Polarization second harmonic generation microscopy provides quantitative enhanced molecular specificity for tissue diagnostics

Abstract

Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p-SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p-SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p-SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second-order χ tensor elements. These values may be used as a bio-marker to detect any structural alterations in pathological tissue for diagnostic purposes. The SHG intensity image (red) in (a) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution (b) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers.

Analyzing the feasibility of discriminating between collagen types I and II using polarization‐resolved second harmonic generation

“…Backward and forward scattered SHG (BSHG and FSHG) were collected using a HyD‐detector and photon multiplier tube, respectively, each combined with a 435 to 455 nm bandpass filter. Control of the laser polarization was enabled by placing a quarter‐wave and half‐wave plate in the laser beam prior to the microscope, as described previously . The control and calibration of the polarization were automated to enhance accuracy and speed by using motorized rotation stages.…”

“…Hence, χ 31 / χ 15 = 1. However, when A was explicitly set equal to 1, the experimental data could not be fitted to the theoretical model outlined below with acceptable fitting performance Therefore, the ratio χ 31 / χ 15 was allowed to vary, as in previous studies .…”

Nonlinear optical microscopy of early stage (ICRS Grade-I) osteoarthritic human cartilage

“…CPL light in the sample plane was produced by introducing an additional optical module (Fig. 1) in the optical path of the microscope [25]. SHG and TPEF images covering areas of 225µm × 225µm were selected in the cartilage and the signal (256 gray levels) was recorded in 512 pixels × 512 pixels matrices.…”

Divided pulse soliton self-frequency shift: a multi-color, dual-polarization, power-scalable, broadly tunable optical source

“…SHG imaging probes the spatial distribution of the second-order nonlinear susceptibility tensor χ 2 [18,19]. Polarized SHG imaging is often necessary to reveal the rich tensorial properties of χ 2 , which contain useful information about molecular orientation or domain structure [20–24]. As an example, a tetragonal barium titanate (BaTiO 3 ) crystal consisting of alternating domains is imaged [25].…”