submitted papers Temperature-Controlled Confocal Raman Microscopy to Detect Phase Transitions in Phospholipid Vesicles CHRISTOPHER B. FOX, GRANT A. MYERS, and JOEL M. HARRIS* Department of Bioengineering, University of Utah, 50 South Central Campus Drive, Salt Lake City, Utah 84112-9202 (C.B.F., J.M.H.); and Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850 (G.A.M., J.M.H.) Optical-trapping confocal Raman microscopy enhances the capabilities of traditional Raman spectroscopy for the analysis of small particles by significantly reducing the sampling volume and minimizing background signal from the particle surroundings. Chemical composition and structural information can be obtained from optically trapped particles in aqueous solution without the need for labeling or extensive sample preparation. In this work, the challenges of measuring temperature- dependent changes in suspended particles are addressed with the development of a small-volume, thermally conductive sample cell attached to a temperature-controlled microscope stage. To demonstrate its function, the gel to liquid-crystalline phase transitions of optically trapped lipid vesicles, composed of pure 1,2-ditridecanoyl-sn-glycero-3-phospho- choline (DTPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), were detected by changes in Raman spectra of the lipid bilayer. The Raman scattering data were found to correlate well with differential scanning calorimetry (DSC) results. Index Headings: Confocal Raman microscopy; CRM; Temperature control; Phospholipid vesicles. INTRODUCTION Raman spectroscopy is an excellent tool for investigating the composition and structure of small particles dispersed in solution; 1–5 the technique involves no labeling and easily accommodates aqueous suspensions with a minimum of sample preparation. When integrated with an epi-illumination confocal microscope, 1,6 Raman spectroscopy is an excellent tool for studying dispersed particles at very low concentrations, including biological samples. With a high numerical aperture (NA) objective, the microscope allows the excitation laser to be tightly focused, creating an optical trap for small particles that have a higher refractive index than their surroundings. 7 The signal-to-noise ratio of Raman scattering detected from an optically trapped particle is enhanced by the collection efficiency of the high NA objective and the low background from the particle surroundings, which is excluded by confocal optics. 8 The detection limits within the ;1 fL confocal detection volume can be less than ten million molecules. 9 Recent studies have employed this technique to observe chemical reactions on optically trapped silica particles 10 and to investigate permeability and structure of optically trapped and surface-adhered polystyrene particles 9,11 and phosphati- dylcholine vesicles. 12–14 In this work, optical trapping confocal Raman microscopy is adapted to studies of temperature-dependent processes in individual micrometer-sized particles by the addition of a small-volume, temperature-controlled sample cell to the inverted microscope. The challenges of avoiding large temperature gradients within the sample are addressed by reducing the size of the solution volume and constructing the cell out of a thermally conductive material (copper). The heat transfer properties of the cell are modeled using finite element methods, and its performance is tested by studying the membrane phase-transition behavior of optically trapped phospholipid vesicles. Phospholipid vesicles, or liposomes, are an active research topic due to their importance as membrane models and drug delivery vehicles. 15 Phospholipid vesicles can be optically trapped and observed for hours, 13 allowing membrane order, permeability, and stability to be assessed by observing changes in Raman spectra. The optical trap permits vesicles to be examined in free solution, avoiding interference from surfac- es. 16 In this study, Raman spectra of optically trapped vesicles were acquired while the temperature was varied to produce gel to liquid-crystalline phase-transition profiles for three phos- phatidylcholines having different transition temperatures to investigate the range of temperatures achievable with the microscope stage. Differential scanning calorimetry (DSC) data on dispersions of lipid vesicles were also acquired to compare with the transition temperature profiles determined by Raman microscopy. Temperature-controlled optical trapping confocal Raman microscopy is found to be a useful method to study phospholipid structure and membrane transition behavior on ,50 lL samples containing lM lipid concentrations. EXPERIMENTAL Reagents and Materials. 1,2-Ditridecanoyl-sn-glycero-3- phosphocholine (DTPC), 1,2-dimyristoyl-sn-glycero-3-phos- phocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phos- phocholine (DPPC) were purchased from Avanti Polar Lipids (Alabaster, AL). An extruder was also purchased from Avanti. Polycarbonate membranes were made by Nucleopore (Pleas- anton, CA). Water was quartz-distilled and then filtered with a Received 1 December 2006; accepted 8 February 2007. * Author to whom correspondence should be sent. E-mail: harrisj@chem. utah.edu. Volume 61, Number 5, 2007 APPLIED SPECTROSCOPY 465 0003-7028/07/6105-0465$2.00/0 Ó 2007 Society for Applied Spectroscopy