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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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