Fast polarization-sensitive second-harmonic generation microscopy based on off-axis interferometry.

Abstract

We propose polarization-sensitive second-harmonic generation microscopy based on off-axis interferometry (OI-PSHG) by recording the complex field of a wide-field second-harmonic generation (SHG) image and performing polarization measurements. With the ability to record the SHG signals associated with different positions simultaneously, the proposed method exhibits a higher imaging frame rate than raster scanning-based SHG microscopy. The molecular orientation (in terms of their symmetric axis) of tendon collagen fibrils and myosin in muscle is resolved in three dimensions from a subset of polarization-resolved SHG holograms. With the present configuration, it takes approximately 0.01 s to acquire an image with 128 × 128 pixels, which is mainly limited by the excitation power density for wide-field illumination. For the same data throughput using pixel-by-pixel scanning, 0.16-s-long acquisition is required, with the pixel dwell time of 10 µs. Offering the ability to perform wide-field imaging and polarization measurements, the present work lays the foundation for fast SHG microscopy using complex deconvolution and harmonic tomography.