Вы находитесь на странице: 1из 3

Journal of Microscopy, Vol. 228, Pt 2 2007, pp.

107109
Received 9 May 2007; accepted 24 June 2007

A study of the human hair structure with a Zernike phase contrast


X-ray microscope
H WA S H I K YO U N & TA E J O O S H I N
Pohang Accelerator Laboratory, POSTECH, 31 San, Hyoja-dong, Nam-ku, Pohang,
KyungBuk 790-784, Korea

Key words. Hair, X-ray microscope, zernike phase contrast, zone plate.
Summary
We have observed the internal structure of human hair
shafts with a transmission Zernike phase contrast hard X-ray
microscope. Due to the high spatial resolution and the high
contrast of the microscope, we could image scales, macrofibrils, medulla and melanin without staining. The structure
of a black hair shaft is compared with that of a white one.
Introduction
The structure of hair has been studied with various techniques
such as a light microscopy, electron microscopy (Swift, 1997;
Jinan et al., 2006), atomic force microscopy (AFM) (Swift
& Smith, 2000; Chen & Bhushan, 2005) and small angle
X-ray (microbeam) scattering (Briki et al., 2000). Among them,
electron microscopy has played a vital role of bridging the gap
in scale between light microscopy and X-ray scattering and has
been crucial in determining the structural model of hair.
However, one critical limitation of electron microscopy is
that samples have to be in vacuum, which can be especially
harsh for wet biosamples. For example, a good hair is said
to be able to absorb 30% of weight in water. Unless one
uses an environmental SEM, these biosamples are typically
dehydrated, leading to distortions in both the sample and the
image. Additional distortions can be caused by staining the
sample. Furthermore, electron microscopy techniques image
either surfaces (SEM, scanning electron microscope) or very
thin films (TEM, transmission electron microscope), so it is
necessary to section a thick sample to image internal structures
within it. Although AFM has been successfully employed to
observe hair in air, it is also a surface sensitive technique.
Method
It has been a dream of life scientists to see samples as they
are in their natural state. X-ray microscopes are close to an
ideal technique in that they can be used to image the inside
Correspondence to: Hwa Shik Youn. Tel: 82 54 279 1531; fax: 82 54 279 1599;
e-mail: hsyoun@postech.ac.kr

C 2007 The Authors
C 2007 The Royal Microscopical Society
Journal compilation 

of a sample in air. And because X-rays see electron density


while going through a sample, transmission X-ray microscopes
give superior contrast even for an unstained sample. In the
X-ray region Zernike phase contrast has been very effective
in observing small variations of electron density in biological
samples (Schmahl et al., 1995; Youn & Jung, 2006). Recently,
the X-ray optics has become mature enough to allow hard
X-ray microscopes to have spatial resolution better than
100 nm.
We have developed a full field hard X-ray microscope with
Zernike phase contrast and have reported preliminary results
on hair structure (Youn & Jung, 2005). The operating energy
of our microscope is 6.95 keV at which the depth of focus of
the X-ray objective zone plate lens is 226 m. This is sufficient
to see the whole width of Asian hair shafts which are about
100 m. X-rays go through a hair shaft perpendicular to its
length and projects the volume on the image plane. The X-ray
image of a sample is magnified 32.5 times by an objective zone
plate and converted into a visible light image using the CsI(Tl)
scintillation screen. This visible image is further enlarged
20 times with a microscope objective and captured with a full
frame CCD camera. The field of view of our X-ray microscope
is about 30 20 m2 , while the whole width of an Asian hair
shaft is about 100 m. So we scan a hair shaft perpendicular to
its length in 28-m steps and four or five frames of images were
patched to show the whole width. The detailed description of
our microscope can be found elsewhere (Youn & Jung).
Since the last report, we have improved the contrast and the
spatial resolution of our microscope resulting in significantly
better images of hair structure. In Fig. 1 we show the layout
of the current optical system in our microscope, in which
the old 12-bit CCD camera has been replaced by a new one
with 16-bit resolution. We have confirmed from our recent
data that density difference as small as 200-nm-thick PMMA
can clearly be visible using our microscope. Also, improved
source stability and sharper focusing have enabled us to take
clearer images. The spatial resolution of our microscope is
better than 100 nm. In the following figures we introduce
the images of human hair shafts taken with our microscope,

108

H . S . YO U N A N D T. J. S H I N

Fig. 1. The layout of the current optical system in the X-ray microscope. The size of each optical component is not to scale. The light (X-rays) is from
a synchrotron radiation source called Pohang Light Source in Korea, which has operating energy of 2.5 GeV. The diameters of the condenser and the
objective zone plate are 4000 and 160 m, respectively. Both the 1.6-m-thick gold zone plates have an outer most zone width of 100 nm. The size of
each pixel in the CCD camera is 20 20 m2 .

in which there has been no image processing except for


normalization.
Results and discussion
Figure 2 shows part of a white hair shaft from a Korean male
in his late thirties. We tiled four image frames with a size of
30 20 m2 to form a single 80 20 m2 image of the
whole width of a shaft, where the width of the shaft is 80 m.
Because the exposure time for each image frame was 20 s, it
took less than 2 min for each 20-m-tall slice. The image is
brighter on both ends due to halo effect (Youn & Jung, 2006),
which overexpresses density variations whenever there is a
very large density gradient. The cuticle with several layers of
well-packed scales is clearly visible around the shaft. They look
significantly better than the preliminary results (Youn & Jung,
2005), which are shown in Fig. 3. We estimate the thickness
of each scale at 300 nm. We found literature on the cortex is
scarce compared to that of the cuticle, which is probably due
to the fact that the hair has been studied mainly with surface

techniques. Inside the cuticle of scales there are cortical cells


with a diameter of 3 to 6 microns (Swift, 1997). Neither our Xray microscope nor AFM (Chen & Bhushan, 2005) succeeded
in seeing the walls of cortical cells. Instead the cortex looks
densely packed with macrofibrils, some of which are clearly
resolved and indicated with white arrows. The diameter of a
typical macrofibril is about 300 nm. We see some halo effect in
the medulla as well, which comes from the large variation in
electron density of the air-filled vacuoles. The density variation
is too large for the scales to be visible here. The width of the
medulla is about 18 m.
In Fig. 4 is shown part of a black hair shaft from another
Korean male in his early thirties. The width of the shaft was
123 m, so we tiled five 30 20 m2 image frames to form
a single image of a 20-m-tall and 123-m-wide slice of a
hair shaft. The exposure time for each image frame was also
20 s. As in Fig. 2 there is the halo effect at both ends of the
shaft. The cuticle with more layers of well-packed scales is also
clearly visible around the shaft. The scales of this hair shaft are
about 400-nm thick. They look less conspicuous than those

Fig. 2. A negative Zernike phase contrast image of a white hair shaft. This image was obtained with an X-ray magnification of 32.5 and a 20 microscope
objective. The exposure time for each image frame was 20 s and each image was normalized by flat field. The outer and the inner diameter of the phase
plate are 114.6 and 85.9 m, respectively. We used a 2.1-m-thick gold phase plate that gives a negative ( = 3 /2) phase contrast at 6.95 keV. The two
tiles at the ends look darker because X-rays see significantly less mass and there has not been any data processing.

C 2007 The Authors
C 2007 The Royal Microscopical Society, Journal of Microscopy, 228, 107109
Journal compilation 

S T U DY O F H U M A N H A I R S T RU C T U R E W I T H X - R AY M I C RO S C O P E

109

Fig. 3. A negative Zernike phase contrast image of a hair shaft. This figure is borrowed from Youn and Jung (2005) and shows a 14-m-tall slice of a hair
shaft. The image was taken with a 12-bit CCD camera, the pixel size of which was 9 9 m. The image was 2 2 binned before normalization.

Fig. 4. A negative Zernike phase contrast image of a black hair shaft. The magnification and the exposure time are the same as Fig. 2.

of Fig. 2, which can be due to the comparatively larger width


of the shaft. This hair looks somewhat more complicated than
the white one above. Melanin is responsible for the colour of
a hair shaft and we would expect to find some in the black
hair sample. Black Korean hair has been known to contain
exclusively eumelanin, a type of melanin that is in the form
of a little rice-like granule. These melanin granules are a
lot shorter than surrounding macrofibrils and the density of
melanin is 30% higher than hair keratin (Swift, 1963), which
improves its visibility under an X-ray microscope. From the
characteristic shape and the size we identify the granules inside
the white ellipse as eumelanin grains, which were not observed
in AFM study (Chen & Bhushan, 2005). In the figure there
are many eumelanin grains of various sizes throughout the
shaft overlapping with the macrofibrils. Because we can see a
transition region of the medulla in this figure, it is not possible
to determine the width. The halo effect is moderate for the
medulla as compared to the white hair shaft of Fig. 2, which
indicates that there is less void volume.
Our X-ray microscope allowed us to image hair shafts in air
without sectioning. Macrofibrils and melanin grains within
the black hair shaft were identified. One can even see swollen
hair with water with this microscope. The spatial resolution of
our X-ray microscope is near the end of the range covered by
small angle X-ray scattering, so a real space image obtained
with this microscope can be a useful complement to SAXS.
Acknowledgements
Hwa Shik Youn is grateful to Dr. Baik Kee Cho at St.
Marys Hospital of the Catholic University of Korea who
encouraged him to get started with hair study. This work
has been partially supported by the Korean MOST (Ministry
of Science and Technology) through the X-ray/Particle-beam
nanocharacterization Program. The research at the beam line
1 B2 is supported by MOST and the POSCO (Pohang Iron and
Steel Company, Pohang, Korea).

References
Briki, F., Busson, B., Kreplak, L., Dumas, P. & Doucet, J. (2000) Exploring
a biological tissue from atomic to macroscopic scale using synchrotron
radiation: Example of hair. J. Cell Mol. Biol. 46, 10051016.
Baltenneck, F., Bernard, B.A., Garson, J.C., Engstrom, P., Riekel, C., Leroy,
F., Franbourg, A. & Doucet, J. (2000) Study of the keratinization process
in human hair follicle by X-ray microdiffraction. J. Cell Mol. Biol. 46,
10171024.
Cao, J., Wijaya, R. & Leroy, F.. (2006) Unzipping the cuticle of the human
hair shaft to obtain micron/nano keratin filaments. Biopolymers. 83,
614618.
Chen, N. & Bhushan, B. (2005) Morphological, nanomechanical and
cellular structural characterization of human hair and conditioner
distribution using torsional resonance mode with an atomic force
microscope. J. Microsc. 220, 96112.
Kagoshima, Y., Ibuki, T., Yokoyama, Y., Tsusaka, Y., Matsui, J., Takai, K. &
Aino, M. (2001) 10 keV X-ray phase-contrast microscopy for observing
transparent specimens. Jpn. J. Appl. Phys. 40, L1190L1192.
Schmahl, G., Rudolph, D., Guttmann, P., Schneider, G., Thieme, J. &
Niemann, B. (1995) Phase-contrast studies of biological specimens with
the X-ray microscope at BESSY. Rev. Sci. Instrum. 66, 12821286.
Swift, J.A. (1963) Fundamentals of human hair science. Ph.D. thesis, Univ.
Leeds.
Swift, J.A. (1997) Morphology and histochemistry of human hair.
Formation and Structure of Human Hair (ed. by P. Joll`es, H. Zahn and
H. Hocker). pp. 149. Birkhauser-Verlag, Berlin.
Swift, J.A. & Smith, J. R. (2000) Atomic force microscopy of human hair.
Scanning 22, 310318.
Yokosuka, H., Watanabe, N., Ohigashi, T., Yoshida, Y., Maeda, S., Aoki,
S., Suzuki, Y., Takeuchi, A. & Takano, H. (2002) Zernike-type phasecontrast hard X-ray microscope with a zone plate at the photon factory.
J. Synchrotron Radiat. 9, 179181.
Youn, H.S. & Jung, S.-W. (2005) Observations of a human hair shaft with
an X-ray microscope. Phys. Med. Biol. 50, 54175420.
Youn, Hwa Shik & Jung, Suk-Won (2006) Proc. 8th Int. Conf. X-ray Microsc.,
IPAP Conf. Series 7 pp. 403406.
Youn, H.S. & Jung, S.-W.(2006) Hard X-ray microscopy with zernike phase
contrast. J. Microsc. 223, 5356.


C 2007 The Authors
C 2007 The Royal Microscopical Society, Journal of Microscopy, 228, 107109
Journal compilation