基于SHG显微镜的胶原微结构准晶体模型探讨

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本文主要探讨了一种基于第二谐波成像（Second Harmonic Generation, SHG）的胶原蛋白微结构模型，特别是在研究光束的圆偏振特性时。SHG是一种非线性光学现象，当分子在外部电场，通常由高强度激光束提供时，会发生极化。这种极化特性不仅取决于物质的内在结构属性，即其第二阶非线性电 susceptibility，还受到入射光的强度和偏振方向的影响。文章指出，在研究胶原蛋白这样的生物材料时，使用圆形偏振光束进行SHG成像是特别有意义的。圆形偏振光具有特殊的极化状态，其电场矢量沿光的传播路径在一个圆周上均匀分布，这使得研究者能够深入探究胶原蛋白微观结构中的周期性和非周期性（quasi-crystalline）特征。SHG成像可以揭示材料内部的精细结构，因为非线性效应依赖于局部的极化响应，这对于理解胶原蛋白的自组织和功能性至关重要。研究者在2010年2月的《中国光学 letters》上发表了这一成果，他们分析了当入射光为圆偏振时，如何通过SHG技术获取有关胶原蛋白微结构的更准确信息。他们不仅提出了实验上的考虑因素，如选择适当的光源参数和数据处理方法，还展示了相关的实验结果以及数值模拟，这些都支持了他们关于圆偏振光下SHG成像对揭示胶原蛋白微结构优势的理论分析。这篇论文的核心内容涵盖了以下几个知识点： 1. **SHG原理**：非线性光学过程，分子在外部电场下的极化，以及它与物质结构、光强度和偏振的关系。 2. **圆偏振光的使用**：在胶原蛋白研究中的重要性，因为它能有效捕捉微结构的细节。 3. **胶原蛋白微结构模型**：基于SHG的观察，尤其是对quasi-crystalline特性的探讨。 4. **实验策略**：如何优化实验条件，如选择合适的光源和分析方法，以得到可靠的SHG图像。 5. **研究成果与数值模拟**：通过实验证据和计算结果来支撑模型的有效性。这项工作对于深入理解胶原蛋白的结构与功能有着重要的科学价值，也为其他生物材料的非线性光学研究提供了新的视角和技术手段。

February 10, 2010 / Vol. 8, No. 2 / CHINESE OPTICS LETTERS 213

A quasi-crystal model of collagen microstructure based on

SHG microscopy

Pu Xu

1,3∗

, Eleanor Kable

, Colin J. R. Sheppard

, and Guy Cox

Australian Key Centre for Microscopy and Microanalysis, University of Sydney, NSW 2006, Australia

Division of Bioengineering, National University of Singapore, Singapore 117576

Key Laboratory of Ecophysics, Department of Physics, Shihezi University, Shihezi 832000, China

∗

E-mail: xupu@tsinghua.org.cn

Received April 13, 2009

Second harmonic generation (SHG) results from molecules which are polarized by an external electric ﬁeld

often provided by an intense laser beam. The polarizability depends on ﬁrstly the intrinsic structural

prop erties of the substance and hence the second-order nonlinear susceptibility, and secondly the intensity

and p olarization direction of the incident light. The polarization characteristics of the beam are therefore of

interest. In this letter, we discuss some considerations in SHG microscopy of collagen when the incoming

b eam is circularly polarized, and present some supporting results as well as a numerical analysis. We

prop ose a quasi-crystal model of collagen microstructure in an eﬀort to further our understanding on this

protein.

OCIS codes: 180.0180, 190.0190, 260.0260.

doi: 10.3788/COL20100802.0213.

When molecules are polarized by an electric ﬁeld exerted

on them, their subsequent dipole radiation contains har-

monic components, namely second, third and higher

harmonic generation

[1]

. The second harmonic generation

(SHG) is generally the strongest of these when the ma-

terial lacks structural centro-symmetry, typical examples

of these include solid noncentrosymmetric crystals

[2]

SHG has been employed in microscopy based on in-

tense laser illumination

[3,4]

, and has produced many

promising results

[5−7]

, including biological/biomedical

applications

[8−11]

. While in p erfect periodical structures

such as single crystals, the polarizability of the substance

is uniform throughout; in partially regular structures or

structures containing regular comp onents whereas these

components are randomly scattered in space, it is nat-

ural to ask how the change in polarizability in spatial

terms inﬂuences the signal output which is used to form

images.

Atoms, when forming molecules in ionic bond or va-

lence bond, often exhibit electric polarities. When this

polarity is in line with the electric ﬁeld of incoming light,

it is polarized most strongly; the degree of polarizabil-

ity will decrease with the increase of the angle between

the external ﬁeld and the internal polarity, and reaches

zero when they become perpendicular. When semi-

regular structures are under a microscope, we would

like molecules of all orientations to be excited. A circu-

larly polarized beam oﬀers electric ﬁeld around 360

◦

and

hence oﬀers an attractive candidate for studying these

semi-regular structures such as many biological speci-

mens.

The microscope used was an inverted microscope

(DMIRBE, Leica, Germany), ﬁtted with a spectromet-

ric confocal head (Leica Microsystems, Germany). The

laser is a coherent Mira Ti: sapphire system, tunable

between 700 and 1000 nm, operating in the femtosecond

regime and pumped by a 5-W solid-state laser (Verdi,

Coherent Scientiﬁc, USA). All additional detectors and

optical equipment were supplied by Leica Microsystems,

with the exception of the additional ﬁlters and dichroics,

supplied by Chroma Inc.

To establish the signiﬁcance of being able to manipu-

late the polarization plane of light entering the medium,

we conducted two sets of experiments on single crystal

and biological samples.

We ﬁxed the relative orientation between the sample

and the linear polarization plane of the laser beam, and

rotated the analyser in front of the receiving optics (but

after the sample). After each such cycle we changed the

relative angle between the sample and the beam, i.e.,

either the sample or the polarization plane of the beam

was rotated and carried out the analyser cycle again.

This sequence was repeated until all combinations of the

two angles were investigated.

The ﬁrst set of images is high χ

(2)

single crystal

of potassium dihydrogen phosphate (KDP) at 100-µm

thickness, as shown in Fig. 1. It can be seen that not

only the sample is best excited when the relative angle

between itself and the incident beam remained at one

constant value, but also the second harmonic (SH) emis-

sion is linearly polarized and the polarization plane is

determined by the orientation of the sample, regardless

of the incident light.

Figure 2 shows the similar phenomenon when a piece

of biological specimen, a sliced bone sample, is im-

aged. Collagen in the bone is a strong generator of the

[8,12−14]

. We extracted four images to show the link-

age between the input and the sample, and the sample

and the output. The upper two images were acquired

with a constant angle between the sample and the in-

cident polarization with two analyser positions allowing

emission to pass at angle of 90

◦

to one another; the

bottom two were acquired with a constant angle be-

tween sample and analyser but using two directions of

1671-7694/2010/020213-04

° 2010 Chinese Optics Letters

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