Lesley Davenport

Reconvolution analysis in time‐resolved fluorescence experiments—an alternative approach: Reference‐to‐excitation‐to‐fluorescence reconvolution

A novel method for reconstructing the apparatus response function required for accurate detailed analysis of nanosecond time‐resolved fluorescence decay data is proposed and tested with properly constructed simulated data sets for a variety of pertinent cases, using the high‐accuracy convolution algorithm previously developed [J. Veceř, A. A. Kowalczyk, and R. E. Dale, Rev. Sci. Instrum. 64, xxx (1993)]. The veracity of recovery of test mono‐ and multiexponential decay responses by this method, involving the use of two appropriately chosen monoexponential reference decay responses, is shown to be essentially identical to that attainable with the ‘‘true’’ apparatus response function. The method is also demonstrated to exhibit significant advantages in a variety of situations over currently the most widely employed method of overcoming extant problems in the direct determination of an appropriate apparatus function: indirect analysis against a monoexponential reference decay response (δ‐function convolution...

A Hand-held Fiber-optic Implement for the Site-specific Delivery of Photosensitizer and Singlet Oxygen

We have constructed a fiber optic device that internally flows triplet oxygen and externally produces singlet oxygen, causing a reaction at the (Z)‐1,2‐dialkoxyethene spacer group, freeing a pheophorbide sensitizer upon the fragmentation of a reactive dioxetane intermediate. The device can be operated and sensitizer photorelease observed using absorption and fluorescence spectroscopy. We demonstrate the preference of sensitizer photorelease when the probe tip is in contact with octanol or lipophilic media. A first‐order photocleavage rate constant of 1.13 h−1 was measured in octanol where dye desorption was not accompanied by readsorption. When the probe tip contacts aqueous solution, the photorelease was inefficient because most of the dye adsorbed on the probe tip, even after the covalent ethene spacer bonds have been broken. The observed stability of the free sensitizer in lipophilic media is reasonable even though it is a pyropheophorbide‐a derivative that carries a p‐formylbenzylic alcohol substituent at the carboxylic acid group. In octanol or lipid systems, we found that the dye was not susceptible to hydrolysis to pyropheophorbide‐a, otherwise a pH effect was observed in a binary methanol‐water system (9:1) at pH below 2 or above 8.

Studies of membrane heterogeneity using fluorescence associative techniques

Fluorescence spectra, decay times and emission anisotropy are capable of providing important information regarding the interaction of probes with biological macromolecules. Combinations of different fluorescence parameters can provide information about heterogeneity of liposomes or biological membranes and about the character of excited-state interactions of the fluorescence probe. Decay-associated spectra (DAS) or anisotropy decay associated spectra (ADAS) can be used to resolve heterogeneous species. These methods have been applied to the study of both pyrene and a pyrene methyl cholesterol adduct (PMC) with dimyristoyl-lecithin vesicles (DML). The data indicate microheterogeneity in the distribution of the probes above the phase transition. Anisotropy decay-associated spectra (ADAS) have been used to study diphenylhexatriene (DPH) in dipalmitoyl-lecithin (DPL) and dimyristoyl-lecithin (DML) vesicles. The results indicate that DPH inhabits more than one rotational environment in the liposome preparations used.

Fluorescence Studies of Membrane Dynamics and Heterogeneity

The cellular morphology of lipid bilayer membranes acting as dynamic boundaries is well established. Their biological function, however, is determined at the molecular level. Thus, techniques sensitive to molecular conformation are required to understand how membranes work.

Long-lived fluorescence probes for studying lipid dynamics: A review.

A great many studies have focused on the heterogeneous packing of lipids in the bilayer matrix. However, less attention has been directed toward the temporal aspects of these lipid-lipid interactions. Studies of lipid packing fluctuations, or ‘gel-fluid’ exchange, using fluorescence probe methodologies have been limited. This limitation arises from thesubmicrosecond time scale over which the fluctuations are expected to occur. Traditionally, dynamic studies of lipid bilayers have been restricted to the nanosecond time regime, and the submicrosecond time ‘window’ has not been explored in any great depth by fluorescence methods, although persistent lipid dynamics has been evident. Probes with long fluorescence lifetimes (several hundred nanoseconds) have the potential to expand this important time ‘window,’ providing information on ‘gel-fluid’ exchange rates and insights into how important biological effectors such as proteins, cholesterol, and anesthetics affect or modulate these fluctuations. Using the long-lived fluorescence probe coronene, combined with time-resolved fluorescence methods geared toward microheterogeneity, we present a view of bilayer dynamics in an alternate time domain. Fluorescence probes are expected to inhabit an equilibrium between fluid and gel environments. Some probes remain in their respective environments throughout their excited-state lifetime, while others reside in surroundings that will change (i.e., ‘melt’). Long-lived fluorescence membrane probes can provide direct estimates of submicrosecond lipid fluctuation or ‘melt’ rates. Simple Landau modeling leads to adistribution of ‘melt’ rates and provides an attractive alternative to a simplercompartmental model where a unique lipid fluctuation of gel-fluid exchange rate is measured. Thedistribution model is probe independent (defined by thermodynamic quantities) and can be applied generally to the rotational motions of fluorescence probes embedded in the lipid bilayer.

Pressure Effects on Submicrosecond Phospholipid Dynamics Using a Long-Lived Fluorescence Probe

The effects of applied external hydrostatic pressure on submicrosecond lipid motions in DPPC4 bilayers have been examined using coronene (a long-lived planar fluorescent molecule) and DPH. Steady-state fluorescence emission anisotropy (EA) values () obtained for probe-labeled DPPC SUVs measured at different fixed temperatures above Tc as a function of increasing hydrostatic pressure reveal pressure-induced lipid phase transition profiles. For coronene-labeled samples, the observed lipid “melt” profiles are broad and shifted to higher midpoint EA pressure values (P1/2) compared with corresponding DPH-labeled SUVs at the same temperature. The data suggest lipid motions occurring on the submicrosecond time scale, detected only by using a long-lived fluorescence probe, which occur well above the normally reported “fluid–gel” lipid phase transition. Slopes of the pressure-to-temperature equivalence plots (dP1/2/dT = 39 bar/K) obtained for DPH-or coronene-labeled DPPC SUVs are identical within experimental error and reflect probe independence. For DPH, the slope of the P1/2(T) plot provides the expected phase transition phospholipid volume change. However, intercept values (at P1/2 = 1 bar) or apparent phase transition temperatures obtained from the equivalence plots for the two probes are not equal. Differences appear to arise due to the very disparate fluorescence lifetime values of the two probes, which result in rotational sensitivity of coronene to gel lipid volume fluctuations occurring during the extended time window provided by coronene fluorescence.