Marcus T. Cicerone

Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy is emerging as a powerful method for imaging materials and biological systems, partly because of its noninvasiveness and selective chemical sensitivity. However, its full potential for species-selective imaging is limited by a restricted spectral bandwidth. Recent increases in bandwidth are promising but still are not sufficient for the level of robust component discrimination that would be needed in a chemically complex milieu found, for example, in intracellular and extracellular environments. We demonstrate a truly broadband CARS imaging instrument that we use to acquire hyperspectral images with vibrational spectra over a bandwidth of 2500 cm(-1) with a resolution of 13 cm(-1).

The Effect of 3D Hydrogel Scaffold Modulus on Osteoblast Differentiation and Mineralization Revealed by Combinatorial Screening

Cells are known to sense and respond to the physical properties of their environment and those of tissue scaffolds. Optimizing these cell-material interactions is critical in tissue engineering. In this work, a simple and inexpensive combinatorial platform was developed to rapidly screen three-dimensional (3D) tissue scaffolds and was applied to screen the effect of scaffold properties for tissue engineering of bone. Differentiation of osteoblasts was examined in poly(ethylene glycol) hydrogel gradients spanning a 30-fold range in compressive modulus ( approximately 10 kPa to approximately 300 kPa). Results demonstrate that material properties (gel stiffness) of scaffolds can be leveraged to induce cell differentiation in 3D culture as an alternative to biochemical cues such as soluble supplements, immobilized biomolecules and vectors, which are often expensive, labile and potentially carcinogenic. Gel moduli of approximately 225 kPa and higher enhanced osteogenesis. Furthermore, it is proposed that material-induced cell differentiation can be modulated to engineer seamless tissue interfaces between mineralized bone tissue and softer tissues such as ligaments and tendons. This work presents a combinatorial method to screen biological response to 3D hydrogel scaffolds that more closely mimics the 3D environment experienced by cells in vivo.

High-speed coherent Raman fingerprint imaging of biological tissues

An imaging platform based on broadband coherent anti-Stokes Raman scattering (BCARS) has been developed which provides an advantageous combination of speed, sensitivity and spectral breadth. The system utilizes a configuration of laser sources that probes the entire biologically-relevant Raman window (500 cm−1 to 3500 cm−1) with high resolution (< 10 cm−1). It strongly and efficiently stimulates Raman transitions within the typically weak “fingerprint” region using intrapulse 3-colour excitation, and utilizes the nonresonant background (NRB) to heterodyne amplify weak Raman signals. We demonstrate high-speed chemical imaging in two- and three-dimensional views of healthy murine liver and pancreas tissues and interfaces between xenograft brain tumours and the surrounding healthy brain matter.

LTB4 Is a Signal-Relay Molecule during Neutrophil Chemotaxis

Neutrophil recruitment to inflammation sites purportedly depends on sequential waves of chemoattractants. Current models propose that leukotriene B(4) (LTB(4)), a secondary chemoattractant secreted by neutrophils in response to primary chemoattractants such as formyl peptides, is important in initiating the inflammation process. In this study we demonstrate that LTB(4) plays a central role in neutrophil activation and migration to formyl peptides. We show that LTB(4) production dramatically amplifies formyl peptide-mediated neutrophil polarization and chemotaxis by regulating specific signaling pathways acting upstream of actin polymerization and MyoII phosphorylation. Importantly, by analyzing the migration of neutrophils isolated from wild-type mice and mice lacking the formyl peptide receptor 1, we demonstrate that LTB(4) acts as a signal to relay information from cell to cell over long distances. Together, our findings imply that LTB(4) is a signal-relay molecule that exquisitely regulates neutrophil chemotaxis to formyl peptides, which are produced at the core of inflammation sites.

Broadband CARS spectral phase retrieval using a time-domain Kramers–Kronig transform

We describe a closed-form approach for performing a Kramers-Kronig (KK) transform that can be used to rapidly and reliably retrieve the phase, and thus the resonant imaginary component, from a broadband coherent anti-Stokes Raman scattering (CARS) spectrum with a nonflat background. In this approach we transform the frequency-domain data to the time domain, perform an operation that ensures a causality criterion is met, then transform back to the frequency domain. The fact that this method handles causality in the time domain allows us to conveniently account for spectrally varying nonresonant background from CARS as a response function with a finite rise time. A phase error accompanies KK transform of data with finite frequency range. In examples shown here, that phase error leads to small (<1%) errors in the retrieved resonant spectra.

Protein and solvent dynamics: How strongly are they coupled?

Analysis of Raman and neutron scattering spectra of lysozyme demonstrates that the protein dynamics follow the dynamics of the solvents glycerol and trehalose over the entire temperature range measured 100-350 K. The proteins fast conformational fluctuations and low-frequency vibrations and their temperature variations are very sensitive to behavior of the solvents. Our results give insight into previous counterintuitive observations that protein relaxation is stronger in solid trehalose than in liquid glycerol. They also provide insight into the effectiveness of glycerol as a biological cryopreservant.

Label-Free Cellular Imaging by Broadband Coherent Anti-Stokes Raman Scattering Microscopy

Raman microspectroscopy can provide the chemical contrast needed to characterize the complex intracellular environment and macromolecular organization in cells without exogenous labels. It has shown a remarkable ability to detect chemical changes underlying cell differentiation and pathology-related chemical changes in tissues but has not been widely adopted for imaging, largely due to low signal levels. Broadband coherent anti-Stokes Raman scattering (B-CARS) offers the same inherent chemical contrast as spontaneous Raman but with increased acquisition rates. To date, however, only spectrally resolved signals from the strong CH-related vibrations have been used for CARS imaging. Here, we obtain Raman spectral images of single cells with a spectral range of 600-3200 cm⁻¹, including signatures from weakly scattering modes as well as CH vibrations. We also show that B-CARS imaging can be used to measure spectral signatures of individual cells at least fivefold faster than spontaneous Raman microspectroscopy and can be used to generate maps of biochemical species in cells. This improved spectral range and signal intensity opens the door for more widespread use of vibrational spectroscopic imaging in biology and clinical diagnostics.

Solid state chemistry of proteins: II. The correlation of storage stability of freeze‐dried human growth hormone (hGH) with structure and dynamics in the glassy solid

This research presents storage stability of human growth hormone, hGH, in lyophilized di-saccharide formulations. Stability via HPLC assay was assessed at 40 and 50 degrees C. Structure of the protein in the solids was assessed by infrared spectroscopy. Molecular mobility was characterized by structural relaxation times estimated from DSC data and by measurement of atomic motion on a nanosecond time scale by neutron scattering. Very large stability differences were observed among the various formulations, with both chemical and aggregation stability showing the same qualitative trends with formulation. Near the T(g), T(g) appeared to be a relevant stability parameter, but for storage well below T(g), stability seems unrelated to T(g). Stability (chemical and aggregation) was weakly correlated with secondary structure of the protein, and there was a partial quantitative correlation between degradation rate and the structural relaxation time. However, at equivalent levels of disaccharide relative to protein, sucrose systems were about a factor of two more stable than trehalose formulations, but yet had greater mobility as measured by structural relaxation time. Secondary structure was equivalent in both formulations. Neutron scattering results documented greater suppression of fast dynamics by sucrose than by trehalose, suggesting that well below T(g), fast dynamics are important to stability.

β-Relaxation governs protein stability in sugar-glass matrices

The stabilizing effect of sugar-glass matrix materials for freeze-drying proteins or nucleic acids has been variously ascribed to the thermodynamic effect of ‘water replacement’ by sugar molecules or to the kinetic effect of slowed α relaxation associated with sugar matrix vitrification. While evidence for each of these hypotheses exists, we show that neither can adequately account for the observed stabilization of proteins embedded in sugar-glasses. Instead, we find firm evidence that protein stability in these glasses is directly linked to high frequency β relaxation processes of the sugar matrix. Specifically, we observe that when the β relaxation time, τβ, of sugar-glasses is increased with antiplasticizing additives, protein stability increases in linear proportion to the increase in τβ, even though these same additives simultaneously decrease the glass transition temperature, Tg, and the α relaxation time, τα, of the sugar matrix materials. Moreover, we find that while sugars ‘replace’ water by stabilizing protein native-like conformation in the dry state, the resulting enhanced protein conformational stability does not have a significant impact on the degradation rate of the proteins in sugar-glasses. We discuss implications of these findings for the fundamental physics of glass formation and for effective engineering of protein stabilizing glasses through the modification of τβ.

Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility*

The purpose of this study is to investigate the impact of sucrose level on storage stability of dried proteins and thus better understand the mechanism of protein stabilization by disaccharides in lyophilized protein products. Five proteins were freeze dried with different amounts of sucrose, and protein aggregation was quantified using Size Exclusion Chromatography. Protein secondary structure was monitored by FTIR. The global mobility was studied using Thermal Activity Monitor (TAM), and fast local dynamics with a timescale of nanoseconds was characterized by neutron backscattering. The density of the protein formulations was measured with a gas pycnometer. The physical stability of the proteins increased monotonically with an increasing content of sucrose over the entire range of compositions studied. Both FTIR structure and structural relaxation time from TAM achieved maxima at about 1:1 mass ratio for most proteins studied. Therefore, protein stabilization by sugar cannot be completely explained by global dynamics and FTIR structure throughout the whole range of compositions. On the other hand, both the fast local mobility and free volume obtained from density decreased monotonically with an increased level of sucrose in the formulations, and thus the local dynamics and free volume correlate well with protein storage stability.