms. Among the suite of experiments that have been developed are those that exploit methyl group probes by recording methyl 1H single quantum (Tugarinov and Kay in J Am Chem Soc 129:9514-9521, 2007) and triple quantum (Yuwen et al. in Angew Chem Int Ed Engl 55:11490-11494, 2016) relaxation dispersion profiles. Here we build upon these by developing a third experiment in which methyl 1H double quantum coherences evolve during a CPMG relaxation element. By fitting single, double, and triple quantum datasets, akin to recording the single quantum dataset at static magnetic fields of Bo, 2Bo and 3Bo, we show that accurate exchange values can be obtained even in cases where exchange rates exceed 10,000 s-1. The utility of the double quantum experiment is demonstrated with a pair of cavity mutants of T4 lysozyme (T4L) with ground and excited states interchanged and with exchange rates differing by fourfold (~ 900 s-1 and ~ 3600 s-1), as well as with a fast-folding domain where the unfolded state lifetime is ~ 80 µs.

Lipotype acquisition during neural development is not recapitulated in stem cell-derived neurons.

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MSALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell-derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell-derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

CPMG Experiments for Protein Minor Conformer Structure Determination.

CPMG relaxation dispersion NMR experiments have emerged as a powerful method to characterize protein minor states that are in exchange with a visible dominant conformation, and have lifetimes between ~0.5 and 5 milliseconds (ms) and populations greater than 0.5%. The structure of the minor state can, in favorable cases, be determined from the parameters provided by the CPMG relaxation dispersion experiments. Here, we go through the intricacies of setting up these powerful CPMG experiments.