Session Y39: Methods in Biological Physics

``Calibration-on-the-spot'': How to calibrate an EMCCD camera from its images

In localization-based microscopy, super-resolution is obtained by analyzing isolated diffraction-limited spots imaged, typically, with EMCCD cameras. To compare experiments and calculate localization precision, the photon-to-signal amplification factor is needed but unknown without a calibration of the camera. Here we show how this can be done \textit{post festum} from just a recorded image. We demonstrate this (i) theoretically, mathematically, (ii) by analyzing images recorded with an EMCCD camera, and (iii) by analyzing simulated EMCCD images for which we know the true values of parameters. In summary, our method of \textit{calibration-on-the-spot} allows calibration of a camera with unknown settings from old images on file, with no other info needed. Consequently, \textit{calibration-on-the-spot} also makes future camera calibrations before and after measurements unnecessary, because the calibration is encoded in recorded images during the measurement itself, and can at any later time be decoded with \textit{calibration-on-the-spot}.   

Live-cell thermometry with nitrogen vacancy centers in nanodiamonds

The ability to measure temperature is typically affected by a tradeoff between sensitivity and spatial resolution. Good thermometers tend to be bulky systems and hence are ill-suited for thermal sensing with high spatial localization. Conversely, the signal resulting from nanoscale temperature probes is often impacted by noise to a level where the measurement precision becomes poor. Adding to the microscopist toolbox, the nitrogen vacancy (NV) center in diamond has recently emerged as a promising platform for high-sensitivity nanoscale thermometry [1,2]. Of particular interest are applications in living cells because diamond nanocrystals are biocompatible and can be chemically functionalized to target specific organelles. Here we report progress on the ability to probe and compare temperature within and between living cells using nanodiamond-hosted NV thermometry. We focus our study on cancerous cells, where atypical metabolic pathways arguably lead to changes in the way a cell generates heat, and thus on its temperature profile. 1.~V. Acosta et al., \textit{Phys. Rev. Lett.~}\textbf{104},~070801~(2010). 2. G. Kucsko et al.,~\textit{Nature~}\textbf{500},~54 (2013).   

Production and NMR signal optimization of hyperpolarized 13C-labeled amino acids

Amino acids are targeted nutrients for consumption by cancers to sustain their rapid growth and proliferation. 13C-enriched amino acids are important metabolic tracers for cancer diagnostics using nuclear magnetic resonance (NMR) spectroscopy. Despite this diagnostic potential, 13C NMR of amino acids however is hampered by the inherently low NMR sensitivity of the 13C nuclei. In this work, we have employed a physics technique known as dynamic nuclear polarization (DNP) to enhance the NMR signals of 13C-enriched amino acids. DNP works by transferring the high polarization of electrons to the nuclear spins via microwave irradiation at low temperature and high magnetic field. Using a fast dissolution method in which the frozen polarized samples are dissolved rapidly with superheated water, injectable solutions of 13C-amino acids with highly enhanced NMR signals (by at least 5,000-fold) were produced at room temperature. Factors that affect the NMR signal enhancement levels such as the choice of free radical polarizing agents and sample preparation will be discussed along with the thermal mixing physics model of DNP. The authors would like to acknowledge the support by US Dept of Defense award no. W81XWH-14-1-0048 and Robert A. Welch Foundation grant no. AT-1877.   

Real-time tracking of dissociation of hyperpolarized $^{\mathrm{89}}$Y-DTPA: a model for degradation of open-chain Gd$^{\mathrm{3+}}$ MRI contrast agents

Gadolinium (Gd) complexes are widely used relaxation-based clinical contrast agents in magnetic resonance imaging (MRI). Gd-based MRI contrast agents with open-chain ligand such as Gd-DTPA, commercially known as magnevist, are less stable compared to Gd complexes with macrocyclic ligands such as GdDOTA (Dotarem). The dissociation of Gd-DPTA into Gd ion and DTPA ligand under certain biological conditions such as high zinc levels can potentially cause kidney damage. Since Gd is paramagnetic, direct NMR detection of the Gd-DTPA dissociation is quite challenging due to ultra-short relaxation times. In this work, we have investigated Y-DTPA as a model for Gd-DPTA dissociation under high zinc content solutions. Using dissolution dynamic nuclear polarization (DNP), the $^{\mathrm{89}}$Y NMR signal is amplified by several thousand-fold. Due to the the relatively long T$_{\mathrm{1}}$ relaxation time of $^{\mathrm{89}}$Y which translates to hyperpolarization lifetime of several minutes, the dissociation of Y-DTPA can be tracked in real-time by hyperpolarized $^{\mathrm{89}}$Y NMR spectroscopy. Dissociation kinetic rates and implications on the degradation of open-chain Gd$^{\mathrm{3+}}$ MRI contrast agents will be discussed.   

Optimization of $^{13}$C dynamic nuclear polarization: isotopic labeling of free radicals

Dynamic nuclear polarization (DNP) is a physics technique that amplifies the nuclear magnetic resonance (NMR) signals by transferring the high polarization of the electrons to the nuclear spins. Thus, the choice of free radical is crucial in DNP as it can directly affect the NMR signal enhancement levels, typically on the order of several thousand-fold in the liquid-state. In this study, we have investigated the efficiency of four variants of the well-known 4-oxo-TEMPO radical (normal 4-oxo-TEMPO plus its $^{15}$N-enriched and/or perdeuterated variants) for use in DNP of an important metabolic tracer [1-$^{\, 13}$C]acetate. Though the variants have significant differences in electron paramagnetic resonance (EPR) spectra, we have found that changing the composition of the TEMPO radical through deuteration or $^{15}$N doping yields no significant difference in $^{13}$C DNP efficiency at 3.35 T and 1.2 K. On the other hand, deuteration of the solvent causes a significant increase of $^{13}$C polarization that is consistent over all the 4-oxo-TEMPO variants. These findings are consistent with the thermal mixing model of DNP.   

Simulated biophysical experimental techniques for chlorhexidine in dmpc/cholesterol systems

We have investigated the use of molecular dynamic simulations and the MARTINI force field to simulate isothermal titration calorimetry and differential scanning calorimetry techniques. The goal of these simulations was to observe how well they can reproduce the concentration effects of the addition of the small molecule chlorhexidine (CHX) into a model DMPC membrane containing varying concentrations of cholesterol. We constructed a coarse grained model for CHX compatible with the MARTINI force field. We were able to mimic an isothermal titration calorimetry experiment by repeatedly adding CHX into a DMPC membrane. With the increased concentration, we observed a decreasing affinity between CHX and the membrane as well as a resulting increase in the reaction time before the system was equilibrated. We then performed a controlled cooling of the membrane with various CHX concentrations to mimic a differential scanning calorimetry experiment. A change in membrane structure accompanied by a spike in the specific heat was measured at specific temperature T$_m$ signaling a phase transition. We then varied the concentration of CHX as well as the addition of varying concentrations of cholesterol to observe trends in the change to T$_m$ due to the addition of CHX and cholesterol.   

Detection of carotenoids present in blood of various animal species using Raman spectroscopy

Raman spectroscopy is simple stable powerful diagnostic tool for body fluids, tissues and other biomolecules. Human blood possesses different kind of carotenoids that play a key role for protecting the cells from damaging by different viral and bacterial diseases. Carotenoids are antioxidative components which are capable to overcome the attack of different free radicals and reactive oxygen species. Carotenoids are not prepared by human body, therefore it is recommended to eat carotenoids enrich vegetable foods. No standard data is available on the concentration of useful carotenoids component in non-vegetable consumed items. In present research work, Raman spectroscopy is used to compare various blood components like plasma, serum, carotenoids present in blood of different animal species like goat, sheep, cow and buffalo consumed by human. Especially beta carotene is investigated. The Raman shift ranges from 600-1700 cm$^{\mathrm{-1}}$ for samples. Different characteristic peaks of the blood components are found which are not characterized before in animal samples.   

Direct simulation of amphiphilic nanoparticle mediated membrane interactions

Membrane fusion is a critical step in the transport of biological cargo through membrane-bound compartments like vesicles. Membrane proteins that alleviate energy barriers for initial stalk formation and eventual rupture of the hemifusion intermediate during fusion generally assist this process. Gold nanoparticles functionalized with a combination of hydrophobic and hydrophilic alkanethiol ligands have recently been shown to induce membrane re-arrangements that are similar to those associated with these fusion proteins. In this work, we utilize molecular dynamics simulation to systematically design nanoparticles that exhibit targeted interactions with membranes. We introduce a method for rapidly parameterizing nanoparticle topology for the MARTINI biomolecular force field to permit long timescale simulation of their interactions with lipid bilayers. We leverage this model to investigate how ligand chemistry governs the nanoparticle's insertion efficacy and the perturbations it generates in the membrane environment. We further demonstrate through unbiased simulations that these nanoparticles can direct the fusion of lipid assemblies such as micelles and vesicles in a manner that mimics the function of biological fusion peptides and SNARE proteins.   

Exposure to TiO$_{\mathrm{2}}$ nanoparticles increases Staphylococcus\textit{ aureus }infection of HeLa cells

TiO$_{\mathrm{2}}$ is one of the most common nanoparticles in industry from food additives to energy generation. Even though TiO$_{\mathrm{2}}$ is also used as an anti-bacterial agent in combination with UV, we found that, in the absence of UV, exposure of HeLa cells to TiO$_{\mathrm{2}}$ nanoparticles largely increased their risk of bacterial invasion. HeLa cells cultured with low dosage rutile and anatase TiO$_{\mathrm{2}}$ nanoparticles (0.1 mg/ml) for 24 hrs prior to exposure to bacteria had 350{\%} and 250{\%} respectively more bacteria infected per cell. The increase was attributed to increased LDH leakage, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO$_{\mathrm{2}}$ particles ingested 40{\%} fewer bacteria, further increasing the risk of infection. In combination, these two factors raise serious concerns regarding the impact of exposure to TiO$_{\mathrm{2}}$ nanoparticles on the ability of organisms to resist bacterial infection.   

Effect of TiO2 nanoparticles on adipose derived stromal cell differentiation, morphology, ECM deposition and its susceptibility to bacterial infections.

The growing annual production of Titanium dioxide (TiO$_{\mathrm{2}})$ nanoparticles is proportional to an increase in the chances of occupational and consumer exposure. Considering, that these nanoparticles are currently being used in multiple personal care products many concerns have arisen about their health impact. Human skin is in constant contact with the external environment and is one of the most important routes of exposure to TiO$_{\mathrm{2}}$. In this study we have investigated the effect of two forms of TiO$_{\mathrm{2}}$, rutile and anatase, on human adipose derived stromal cells (ADSCs). Here, we focus on the~effects of TiO$_{\mathrm{2}}$ exposure on intracellular lipid accumulation and expression of adipogenic markers; on whether different forms of TiO$_{\mathrm{2}}$ have similar effects on cell function; and whether nanoparticle localization inside cells correlates with loss of cell function. In addition presence of bacteria on the skin is taken into account in its complex interaction with ADSCs and TiO$_{\mathrm{2}}$ nanoparticles. Altogether, the present study indicates that nanosized TiO$_{\mathrm{2}}$~particles adversely effects the differentiation of ADSCs, have profound effects on cell function and increase the rate of bacterial infection.   

Effect of cell donor age on the cellular response to nanoparticle exposure.

As human age there are many significant changes that occur in the skin. Here we investigate how the age-dependent changes in dermal fibroblast mechanics affect cell response to the AuNPs nanoparticles. To analyze these processes we exposed cells from donors of different age groups to AuNPs of two different sizes. Our results indicate that there are significant changes in cell rigidity with age, which in turn lead to different penetration rates of AuNPs through cell membrane and overall nanoparticle toxicity. Cell proliferation results revealed that all cell groups exposed to the same concentration of AuNPs had a very similar decrease in cell proliferation and similar impact on cell morphology. However, recovery data demonstrated that the rate of recovery from the damage is much faster for neonatal cells as compared to 30- and 80-years old cell group. Therefore, we conclude that nanoparticle uptake depends on cell membrane mechanics that in turn is a function of cell donor age.   

Mechanics of Cellulose Synthase Complexes in Living Plant Cells

The polymer cellulose is one of the major components of the world's biomass with unique and fascinating characteristics such as its high tensile strength, renewability, biodegradability, and biocompatibility. Because of these distinctive aspects, cellulose has been the subject of enormous scientific and industrial interest, yet there are still fundamental open questions about cellulose biosynthesis. Cellulose is synthesized by a complex of transmembrane proteins called ``Cellulose Synthase A'' (CESA) in the plasma membrane. Studying the dynamics and kinematics of the CESA complex will help reveal the mechanism of cellulose synthesis and permit the development and validation of models of CESA motility. To understand what drives these complexes through the cell membrane, we used total internal reflection fluorescence microscopy (TIRFM) and variable angle epi-fluorescence microscopy to track individual, fluorescently-labeled CESA complexes as they move in the hypocotyl and root of living plants. A mean square displacement analysis will be applied to distinguish ballistic, diffusional, and other forms of motion. We report on the results of these tracking experiments.   

Electronic measurements in an alternating magnetic field (AMF) for studying magnetic nanoparticle hyperthermia

Magnetic nanoparticle hyperthermia is a promising cancer treatment in which magnetic nanoparticles are injected into a tumor and then exposed to an alternating magnetic field (AMF). This process releases heat and damages tumor cells, but the exact mechanisms behind the effectiveness of this therapy are still unclear. Accurate sensors are required to monitor the temperature and, potentially, other parameters such as magnetic field or mechanical stress during clinical therapy or lab research. Often, optical rather than electronic temperature sensors are used to avoid eddy current self-heating in conducting parts in the AMF. However, eddy current heating is strongly dependent on the size and geometry of the conducting part, thus micro- and nano-scale electronics are a promising possibility for further exploration into magnetic nanoparticle hyperthermia. This presentation quantitatively discusses the eddy current self-heating of thin wires (thermocouples) and will also present a proof of concept thin film resistive thermometer and magnetic field sensor along with measurements of their eddy current self-heating. The results show that electronic measurements are feasible in an AMF with both thin wires and patterned thin film sensors under certain conditions.   

Non-invasive measurement of the blood pressure pulse using multiple PPGs

Heart disease, the leading cause of death in the US, may be spotted early on by looking at photoplethysmogram (PPG) data. This experiment explores a new method of continuously monitoring the blood pressure pulse with PPG data. In contrast to the traditional sphygmomanometer (cuff) method, which yields only the systolic and diastolic pressure during measurement, this method tracks the blood pressure pulse wave in a non-invasive continuous manner. This procedure allows for fast, inexpensive, and detailed analysis of the patient's blood pressure implementable on a large scale. We also explore the second derivative of the PPG data. In combination with the above method, the patient's heart risk can be effectively detected.