- Browse by Subject
Browsing by Subject "permeability"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Biophysics of Zebrafish (Danio rerio) Sperm(Elsevier, 2009-02) Hagedorn, M.; Ricker, J.; McCarthy, M.; Meyers, S.A.; Tiersch, T.R.; Varga, Z. M.; Kleinhans, F.W.; Physics, School of ScienceIn the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (Vo) of 12.1 ± 0.2 μm3 (SEM), a water permeability (Lp) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) um/min/atm, and a cryoprotectant permeability (Ps) of 0.10 +/− 0.01 (SEM) × 10−3 cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0°C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.Item Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy(Taylor & Francis Online, 2014-03-01) Sandoval, Ruben M.; Wang, Exing; Molitoris, Bruce A.; Department of Medicine, School of MedicineMaximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required.