Comparison of Different Polarization Sensitive Second Harmonic Generation Imaging Techniques

Abstract

Polarization sensitive second harmonic generation (pSHG) microscopy is an imaging technique able to provide, in a non-invasive manner, information related to the molecular structure of second harmonic generation (SHG) active structures, many of which are commonly found in biological tissue. The process of acquiring this information by means of pSHG microscopy requires a scan of the sample using different polarizations of the excitation beam. This process can take considerable time in comparison with the dynamics of in vivo processes. Fortunately, single scan polarization sensitive second harmonic generation (SS-pSHG) microscopy has also been reported, and is able to generate the same information at a faster speed compared to pSHG. In this paper, the orientation of second harmonic active supramolecular assemblies in starch granules is obtained on by means of pSHG and SS-pSHG. These results are compared in the forward and backward directions, showing a good agreement in both techniques. This paper shows for the first time, to the best of the authors' knowledge, data acquired using both techniques over the exact same sample and image plane, so that they can be compared pixel-to-pixel.

Keywords: Medical and biological imaging; Nonlinear Microscopy; Polarization; Second harmonic generation.

Figure 1

Schematic diagram of the optical setup: (a) The polarization sensitive second harmonic generation (pSHG) microscopy setup, where z’ is the orientation of the supramolecular assembly, which for the case of starch can be seen in Figure 2, ϕ is the angle of orientation of the assembly, and α is the orientation of the polarization of the excitation beam, (b) the single scan polarization sensitive second harmonic generation (SS-pSHG) microscopy setup and (c) the detection unit to collect the second harmonic generation (SHG) signal in three directions in the SS-pSHG setup. The same detection unit is used in the forward and backward directions.

Figure 2

Schematic drawing of the internal structure of a starch granule, where z is the axis along which the cylindrical supramolecular assembly is oriented [27].

Figure 3

Forward and backward pSHG images acquired from starch granules. The images in both directions are obtained simultaneously in the same scan. Images are 70 × 70 pixels, with a pixel size of ~195 nm. Scale bar is 5 µm, and the acquisition time for these images was ~1.5 min. (a) Average intensity of the SHG images generated by 9 linear polarized excitation beams in forward direction. (b) Amylopectin supramolecular assembly orientations, φ, calculated from data in (a). (c) Average of the 9 intensity SHG images generated in the backward direction. (d) Assembly orientation values, φ, calculated from data in (c).

Figure 4

(a) Subtraction of the orientation of the amylopectin supramolecular assembly values, φ_s, calculated using data acquired in the forward, Figure 3b, and backward, Figure 3d, directions. (b) Histogram of φ_s values in (a).

Figure 5

pSHG and SS-pSHG images acquired from starch granules in the forward direction. Images are 135 × 115 pixels, with pixel size of ~ 86 nm. Scale bar is 5 µm. (a) Average of forward SHG intensity images acquired using 9 different linear polarization of the excitation beam. Acquisition time for this pSHG image was 1.5 min. (b) Amylopectin supramolecular assembly orientation, φ, calculated from data in (a). (c) Average of forward SHG intensity images acquired from 0°,45° and 90° channels in SS-pSHG. Acquisition time for this image was ~7.5 s. (d) Assembly orientation values, φ, calculated from data in (c).

Figure 6

(a) Subtraction of the φ values obtained by means of pSHG and SS-pSHG in the forward direction shown in Figure 4b,d. (b) Histogram of φ_s values shown in (a).

Figure 7

pSHG and SS-pSHG images acquired from starch granules in the backward direction. Images are 70 × 70 pixels, with a pixel size of ~195 nm. Scale bar is 5 µm. (a) Average of backward SHG intensity images acquired using 9 different linear polarizations of the excitation beam. Acquisition time for this pSHG image was 1.5 min. (b) Supramolecular assembly orientations, φ, calculated from data in (a). (c) Average of the backward SHG intensity images acquired from 0°, 45° and 90° channels in SS-pSHG. Acquisition time for this SS-pSHG image was ~7.5 s. (d) Assembly orientation values, φ, values calculated from data in (c).

Figure 8

(a) Subtraction of the φ values obtained by means of pSHG and SS-pSHG in the backward direction shown in Figure 6b,d. (b) The histogram of φ_s values.