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Applications of Laser Tweezing to the Study of Liposomal Membranes

In collaboration with Dr Andrew Ward, we are seeking to apply optical entrapment (laser tweezing) methodologies to the study of liposomal membranes. Laser tweezing allows us to observe a single liposome or pair of interacting liposomes for a prolonged period of time, and therefore observe slow membrane phenomena such as lipid exchange.
In initial work we used liposomes whose contents or membranes were labelled with fluorescent markers, and were able to hold trapped liposomes for periods in excess of 1 h. Some problems arising from photobleaching were encountered however. In more recent work, we have turned to Raman spectroscopy in order to study membrane structure. Raman spectroscopy offers the large advantage that no labelling is required in order to obtain spectra. We have demonstrated that it is feasible to obtain Raman spectra from individual trapped liposomes (Figure 1), and then use these for analytical purposes in order to examine membrane composition.1

Raman spectra obtained from single liposomes of DMPC or DOPC

Figure 1 Raman spectra obtained from single trapped liposomes (1 micron diameter) composed of pure DMPC (A) or DOPC (B) in phosphate buffered saline at pH7.2.

Key signals in the DMPC spectrum are at 1060, 1085 and 1125 cm-1 (C-C skeletal stretching vibrations), 1296 cm-1 (CH2 twist) and 1445 cm-1 (CH2 bend). In the DOPC spectrum, additional signals of note are found at 1264 cm-1 and 1655 cm-1 (cis C=C stretching vibration).

By using the spectra above as reference spectra, we are able to analyse the composition of membranes. An example is shown below for the partitioning of hexafluoroisopropanol (HFIP) into DMPC membranes (Figure 2). From the data it is apparent that HFIP interacts favourably with DMPC membranes, considering the signal strength from trapped liposomes in the presence of low HFIP concentrations.

Raman spectra obtained for single DMPC liposomes incubated in the presence of HFIP

Figure 2 Raman spectra obtained from a 2.5% solution of hexafluoroisopropanol (HFIP) in phosphate buffered saline (A) and from single trapped liposomes (1 micron diameter) composed of pure DMPC (B) in phosphate buffered saline at pH7.2 in the presence of 0.25% hexafluoroisopropanol (HFIP).2

Key signals in the HFIP spectrum are at 740 and 846 cm-1. Signals from HFIP in the HFIP/DMPC mixture are indicated by arrows.

High-Resolution Microscopy of Peptides Binding to Membranes

In collaboration with Professor Ritu Kataky (University of Durham), we are seeking to obtain high resolution images of synthetic peptides binding to the surface of lipid membranes and assembling into transmembrane channels. In particular, we are interested in characterising the assembly process and identifying the specific lipid molecules that preferentially associate with the assembling channel. Our microscopic techniques, Scanning Electrochemical Microscopy (SECM) and the Scanning Kelvin Probe Microscopy (SKP), are well suited to this end. By inserting unusual amino acids at particular sites in a peptide template, and determining the subsequent effects on the assembly process, we hope to establish structure-function relationships for peptide insertion into lipid bilayers.

  1. "Analysis of Liposomal Membrane Composition Using Raman Tweezers", John M. Sanderson and Andrew D. Ward, Chem. Commun., 2004, 1120.
  2. "The Formation of Micellar Aggregates Following the Addition of Hexafluoroisopropanol to Phospholipid Membranes", Sue M. Ennaceur and John M. Sanderson, Langmuir, 2005, 21, 552-561.