Hyuk-Sang Kwon

Three-Dimensional Cellular Deformation Analysis with a Two-Photon Magnetic Manipulator Workstation

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.

Resolution enhancement in standing-wave total internal reflection microscopy: a point-spread-function engineering approach

The theoretical basis for resolution enhancement in standing-wave total internal reflection microscopy (SW-TIRM) is examined. This technique relies on the formation of an excitation field containing super-diffraction-limited spatial-frequency components. Although the fluorescence generated at the object planes contains high-frequency information of the object distribution, this information is lost at the image plane, where the detection optics acts as a low-pass filter. From the perspective of point-spread-function (PSF) engineering, one can show that if this excitation field is translatable experimentally, the high-frequency information can be extracted from a set of images where the excitation fields have different displacement vectors. We have developed algorithms to combine this image set to generate a composite image with an effective PSF that is equal to the product of the excitation field and the Fraunhofer PSF. This approach can easily be extended to incorporate nonlinear excitation modalities into SW-TIRM for further resolution improvement. We theoretically examine high-resolution imaging based on the addition of two-photon, pump-probe, and stimulated-emission depletion methods to SW-TIRM and show that resolution better than 1/20 of the emission wavelength may be achievable.

Resolving myoarchitectural disarray in the mouse ventricular wall with diffusion spectrum magnetic resonance imaging.

The myoarchitecture of the ventricular wall provides a structural template dictating tissue-scale patterns of mechanical function. We studied whether myofiber tract imaging performed with MR diffusion spectrum imaging (DSI) tractography has the capacity to resolve abnormalities of ventricular myoarchitecture in a model of congenital hypertrophic cardiomyopathy (HCM) associated with the ablation of myosin binding protein-C (MyBP-C). Homozygous MyBP-C knockout mice were generated by deletion of exons 3–10 from the endogenous MyBP-C gene. Fiber alignment in the left ventricular wall of wild type mice was depicted through DSI tractography (and confirmed by multi-slice two-photon microscopy) as a set of helical structures whose angles display a continuous transition from negative in the subepicardium to positive in the subendocardium. In contrast, the hearts obtained from the MyBP-C knockouts displayed substantial myoarchitectural disarray, characterized by a loss of voxel-to-voxel orientational coherence for fibers principally located in the mid-myocardium-subendocardium and impairment of the transmural progression of helix angles. These results substantiate the use of DSI tractography in determining myoarchitectural disarray in models of cardiomyopathy and suggest a biological association between myofilament expression, cardiac fiber alignment, and torsional rotation in the setting of congenital HCM.

Experimental Investigation of Pulsatility Effect on the Deformability and Hemolysis of Blood Cells

In this study, we investigated the differences between pulsatile cardiopulmonary bypass (CPB) procedure and nonpulsatile CPB procedure in terms of their effects on hemolysis and deformability of red blood cells (RBCs) under various shear stress conditions. In order to research the effects on hemolysis and deformability, four parameters--free hemoglobin (fHb) concentration, normalized index of hemolysis (NIH), deformability index (DI) of RBCs, and elongation index of RBCs--have been deeply investigated. For these investigations, two randomly assigned adult mongrel dog groups-nonpulsatile group (NP, n = 6) and pulsatile group (P, n = 6)--were examined. According to our results, both types of perfusion did not show any statistical differences in terms of the concentrations of fHb as well as NIH. In addition, there were no significant differences in RBC deformability between perfusion types within an operation time of 3 h. Therefore, our studies suggest that pulsatile perfusion has no significant difference from nonpulsatile perfusion in terms of hemolysis and deformability of RBCs.

Integrated one- and two-photon imaging platform reveals clonal expansion as a major driver of mutation load

The clonal expansion of mutant cells is hypothesized to be an important first step in cancer formation. To understand the earliest stages of tumorigenesis, a method to identify and analyze clonal expansion is needed. We have previously described transgenic Fluorescent Yellow Direct Repeat (FYDR) mice in which cells that have undergone sequence rearrangements (via homologous recombination events) express a fluorescent protein, enabling fluorescent labeling of phenotypically normal cells. Here, we develop an integrated one- and two-photon imaging platform that spans four orders of magnitude to permit rapid quantification of clonal expansion in the FYDR pancreas in situ. Results show that as mice age there is a significant increase in the number of cells within fluorescent cell clusters, indicating that pancreatic cells can clonally expand with age. Importantly, >90% of fluorescent cells in aged mice result from clonal expansion, rather than de novo sequence rearrangements at the FYDR locus. The spontaneous frequency of sequence rearrangements at the FYDR locus is on par with that of other classes of mutational events. Therefore, we conclude that clonal expansion is one of the most important mechanisms for increasing the burden of mutant cells in the mouse pancreas.

Multiscale structural analysis of mouse lingual myoarchitecture employing diffusion spectrum magnetic resonance imaging and multiphoton microscopy

The tongue consists of a complex, multiscale array of myofibers that comprise the anatomical underpinning of lingual mechanical function. 3-D myoarchitecture was imaged in mouse tongues with diffusion spectrum magnetic resonance imaging (DSI) at 9.4 T (b(max) 7000 smm, 150-microm isotropic voxels), a method that derives the preferential diffusion of water/voxel, and high-throughput (10 fps) two-photon microscope (TPM). Net fiber alignment was represented for each method in terms of the local maxima of an orientational distribution function (ODF) derived from the local diffusion (DSI) and 3-D structural autocorrelation (TPM), respectively. Mesoscale myofiber tracts were generated by alignment of the principal orientation vectors of the ODFs. These data revealed a consistent relationship between the properties of the respective ODFs and the virtual superimposition of the distributed mesoscale myofiber tracts. The identification of a mesoscale anatomical construct, which specifically links the microscopic and macroscopic spatial scales, provides a method for relating the orientation and distribution of cells and subcellular components with overall tissue morphology, thus contributing to the development of multiscale methods for mechanical analysis.

Three-dimensional cardiac architecture determined by two-photon microtomy

Cardiac architecture is inherently three-dimensional, yet most characterizations rely on two-dimensional histological slices or dissociated cells, which remove the native geometry of the heart. We previously developed a method for labeling intact heart sections without dissociation and imaging large volumes while preserving their three-dimensional structure. We further refine this method to permit quantitative analysis of imaged sections. After data acquisition, these sections are assembled using image-processing tools, and qualitative and quantitative information is extracted. By examining the reconstructed cardiac blocks, one can observe end-to-end adjacent cardiac myocytes (cardiac strands) changing cross-sectional geometries, merging and separating from other strands. Quantitatively, representative cross-sectional areas typically used for determining hypertrophy omit the three-dimensional component; we show that taking orientation into account can significantly alter the analysis. Using fast-Fourier transform analysis, we analyze the gross organization of cardiac strands in three dimensions. By characterizing cardiac structure in three dimensions, we are able to determine that the alpha crystallin mutation leads to hypertrophy with cross-sectional area increases, but not necessarily via changes in fiber orientation distribution.

Bead Packing and Release Using Flexible Polydimethylsiloxane Membrane for Semi-Continuous Biosensing

The continuous or semi-continuous biosensing of systemic inflammatory responses is important both during and after cardiopulmonary bypass (CPB) procedures. A bead packing and release method, which is able repetitively to capture and release receptor-coated beads within microfluidic channels, is herein advanced for use in semi-continuous biosensing. The receptor-coated beads are compacted and concentrated at specific locations in the device using an elastomeric valve. This concentration creates a localized bioreactor in which the binding of the antigen with the functionalized beads can be made more effective. After the reaction and detection have taken place, the beads can be released and a new assay carried out. We demonstrated the operation of our device using streptavidin-coated beads and biotin-4-fluorescein (B4F). The high sensitivity of the device allows it to detect a B4F concentration of 50 pg/mL after an incubation time of 5 min. We also tested our device in the semi-continuous immunoassay of interleukin (IL)-6, which is one of the proinflammatory cytokines. The assay demonstrated the linear dependence of the intensity of fluorescence at concentrations of IL-6 from 10 to 250 pg/mL, which is a physiologically important range for CPB procedures.

Lensed fiber-optic probe design for efficient photon collection in scattering media.

Measurement of bioluminescent or fluorescent optical reporters with an implanted fiber-optic probe is a promising approach to allow real-time monitoring of molecular and cellular processes in conscious behaving animals. Technically, this approach relies on sensitive light detection due to the relatively limited light signal and inherent light attenuation in scattering tissue. In this paper, we show that specific geometries of lensed fiber probes improve photon collection in turbid tissue such as brain. By employing Monte Carlo simulation and experimental measurement, we demonstrate that hemispherical- and axicon-shaped lensed fibers increase collection efficiency by up to 2-fold when compared with conventional bare fiber. Additionally we provide theoretical evidence that axicon lenses with specific angles improve photon collection over a wider axial range while conserving lateral collection when compared to hemispherical lensed fiber. These findings could guide the development of a minimally-invasive highly sensitive fiber optic-based light signal monitoring technique and may have broad implications such as fiber-based detection used in diffuse optical spectroscopy.

Two-photon microscopy using an Yb 3+ -doped fiber laser with variable pulse widths

Most of the two-photon fluorescence microscopes are based on femtosecond Ti:Sapphire laser sources near the 800 nm wavelength. Here, we introduce a new confocal two-photon microscope system using a mode-locked Yb(3+)-doped fiber laser. The mode-locked fiber laser produces 13 ps pulses with large positive chirping at a repetition rate of 36 MHz with an average power of 80 mW. By using an external grating pair pulse compressor, the pulse width and the frequency chirping of the laser output are controlled for optimum two-photon excitation. For a given objective lens, the optimum condition was obtained by monitoring the two-photon-induced-photocurrent in a GaAsP photodiode at the sample position. The performance of this pulse width optimized two-photon microscope system was demonstrated by imaging Vybrant DiI-stained dorsal root ganglion cells in 2 and 3 dimensions.