Life Science : Medical Biology

Three-Dimensional Visualization of a Human Chromosome
Using Coherent X-Ray Diffraction
When we want to know the structure of a protein orits complex, we have to make its crystals in advance.
If we are lucky, we will obtain the crystals and then
X-ray crystallography will yield the structure of theprotein or its complex with about 1 Å resolution.
However, when we want to know the detailed structureof much bigger objects such as cellular organelles,
for example, chromosomes or nuclei, what should
we do? Such objects with varying sizes and shapes
do not form crystals for X-ray crystallography. Moreover,they are too thick for electron microscopy. Coherent
X-ray diffraction microscopy (XDM) is the method toachieve it [1].
When a noncrystalline object is illuminated bycoherent, monochromatic X-rays having a well-definedphase, a continuous diffraction pattern called speckles
occurs (Fig. 1(b)). In the case of light microscopy, thiskind of diffraction from an object is converted into areal-space image by lenses. Unfortunately, we do nothave good optical lenses for X-rays. Recently, it
became possible to convert the diffraction into a realspace image by computational calculation, instead oflenses [2]. If the diffraction speckle pattern is recorded
finely enough to satisfy the “oversampling condition,”
which is related to the Shannon sampling theorem [3],the structure can be reconstructed by an “iterativephase retrieval method,” a process by which computersfind a structure to fit the diffraction pattern [2]. That is,
XDM is an emerging technique for “lensless” highresolution X-ray microscopy.
We observed a human chromosome using XDM[1]. Chromosomes are essential organelles for thetransmission of replicated genomic DNA into twodaughter cells during cell division [4]. Although morethan 100 years have passed since chromosomes
were first observed, how a long string of genomic
DNA is packaged into compact chromosomes remains
unclear.
A schematic of XDM measurement of a humanchromosome at beamline BL29XUL is shown inFig. 1(a). X-rays at an energy of 5 keV passed
through a 20-µ m-diameter pinhole to illuminate thetarget chromosome only, which was stuck to a thinsilicon nitride membrane. The coherent diffraction(speckles) from the chromosome was recorded with
an X-ray direct-detection charge-coupled device(CCD) 1.32 m downstream from the sample. A
beamstop before the CCD blocked the unscattereddirect X-ray beam.

From the coherent diffraction pattern (Fig. 1(b)),
a human chromosome image was reconstructed. A
two-dimensional (2D) reconstruction of the humanchromosome is shown in grayscale (Fig. 2(a)) andin color (Fig. 2(b)). Note that the intensities areproportional to the projection of the electron density.
The spatial resolution of the 2D reconstruction is38 nm. The most striking feature in the reconstructionis the high-intensity region near the chromosome
axes with a width of ~200 nm (Fig. 2(b)). The axial
structure has so far been detected only using
immunoelectron microscopy or fluorescencemicroscopy on labeling condensin and topoisomerase
IIα, which are essential proteins for the chromosome
assembly process [4].
Diffrac(a)tion patternBeam stop

SampleCCD

Guard slit
Pinholey-ratXenherCo

Human chromosome(b) Coherent Diffraction Intensity (arb. units)100

10110210310410520qy (μm-1)

100–10–20–20–100qx (μm-1)1020Fig. 1. (a) Schematic of XDM measurementof an unstained human chromosome
at BL29XUL. (b) Coherent diffraction(speckles) from the chromosome.

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For three-dimensional (3D) reconstruction, we
obtained data at different incident angles ranging
from –70° to +70°(38 diffraction data sets). A
reconstructed 3D electron density map of thechromosome is shown in Fig. 2(c). The reconstructedchromosome image also has the highest electron
density around the centromere. This can be explainedby the fact that the centromere region is morecondensed than other chromosomal regions. We
estimate the spatial resolution of the 3D reconstruction
to be 120 nm. This is the first 3D electron density
mapping of an unstained cellular organelle using
XDM.
Since the spatial resolution of microscopy isdetermined by the wavelength, hard X-rays canachieve atomic resolution in principle. With thirdgeneration synchrotron radiation X-rays, the spatial
resolution is often limited by the radiation damage or
the X-ray intensity. Both limitations may be removed
or lowered dramatically with the X-ray free electron
lasers (XFELs) to produce high peak-brilliance coherent
hard X-rays, which is currently being developed [6].
X-ray diffraction resolved the structure of DNA almost
50 years ago [5] and it resolved that of thenucleosome, where DNA is wrapped around histone
proteins, 10 years ago [4]. XFELs will enable adramatic improvement in the spatial resolution of
XDM and would accelerate research for the atomic
structure analysis of chromosomes and other cellularorganelles, including macromolecular complexes [6].

(a) 2D-image
00.5

1(c) 3D-image
00.5

11 μm
00.5

1(b)

500 nm

200 nmFig. 2. A two-dimensional reconstruction of the human chromosome is shownin grayscale (a) and in color (b). Note that the intensities are proportional tothe projection of the electron density. The centromere region is indicated by anarrow in (b). (c) Reconstructed 3D electron density map of the chromosome.
Cross-sectional images of the chromosome at 409 nm intervals are shown.

References[1] Y. Nishino, Y. Takahashi, N. Imamoto, T. Ishikawa andK. Maeshima: Phys. Rev. Lett. 102 (2009) 18101.
[2] J. Miao et al.: Nature 400 (1999) 342.
[3] D. Sayre: Acta Cryst. 5 (1952) 843.
[4] K. Maeshima and M. Eltsov: J. Biochem. 143 (2008)
145.
[5] J.D. Watson and F.H.C. Crick: Nature 171 (1953) 737.
[6] T. Shintake et al.: Nat. Photonics 2 (2008) 555.

Kazuhiro Maeshimaa,* and Yoshinori Nishinob,†a Structural Biology Center, National Institute of Genetics
b SPring-8 / RIKEN*E-mail: kmaeshim@lab.nig.ac.jp† Present address: Research Institute for Electronic Science,

Hokkaido University

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