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.

Digital polarimetric second harmonic generation microscopy of partially oriented fiber structures

“…Changing handedness of the fibers does not change SHG intensity and R ratio, but changes the sign of C ratio (Figs. 3,5,8,9,11,12,13,14). The sign and magnitude of C ratio is influenced by tilt angle and polarity of the fibers.…”

“…Second harmonic generation (SHG) microscopy is an indispensable ultrastructure characterization technique for biological samples containing non-centrosymmetric fibrillar structures such as collagen (1)(2)(3)(4)(5), myosin (6,7), or starch granules (8)(9)(10). The fibrillar structures are partially oriented in the biological samples, thus polarimetric SHG microscopy can be used to extract information about molecular organization in each focal volume of the imaged structures (3,4,6,(11)(12)(13).…”

“…The reduced polarimetry techniques may employ linear incident polarization states (2,11,12,14), or incident and outgoing linear polarizations (3,(19)(20)(21)(22)(23). Circular polarization states are also used for the in-image-plane orientation independent measurements, revealing the susceptibility ratios (8,17). The polarimetric nonlinear microscopy uses high numerical aperture (NA) objectives for fundamental beam focusing into a tiny voxel.…”

Polarization-Sensitive Second Harmonic Generation Microscopy for Investigations of Diseased Collagenous Tissues

“…The clever use of fewer input polarizations could also be used, such as in Stokes-Mueller SHG [67] which uses four polarizations in a PI-SHG setup, or advanced polarimeters with multiple detectors can be used for simultaneous detection of the SHG polarization states [68]. In another technique, polarimetry analysis can also be performed using circularly polarized laser excitation, only requiring a single scan [53,69]. Fourier techniques could be used for faster data analysis [16,23,24], while imaging rates can be increased using higher repetition rate pulsed lasers combined with faster scanners such as spinning mirrors, or by increasing the field of view using a wide-field imaging approach [70].…”

“…The simplest PSA consists of a linear polarizer in a motorized rotation mount. SHG images are typically captured with at least 8 PSG angles and 8 PSA angles, hence at least 64 images are recorded for the technique, resulting in ∼20 min acquisition times in comparison to PI-SHG which takes ∼1.5 min [53]. The additional dimensionality of the data is thought to produce higher accuracy fitting, although at the time of writing this manuscript no study has performed the comparison with the other techniques.…”