Detecting Phase Transitions in Phosphatidylcholine Vesicles by Raman Microscopy and Self-Modeling Curve Resolution

Abstract

The study of phospholipid phase transitions is important for understanding drug- and protein-membrane interactions as well as other phenomena such as trans-membrane diffusion and vesicle fusion. A temperature-controlled stage on a confocal Raman microscope has allowed phase transitions in optically trapped phospholipid vesicles to be monitored. Raman spectra were acquired and analyzed using self-modeling curve resolution, a multivariate statistical analysis technique. This method revealed the subtle spectral changes indicative of sub- and pretransitions and main transitions in vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The Raman scattering results were compared to differential scanning calorimetry (DSC) experiments and found to be in good agreement. This method of observing lipid phase transition profiles requires little sample preparation and a minimal amount of lipid (<or=0.1 nmol) for an experiment. The conformational changes of the phospholipid molecules occurring during phase transitions are elucidated from the Raman spectroscopy results. The evolution of chain decoupling, rotational disorder, and gauche defects in the lipid acyl chains as a function of temperature is described.

Chemical Imaging with Frequency Modulation Coherent Anti-Stokes Raman Scattering Microscopy at the Vibrational Fingerprint Region

“…DPPC has two completely saturated alkyl chains, and its membrane is in a gel phase at room temperature. 34 Figure 9a shows the FM-CARS spectrum of a thick bulk DPPC film. Note that the CARS spectrum of DPPC membrane can be significantly different from this bulk spectrum.…”

“…In the literature, the 1060 and 1130 cm -1 peaks are assigned to out-of-phase and in-phase C-C stretching vibrations of all-trans conformers, respectively. 32,34 The narrow 1100 cm -1 peak is the C-C stretching mode of conformers with isolated gauche defects. 34,35 Interestingly, we see distinct polarization dependence of CARS images in this frequency region.…”

“…32,34 The narrow 1100 cm -1 peak is the C-C stretching mode of conformers with isolated gauche defects. 34,35 Interestingly, we see distinct polarization dependence of CARS images in this frequency region. Figure 9b shows the CARS mages at 1060, 1100, and 1130 cm -1 .…”

Raman spectra measurements on DEPC liposome and cell membrane of living neuron under xenon pressure

“…These peaks were assigned as the methylene C-C stretching mode of the trans and gauche conformations in the lipid acyl chains, respectively, and the relative intensity ratio of these peaks are known to be a measure of the ordering of the lipid tails in the liposomes. 29 Unfortunately, most of peaks around 1000 cm -1 had low signal/noise ratio due to the background noise from the silica glass plate used at the bottom of the glass dish, and we could not analyze the changes in peak intensity quantitatively. However, we identified that these peaks shifted to slightly lower wave numbers during the experiments.…”

Vibrational spectroscopic studies of newly developed synthetic biopolymers

“…27,41 Several techniques, including variable-temperature infrared and Raman spectroscopy, have been extensively applied in order to monitor the change of the inter-and intramolecular order of lipid molecules during thermal transitions. 27,[41][42][43][44][45][46] In temperature-dependent infrared spectroscopy, the shifts in the vibrational peak positions in the CÀ ÀH stretching region as a function of temperature is utilized to derive the phase transition temperature profiles of PEGylated lipids. Furthermore, the frequencies corresponding to the CÀ ÀH stretching vibrational region (3000-2800 cm 21 ) can be used as indicators of order-disorder transitions of acyl chains because of their strong sensitivities to the conformation of the hydrocarbon chain.…”