Three‐dimensional structural imaging of starch granules by second‐harmonic generation circular dichroism

Abstract

SummaryChirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best-known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second-harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second-harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that secondharmonic generation, as a second-order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser-scanning microscope. In this work, we combine the axial resolving power of second-harmonic generation and chiral sensitivity of second-harmonic generation circular dichroism to realize three-dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second-harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second-harmonic generation circular dichroism response in complicated three-dimensional biological systems.Correspondence to: Shi-Wei Chu, 1, Sec 4, Roosevelt Rd., Taipei 10617, Taiwan. Tel: +88-623-366-5131; fax: +88-622-363-9984; e-mail: swchu@phys.ntu.edu.twThe sample we use is starch granules whose secondharmonic generation-active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second-harmonic generation for right-handed circularly polarized excitation is significantly different from second-harmonic generation for left-handed one, offering excellent second-harmonic generation circular dichroism contrast that approaches 100%. In addition, three-dimensional visualization of secondharmonic generation circular dichroism distribution with sub-micrometer spatial resolution is realized. We observed second-harmonic generation circular dichroism sign change across the starch granules, and the result suggests that in thick biological tissue, second-harmonic generation circular dichroism arises from macroscopic molecular packing. Our result provides a new method to visualize the organization of three-dimensional structures of starch granules. The second-harmonic generation circular dichroism imaging method expands the horizon of nonlinear chiroptical studies from simplified surface/solution environments to complicated biological tissues.

Investigating starch gelatinization through Stokes vector resolved second harmonic generation microscopy

“…From the observation of the electron diffraction data of potato starch fragments, Oostergetel and van Bruggen1 concluded that semi-crystalline domains form a network of left handed superhelices of diameter 18 nm and pitch 10 nm. It is consistent with previous reports identifying that amylopectin molecules in starch granules are the source of the SH signal1417. It is noted that the strong SH signal arises due to the fraction of different handed amylopectin molecules being unequal, in turn producing a non-centrosymmetric structure18.…”

“…It is noted that the strong SH signal arises due to the fraction of different handed amylopectin molecules being unequal, in turn producing a non-centrosymmetric structure18. The effect of the molecular arrangement of the concentric shell like structure of starch granules is observed in the SH intensity pattern and in various polarization components1217. Figure 2 compares the SH signal (S 0 ), Stoke vector (S 3 ) and DOCP of dry and hydrated starch granules at room temperature.…”

“…The origin of chirality in starch has been determined using second-harmonic-generation circular dichroism (SHG-CD) microscopy17 with high contrast and improved axial resolution. Figure 5 shows the pixel distribution of DOCP values in hydrated and dry starch upon heating.…”

“…A strong SH signal from semi-crystalline amylopectin chains which are assumed to lie in the amorphous lamellae (amylose) form radially distributed amorphous growth rings in starch1415. A Stokes vector based SHG microscopy scheme is distinct from several other previously demonstrated polarization resolved approaches in that it uses a large number of images rather than a single shot measurement11161718. The complete polarization states of the SH light of starch granules were characterized from SHG Stokes micrographs1113.…”

Polarimetric second harmonic generation microscopy: An analytical tool for starch bioengineering

“…Since the 1990s, newly developed commercial low noise, high stability, solid‐state ultrafast pulsed lasers enable high quality laser scanning nonlinear microscopy . The following lasers have been used for SHG microscopy of starch: Ti:Sapphire at 800–854 nm , Yb:KGW at around 1030 nm , Yb fiber laser at 1040 nm , Nd:YAG at 1064 nm , and a Cr:Forsterite at 1230 nm , providing a pulse energy of at least several nJ. Laser wavelengths ranging from 800 to 1230 nm result in SHG signal wavelength falling within the high sensitivity range of PMT detectors (400–650 nm).…”