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
1
, Colin J. R. Sheppard
2
, and Guy Cox
1
1
Australian Key Centre for Microscopy and Microanalysis, University of Sydney, NSW 2006, Australia
2
Division of Bioengineering, National University of Singapore, Singapore 117576
3
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
SH
[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
c
° 2010 Chinese Optics Letters
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