Computer-based tracking of single sperm

This paper describes a robust single sperm tracking algorithm (SSTA) that can be used in laser optical trapping and sperm motility studies. The algorithm creates a region of interest (ROI) centered about a sperm selected by the user. SSTA contrast enhances the ROI image and implements a modified four-class thresholding method to extract the tracked sperm as it transitions in and out of focus. The nearest neighbor method is complemented with a speed-check feature to aid tracking in the presence of additional sperm or other particles. SSTA has a collision-detection feature for real or perceived collision or near-miss cases between two sperm. Subsequent postcollision analysis employs three criteria to distinguish the tracked sperm in the image. The efficacy of SSTA is validated through examples and comparisons to commercially available computer-aided sperm tracking systems

Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment—twodimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSCderived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECMpromoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease

Concentration Independent Modulation of Local Micromechanics in a Fibrin Gel

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues

Internet-based robotic laser scissors and tweezers microscopy.

We have engineered a robotic laser ablation and tweezers microscope that can be operated via the internet using most internet accessible devices, including laptops, desktop computers, and personal data assistants (PDAs). The system affords individual investigators the ability to conduct micromanipulation experiments (cell surgery or trapping) from remote locations (i.e., between the US and Australia). This system greatly expands the availability of complex and expensive research technologies via investigator-networking over the internet. It serves as a model for other "internet-friendly" technologies leading to large scale networking and data-sharing between investigators, groups, and institutions on a global scale. The system offers three unique features: (1) the freedom to operate the system from any internet-capable computer, (2) the ability to image, ablate, and/or trap cells and their organelles by "remote-control," and (3) the security and convenience of controlling the system in the laboratory on the user's own personal computer and not on the host machine. Four "proof of principle" experiments were conducted: (1) precise control of microscope movement and live cell visualization, (2) subcellular microsurgery on the microtubule organizing center of live cells viewed under phase contrast and fluorescence microscopy, (3) precise targeting of multiple sites within single red blood cells, and (4) optical trapping of 10 microm diameter polystyrene microspheres