Young Jong Lee

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.

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.

Quantitative image analysis of broadband CARS hyperspectral images of polymer blends.

We demonstrate that broadband coherent anti-Stokes Raman scattering (CARS) microscopy can be very useful for fast acquisition of quantitative chemical images of multilayer polymer blends. This is challenging because the raw CARS signal results from the coherent interference of resonant Raman and nonresonant background and its intensity is not linearly proportional to the concentration of molecules of interest. Here we have developed a sequence of data-processing steps to retrieve background-free and noise-reduced Raman spectra over the whole frequency range including both the fingerprint and C-H regions. Using a classical least-squares approach, we are able to decompose a Raman hyperspectral image of a tertiary polymer blend into quantitative chemical images of individual components. We use this method to acquire 3-D sectioned quantitative chemical images of a multilayer polymer blend of polystyrene, styrene-ethylene/propylene copolymer, and polypropylene that have overlapping spectral peaks.

Synergistic roles for lipids and proteins in the permanent adhesive of barnacle larvae

Thoracian barnacles rely heavily upon their ability to adhere to surfaces and are environmentally and economically important as biofouling pests. Their adhesives have unique attributes that define them as targets for bio-inspired adhesive development. With the aid of multi-photon and broadband coherent anti-Stokes Raman scattering microscopies, we report that the larval adhesive of barnacle cyprids is a bi-phasic system containing lipids and phosphoproteins, working synergistically to maximize adhesion to diverse surfaces under hostile conditions. Lipids, secreted first, possibly displace water from the surface interface creating a conducive environment for introduction of phosphoproteins while simultaneously modulating the spreading of the protein phase and protecting the nascent adhesive plaque from bacterial biodegradation. The two distinct phases are contained within two different granules in the cyprid cement glands, implying far greater complexity than previously recognized. Knowledge of the lipidic contribution will hopefully inspire development of novel synthetic bioadhesives and environmentally benign antifouling coatings.

Multicomponent Chemical Imaging of Pharmaceutical Solid Dosage Forms with Broadband CARS Microscopy

We compare a coherent Raman imaging modality, broadband coherent anti-Stokes Raman scattering (BCARS) microscopy, with spontaneous Raman microscopy for quantitative and qualitative assessment of multicomponent pharmaceuticals. Indomethacin was used as a model active pharmaceutical ingredient (API) and was analyzed in a tabulated solid dosage form, embedded within commonly used excipients. In comparison with wide-field spontaneous Raman chemical imaging, BCARS acquired images 10× faster, at higher spatiochemical resolution and with spectra of much higher SNR, eliminating the need for multivariate methods to identify chemical components. The significant increase in spatiochemical resolution allowed identification of an unanticipated API phase that was missed by the spontaneous wide-field method and bulk Raman spectroscopy. We confirmed the presence of the unanticipated API phase using confocal spontaneous Raman, which provided spatiochemical resolution similar to BCARS but at 100× slower acquisition times.

Vibrational dephasing time imaging by time-resolved broadband coherent anti-Stokes Raman scattering microscopy

Time delay control in broadband coherent anti-Stokes Raman scattering (CARS) allows acquisition of time-resolved CARS images free of nonresonant background (NRB). We demonstrate that, in some cases, CARS image contrast is not chemical contrast but simply due to differences in NRB. Time-resolved CARS is used to rectify this by eliminating the NRB. We also construct a vibrational dephasing time image from a sequence of time-resolved CARS images of polystyrene beads in toluene. In doing so, we demonstrate the potential of imaging local molecular interactions between molecules and their surrounding in a structured medium.

Optimized continuum from a photonic crystal fiber for broadband time-resolved coherent anti-Stokes Raman scattering.

We demonstrate an optimization of continuum generation in a commercially available photonic crystal fiber and show that this continuum can be used to simultaneously measure vibrational dephasing times over an unprecedented frequency range of Raman modes. The dephasing time measurement is based on 2-pulse 3-color coherent anti-Stokes Raman scattering (CARS), and requires a continuum pulse that is coherent over a broad spectral bandwidth. We demonstrate that a continuum with the required characteristics can be generated from a photonic crystal fiber by appropriately conditioning the chirp of the excitation pulse and controlling its pulse energy. We are able to simultaneously measure vibrational dephasing times of multiple Raman modes (covering 500 cm(-1) to 3100 cm(-1)) of acetonitrile and benzonitrile using the optimized continuum with broadband time-resolved CARS.