Effect of the Pulse Width of Flash X-Ray
on the X-Ray Images of Doublet Microtubules
H. Shimizu, T. Majima, T. Tomie, K. Miura, M. Yamada, T. Kanayama
Electrotechnical Laboratory, Umezono 1-1-4, Tsukuba, Ibaraki 305, Japan
E-mail: h.shimiz@etlrips.etl.go.jp
Abstract. Although flash exposure is essential for high resolution imaging,
there have been no experimental reports on the limit of the maximum
exposure time. Here, we present the effects of the pulse width of the flash Xray on the images of doublet microtubules of 40 nm in diameter, which are
subsystems of axoneme in flagella in sperm and other cells. The result
suggests that 0.5 ns exposure time would be sufficient for imaging the
microtubules and 10 ns exposure time would be a little bit longer than the
maximum exposure time for good imaging of 40 nm structure.
1 Introduction
The resolution of optical images would be limited by the shot noise derived from the
number fluctuation of photons that make the image formation. This scheme would
be approved in all X-ray microscopes (XRMs) such as contact, scanning and
imaging XRM. We can distinguish a specific structure of specimen only if the
numbers of the detected photons corresponding the region is apart far from the
numbers corresponding to the surroundings; the difference between the average
photon numbers of the region and the surroundings should be bigger than the
fluctuations of their photon numbers.
Following the above consideration, there are some theoretical reports mentioning
how much dose should be needed for high resolution. One claims that 3*108 Gy[1]
dose is required for 20 nm resolution in Herpes viron and the other estimates 105
Gy[2] dose for 50 nm resolution. Although those values were very different, it is
impossible for biological specimen to survive under the above high dose conditions.
If we are to observe living states in high resolution, flash exposure is inevitable.
Flash exposure of milliseconds duration will be sufficient to avoid the biological
motion. However, much shorter exposure time are estimated when the thermal
expansions following the X-ray absorption are taken into account. A few ps[1] and a
few tens ps[3] are claimed for the resolution of 10 nm and 30 nm, respectively.
There has been no experiment answering the question how short pulse is needed.
2 Specimen and Experimental Set-up
The sperms of barnacle and sea urchin were used for specimen. They have
axonemes, subcellular organs, in their flagella. The axoneme is well known to has
9 + 2 microtubule system. It consists of nine doublet microtubules, two central
microtubules, dynine arms and connecting structures such as radial spokes and nexin
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links. The dynine arms attach to the doublet microtubules. The arms can slide on the
doublet microtubules and generate flagellating force. The size of the doublet
microtubules is 40 nm in diameter.
Although we used the sperm of barnacle in an intact form, we processed the
sperm of sea urchin with detergent (Triton X100) to remove its membrane. The
demembranated sperm can even flagellate in a solution containing suitable amount
of adenosinetriphosphate (ATP)[4]. If the solution contains ATP and digesting
enzyme such as Tripsin, the connecting structures begin to be digested and the
microtubules with dynine arms begin to slide and move away by themselves[5].
Thus we can get the separated microtubules one by one at last. We used two kinds of
specimen; one was the intact sperm of barnacle and the other was the sperm of sea
urchin treated by detergent, ATP and Tripsin.
A drop of the specimen was placed on a silicon wafer spin-coated with
polymethylmethacrylate (PMMA) X-ray resist of 0.5 mm in thickness. The sample
covered by a silicon nitride membrane of 0.1 µm in thickness, was moved into a Xray exposure chamber and exposed to a X-ray pulse from a yttrium plasma
produced by a laser pulse. The schematic diagram of the exposure chamber is shown
in Fig. 1.
Fig. 1. Schematic diagram of the exposure system.
The exposure was performed using two different pumping lasers. One was a
glass laser (0.5 ns in width) and the other was a KrF laser (10 ns in width). The Xray pulses are expected to be 0.5 ns and 10 ns in width, respectively. The X-ray
energy was a few tens mJ/cm2 in the every experiment. Although we exposed the
intact sperm of barnacle using two kinds of lasers, the sperm of sea urchin was
exposed using only the KrF laser. The main contribution to the resist exposure
comes from X-ray of the wavelength band called the water window. A X-ray image
Effect of the Pulse Width of Flash X-Ray on the X-Ray Images
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is obtained as a relief of resist after appropriate development. The relief pattern is
visualized by atomic force microscopy (AFM) measurement [6].
3 Results and Discussions
The axonemes and the nucleus were clearly distinguished in every images, as they
have different relief heights caused by the difference of X-ray absorption ratio
between them.
Fig. 2. X-ray image of sperm of barnacle. Doublet microtubules of 40 nm diameter are clearly
found to be spread out. This image was obtained with use of 0.5 ns laser pulse in width.
Figure 2 shows an image of barnacle sperm obtained with use of 0.5 ns pulse.
Doublet microtubules of 40 nm diameter are clearly found to be spread out in
accidentally damaged specimens, although the diameter of microtubules in the X-ray
image was nearly twice of the real size. From the AFM examination of the
developed resists without specimens, we found that the surface images have the
roughness resulting from the cluster size of resist molecules and the tip radius of
AFM. Both of them are considered to have enlarged the diameter of doublet
microtubules in the visualized image.
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Fig. 3. X-ray image of sperm of barnacle obtained with use of 10 ns laser pulse in width.
X-ray images of barnacle sperm and sea urchin sperm are shown in Fig. 3 and
4, respectively. They were both obtained with use of 10 ns pulse. Although it is hard
to identify the microtubules in Fig. 3 in contrast to Fig. 2, we can see several thin
and curved structures are running separately from the nucleus in Fig. 4. They would
be identified to be images of microtubules.
Fig. 4. X -ray image of sperm of sea urchin. The sperm was treated by detergent, ATP and
Tripsin. Several thin and curved structures are running separately from the nucleus.
The image was obtained with use of 10 ns laser pulse in width.
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These results suggest that 0.5 ns exposure time would be sufficient for imaging
the microtubules and 10 ns exposure time would be near the marginal region for
good imaging. The maximum exposure time for imaging the 40 nm structure would
be exist between 0.5 ns and a few ns. The estimated time is approximately a factor
10 larger than former estimation [3] given theoretically by London et al. The
inconsistency partly comes from the possibility that our images might not have 40
nm resolution, although we could identify the microtubules in the images. We need
more experiments for farther discussions to clarify the maximum exposure time for
good resolution.
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