Optical second harmonic generation microscopy: application to the sensitive detection of cell membrane damage.

ABSTRACT

Optical second harmonic generation (SHG) is a nonlinear optical process which is sensitive to the symmetry of media. SHG microscopy allows for selective probing of a non-centrosymmetric area of sample. This type of nonlinear optical microscope was first used to observe ferroelectric domains and has been applied to various specimens including the biological samples to date. Imaging of the endogenous SHG of biological tissue has been utilized for the selective observation of filament systems in tissues such as collagen, myosin, and microtubules, which exhibit a polar structure. The cellular membrane can be selectively observed by the SHG microscope through membrane staining with amphiphilic polar dye molecules. It has been reported that, by imaging exogenous SHG of the membrane, sensitive detection of membrane damage could be realized using the SHG microscope. Because the staining dye is fluorescent, both SHG and two-photon excited fluorescence (TPF) images can be obtained simultaneously. How the SHG intensity depends on the molecular alignment of the polar dye molecules that reflects the ordering of lipid molecules in the plasma membrane and the necessity of the normalization of the SHG intensity by the TPF intensity is discussed. Furthermore, the assessment of the membrane damage induced by exposing polycation to HeLa cells has been compared with the conventional cytotoxicity and cell viability tests to demonstrate the higher sensitivity of the present SHG-based assay.