Noninvasive analysis of metabolism at the single cell level will have many applications in evaluating cellular physiology. One clinically relevant application would be to determine the metabolic activities of embryos produced through assisted reproduction. There is increasing evidence that embryos with greater developmental capacity have distinct metabolic profiles. One of the standard techniques for evaluating embryonic metabolism has been to evaluate consumption and production of several key energetic substrates (glucose, pyruvate, and lactate) using microfluorometric enzymatic assays. These assays are performed manually using constriction pipets, which greatly limits the utility of this system. Through multilayer soft-lithography, we have designed a microfluidic device that can perform these assays in an automated fashion. Following manual loading of samples and enzyme cocktail reagents, this system performs sample and enzyme cocktail aliquotting, mixing of reagents, data acquisition, and data analysis without operator intervention. Optimization of design and operating regimens has resulted in the ability to perform serial measurements of glucose, pyruvate, and lactate in triplicate with submicroliter sample volumes within 5 min. The current architecture allows for automated analysis of 10 samples and intermittent calibration over a 3 h period. Standard curves generated for each metabolite have correlation coefficients that routinely exceed 0.99. With the use of a standard epifluorescent microscope and CCD camera, linearity is obtained with metabolite concentrations in the low micromolar range (low femtomoles of total analyte). This system is inherently flexible, being easily adapted for any NAD(P)H-based assay and scaled up in terms of sample ports. Open source JAVA-based software allows for simple alterations in routine algorithms. Furthermore, this device can be used as a standalone device in which media samples are loaded or be integrated into microfluidic culture systems for in line, real time metabolic evaluation. With the improved throughput and flexibility of this system, many barriers to evaluating metabolism of embryos and single cells are eliminated. As a proof of principle, metabolic activities of single murine embryos were evaluated using this device.Metabolomics represents an important approach to studying functional biological systems and provides valuable complementary information to that obtained from gene and protein expression studies. The development of new tools and techniques for the analysis of cellular metabolites (specifically various nutrients and waste molecules generated by living cells) has found important application in exploring directed evolution, drug toxicity, and cancer. 1,2 Metabolomic studies of single cells are advantageous because the complexities associated with cellular heterogeneity can be avoided. For studies of organisms composed of a single or small number of cells, such as embryos during early development, single cell sensitivity is an absolute necessity.There is ...
Neutrophil and macrophage (M) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.
We demonstrate a technique for investigating the energetics of flagella or cilia. We record the planar beating of tethered mouse sperm at high resolution. Beating waveforms are reconstructed using proper orthogonal decomposition of the centerline tangent-angle profiles. Energy conservation is employed to obtain the mechanical power exerted by the dynein motors from the observed kinematics. A large proportion of the mechanical power exerted by the dynein motors is dissipated internally by the motors themselves. There could also be significant dissipation within the passive structures of the flagellum. The total internal dissipation is considerably greater than the hydrodynamic dissipation in the aqueous medium outside. The net power input from the dynein motors in sperm from Crisp2-knockout mice is significantly smaller than in wildtype samples, indicating that ion-channel regulation by cysteine-rich secretory proteins controls energy flows powering the axoneme.
While much is known about the microstructure of sperm flagella, the mechanisms behind the generation of flagellar beating patterns by the axoneme are still not fully understood. We demonstrate a technique for investigating the energetics of flagella or cilia. We record the planar beating of tethered wildtype and Crisp2-knockout mouse sperm at high-speed and high-resolution and extract centerlines using digital image processing techniques. We accurately reconstruct beating waveforms using a Chebyshev-polynomial based Proper Orthogonal Decomposition of the centerline tangent-angle profiles. External hydrodynamic forces and the internal resistance from the passive flagellar material are calculated from the observed kinematics of the beating patterns using a Soft, Internally-Driven Kirchhoff-Rod (SIDKR) model. Energy conservation is employed to further compute the flagellar energetics. We thus obtain the distribution of mechanical power exerted by the dynein motors without any further assumptions about mechanisms regulating axonemal function. We find that, in both the mouse genotypes studied, a large proportion of the mechanical power exerted by the dynein motors is dissipated internally, within the passive structures of the flagellum and by the motors themselves. This internal dissipation is considerably greater than the hydrodynamic dissipation in the aqueous medium outside. The net power input from the dynein motors in sperm from Crisp2-knockout mice is significantly smaller than in corresponding wildtype samples. The reduced power is correlated with slower beating and smaller amplitudes. These measurements of flagellar energetics indicate that the ion-channel regulating cysteine-rich secretory proteins (CRISPs) may also be involved in regulating mammalian sperm motility.
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