Calcium Imaging in Freely Behaving <em>Caenorhabditis elegans</em> with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

National Institute for Quantum and Radiological Science and Technology

JST, PRESTO

Physical forces are transduced into chemical reactions, thereby ultimately making a large impact on the whole-animal level phenotypes such as homeostasis, development and behavior. To understand mechano-chemical transduction, mechanical input should be quantitatively delivered with controllable vibration properties⁻frequency, amplitude and duration, and its chemical output should be noninvasively quantified in an unconstrained animal. However, such an experimental system has not been established so far. Here, we develop a noninvasive and unconstrained mechanochemical imaging microscopy. This microscopy enables us to evoke nano-scale nonlocalized vibrations with controllable vibration properties using a piezoelectric acoustic transducer system and quantify calcium response of a freely moving  at a single cell resolution. Using this microscopy, we clearly detected the calcium response of a single interneuron during  escape response to nano-scale vibration. Thus, this microscopy will facilitate understanding of in vivo mechanochemical physiology in the future.

Noninvasive Mechanochemical Imaging in Unconstrained .

Fluorescent nanodiamonds (FNDs) have been attracting much attention as promising therapeutic agents and probes for bioimaging and nanosensing. For their biological applications, several hydrophilizing methods to enhance FND colloidal stability have been developed to suppress their aggregation and the nonspecific adsorption to biomolecules in complex biomedical environments. However, these methods involve several complicated synthetic and purification steps, which prohibit the use of FNDs for bioapplications by biologists. In this study, we describe a simple one-pot FND hydrophilization method that comprises coating of the surface of the nanoparticles with COOH-terminated hyperbranched polyglycerol (HPG-COOH). HPG-COOH-coated FNDs (FND-HPG-COOHs) were found to exhibit excellent dispersibility under physiological conditions despite the thinness of the 5 nm HPG-COOH layer. Biotinylated FND-HPG-COOHs specifically captured avidin molecules in the absence of nonspecific protein adsorption. Moreover, we demonstrated that FND-HPG-COOHs conjugated with antibodies can be used to selectively target integrins in fixed HeLa cells. In addition, intracellular temperature changes were measured via optically detected magnetic resonance using FND-HPG-COOHs conjugated with mitochondrial localization signal peptides. Our one-pot synthetic method will encourage the broad use of FNDs among molecular and cellular biologists and pave the way for extensive biological and biomedical applications of FNDs.

One-Pot Synthesis of Highly Dispersible Fluorescent Nanodiamonds for Bioconjugation.

Nanodiamonds (NDs) provide a unique multitasking system for drug delivery and fluorescent imaging in biological environments. Owing to their quantum properties, NDs are expected to be employed as multifunctional probes in the future for the accurate visualization of biophysical parameters such as temperature and magnetic fields. However, the use of NDs for the selective targeting of the biomolecules of interest within a complicated biological system remains a challenge. One of the most promising solutions is the appropriate surface design of NDs based on organic chemistry and biochemistry. The engineered NDs have high biocompatibility and dispersibility in a biological environment and hence undergo cellular uptake through specific pathways.

Nanodiamonds for bioapplications-specific targeting strategies.

The rotation of an object cannot be fully tracked without understanding a set of three angles, namely, roll, pitch, and yaw. Tracking these angles as a three-degrees-of-freedom (3-DoF) rotation is a fundamental measurement, facilitating, for example, attitude control of a ship, image stabilization to reduce camera shake, and self-driving cars. Until now, however, there has been no method to track 3-DoF rotation to measure nanometer-scale dynamics in biomolecules and live cells. Here we show that 3-DoF rotation of biomolecules can be visualized via nitrogen-vacancy centers in a fluorescent nanodiamond using a tomographic vector magnetometry technique. We demonstrate application of the method to three different types of biological systems. First, we tracked the rotation of a single molecule of the motor protein F1-ATPase by attaching a nanodiamond to the γ-subunit. We visualized the 3-step rotation of the motor in 3D space and, moreover, a delay of ATP binding or ADP release step in the catalytic reaction. Second, we attached a nanodiamond to a membrane protein in live cells to report on cellular membrane dynamics, showing that 3D rotational motion of the membrane protein correlates with intracellular cytoskeletal density. Last, we used the method to track nonrandom motions in the intestine of . Collectively, our findings show that the method can record nanoscale 3-DoF rotation in vitro, in cells, and even in vivo. 3-DoF rotation tracking introduces a new perspective on microscopic biological samples, revealing in greater detail the functional mechanisms due to nanoscale dynamics in molecules and cells.

Tracking the 3D Rotational Dynamics in Nanoscopic Biological Systems.

Fluorescent nanodiamonds containing nitrogen-vacancy centers have attracted attention as nanoprobes for temperature measurements in microenvironments, potentially enabling the measurement of intracellular temperature distributions and temporal changes. However, to date, the time resolution and accuracy of the temperature determinations using fluorescent nanodiamonds have been insufficient for wide-field fluorescence imaging. Here, we describe a method for highly accurate wide-field temperature imaging using fluorescent nanodiamonds for optically detected magnetic resonance (ODMR) measurements. We performed a Monte Carlo simulation to determine the optimal frequency sweep range for ODMR temperature determination. We then applied this sweep range to fluorescent nanodiamonds. As a result, the temperature determination accuracies were improved by a factor ~1.5. Our result paves the way for the contribution of quantum sensors to cell biology for understanding, for example, differentiation in multicellular systems.

Ryuji Igarashi

Institute for Quantum Life Science,
National Institute for Radiological Sciences

National Institute for Quantum and Radiological Science and Technology

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Ryuji Igarashi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute for Quantum Life Science,National Institute for Radiological Sciences

National Institute for Quantum and Radiological Science and Technology

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Institute for Quantum Life Science,
National Institute for Radiological Sciences