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Biophys. J. BioFAST: First Published August 26, 2005. doi:10.1529/biophysj.105.065607
© 2005 by the Biophysical Society.

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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Quantitative Coherent Anti-Stokes Raman Scattering Imaging of Lipid Distribution of Co-existing Domains

Li Li 1, Haifeng Wang 1 and Ji-Xin Cheng 2*

1 Purdue University
2 Purdue.edu

* To whom correspondence should be addressed. E-mail: jcheng{at}purdue.edu.

Submitted on April 30, 2005
Revised on June 18, 2005
Accepted on 9 August 2005


  Abstract
We demonstrate quantitative vibrational imaging of specific lipid molecules in single bilayers using laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with a lateral resolution of 0.25 µm. A lipid is spectrally separated from other molecules by using deuterated acyl chains which provide a large CARS signal from the symmetric CD2 stretch vibration around 2100 cm-1. Our temperature control experiments show that d62-DPPC has similar bilayer phase segregation property as DPPC when mixing with DOPC. By using epi-detection and optimizing excitation and detection conditions, we are able to generate a clear vibrational contrast from d62-DPPC of 10% molar fraction in a single bilayer of DPPC/d62-DPPC mixture. We have developed and experimentally verified an image analysis model that can derive the relative molecular concentration from the difference of the two CARS intensities measured at the peak and dip frequencies of a CARS band. With the above strategies, we have measured the molar density of d62-DPPC in the co-existing domains inside the DOPC/d62-DPPC (1:1) supported bilayers incorporated with 0% to 40% cholesterol. The observed interesting changes of phospholipid organization upon addition of cholesterol to the bilayer are discussed.

Key Words: CARS microscopy, bilayer, cholesterol, imaging, lipid domains, membrane




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Copyright © 2005 by the Biophysical Society.