R P Haugland

Electronic excitation energy transfer as function of distance measured, noting energy transfer process use as spectroscopic ruler

1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a cationic analogue of diphenylhexatriene (DPH), has photophysical properties that are generally similar to those of DPH. In solution the fluorescence lifetime (tau) of TMA-DPH is short (less than 1.5 ns), but tau increases to approximately 7 ns when the probe is embedded in lipid bilayers at temperatures less than the thermal transition temperature (Tc) of the lipid. The cationic charge ensures that the probe is anchored at the lipid-water interface, most likely with the DPH moiety intercalated between the upper portions of the fatty acyl chains. The profiles of changes in steady-state anisotropies (rss) and limiting hindered anisotropies (r infinity) are similar for both TMA-DPH and DPH embedded in lipid bilayers, but r infinity values for TMA-DPH even at T much greater than Tc are generally greater than 0.14, e.g., at 35 degrees C in 1,2-dimyristoylglycero-3-phosphocholine (DMPC) (cf. 0.03 for DPH in DMPC at 35 degrees C). Electrostatic interactions of the cationic probe with head groups of phospholipids do not appear to significantly influence the apparent dynamics of the probe. TMA-DPH should prove useful in the study of the dynamics of phospholipid monolayers, e.g., in native or reconstituted lipoproteins.

A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.

A new membrane-permeant DNA stain, SYBR-14, was used in combination with propidium iodide (PI) to estimate the proportion of living sperm in bovine semen. The SYBR-14 stained living sperm while PI only stained degenerate cells that had lost their membrane integrity. Staining with SYBR-14 resulted in the nuclei of living sperm fluorescing bright green. Aliquots containing nearly all living bovine sperm were prepared using glass wool/Sephadex filtration to remove dead and damaged cells. A portion of this filtered sample was killed by unprotected freeze-thawing and used to provide mixed aliquots containing known ratios of living and dead sperm. Flow cytometry was used to assess the green and red fluorescence of these mixtures. The percentages of living sperm, as determined by the log of green fluorescence, were 85.1, 68.8, 39.8, 20.7, and 1.4 for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 of the filtered, killed mixtures. Also, bovine semen was diluted 1:60 in HEPES-0.1% bovine serum albumin and incubated for 0, 3, 6, and 24 hours at 36 degrees C to assess changes in cell viability. As cell death occurred during this incubation period, a relatively rapid transition of staining from green to red occurred as sperm died. Three replicates of cryopreserved sperm from six bulls were also examined using SYBR-14 and PI to assess the proportion of living and dead cells. Flow cytometric analyses of these samples, which had been processed and stored in homogenized milk, indicated that this stain combination was useful in assessing the quality of cryopreserved sperm. The combination of SYBR-14 and PI was determined to be an effective tool for assessing the viability of fresh or cryopreserved sperm.