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Application of soft X-ray microscopy toenvironmental microbiology of hydrosphere
To cite this article: K Takemoto et al 2017 J. Phys.: Conf. Ser. 849 012010- Quantitative study of mammalian cells by
scanning transmission soft X-ray
microscopy
K Shinohara, T Ohigashi, S Toné et al.
- EVIDENCE FOR THE PRISTINEPRESENCE OF A LUNARHYDROSPHERE
J. J. Gilvarry- The history and future of X-ray microscopy
J Kirz and C Jacobsen

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X-Ray Microscopy Conference 2016 (XRM 2016)
IOP Conf. Series: Journal of Physics: Conf. Series 1234567890
849 (2017) 012010

IOP Publishingdoi:10.1088/1742-6596/849/1/012010

Application of soft X-ray microscopy to environmental
microbiology of hydrosphereK Takemoto1, M Yoshimura2, T Ohigashi3, Y Inagaki3, H Namba2 andH Kihara2, 4
1

Kansai Medical University, 2-5-1 Shin-machi, Hirakata, JapanThe SR Center, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Japan
3
Institute for Molecular Science, 38 Nishigonaka, Myodaiji, Okazaki, Japan
4
Himeji-Hinomoto College, 890 Koro, Kodera-cho, Himeji, Japan
E-mail: takemoto@hirakata.kmu.ac.jp
2

Abstract. Microstructures of unprocessed filamentous cyanobacterium, Pseudanabaena foetidasp., producing a musty smell were observed using soft X-ray microscopy. Carbon-enriched
structures and granules as well as oxygen-enriched granules which have been already reported
were observed. Except for early log growth phase, the oxygen-enriched granules were observed.
However, the carbon-enriched structures were observed throughout log growth phase. The result
suggests there is a relationship between the oxygen-enriched granules and 2-methylisoborneol(2-MIB) productivity, since the 2-MIB productivity of each cell is increased depending on theculture period in log growth phase.

1. Introduction
In environmental microbiology of hydrosphere, the needs for observation of microbes and the mucous
matter are high.
Lake Biwa is the most important water reservoir in Kyoto-Osaka-Kobe region in Japan. The higheconomic growth in the 1960s caused severe environmental problems in the lake such as eutrophication,
losses in biodiversity, degradation of ecosystems. Although the water quality was gradually improvedby cooperation with other stakeholders such as governments, local governments, citizens, the biota
began to exhibit correspondingly drastic negative changes.
In 1969, a problem of musty smell in tap water has occurred due to sudden propagation of a certain
green filamentous cyanobacterium. The filamentous cyanobacterium was reported as Phormidium tenue(Menegh.) Gomont and 2-methylisoborneol (2-MIB) was identified as a causative substance [1].
However, results of our soft X-ray microscopy (XM) observation suggested the necessity ofreidentifying of it [2, 3]. In 2016, as a result of morphological and genetical analysis, the cyanobacterium
was proposed as a new planktonic species producing 2–MIB: Pseudanabaena foetida Niiyama, Tuji etIchise sp. nov. [4]. Now, the study how the microstructure and culturing conditions are related to the 2MIB productivity is proceeding. The aim of this study is to elucidate the microstructure related to the 2MIB productivity of P. foetida sp. using the XM.
2. Materials and methods
2.1. Phytoplankton Material
Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distributionof this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd
1

X-Ray Microscopy Conference 2016 (XRM 2016)
IOP Conf. Series: Journal of Physics: Conf. Series 1234567890
849 (2017) 012010

IOP Publishingdoi:10.1088/1742-6596/849/1/012010

P. foetida sp. was collected from Lake Biwa and subcultured. The culture was maintained on CT
medium (pH 8.0) under a 12 h light/12 h dark cycle at 20 °C. The cultures were illuminated withfluorescent lamps, which provided about 85 μmol photons m-2 s-1 of photosynthetically active radiation.
2.2. X-ray microscopy
Phytoplankton was observed by soft XM using the BL-12 microscope at the SR Center of Ritsumeikan
University [5, 6]. The microscope was equipped with Fresnel zone plate condenser and objective with
53.7 nm and 36 nm outer zone widths respectively. The images were recorded using an X-ray CCD
camera (C4880-21-24WD: Hamamatsu Photonics K.K.). The achievable spatial resolution wasapproximately 70 nm based on the knife-edge estimation (20 - 80%) at 517 eV (see e.g. [7]).
Projection images were collected using exposure times ranging from 60 to 120 s. Background images
were also collected and each image was divided by the background image. Cryogenic imaging is allowedby using the indirect contact cryogenic system which enables cooling to around -160 °C without frost[8].
2.3. Sample preparation and observation
For 2D imaging, the cells suspension dropped onto a 100 nm thickness silicon nitride membrane window(Silson Ltd.). After air-dried, the samples were observed by XM at 620 eV and 532 eV at room
temperature.
For 3D imaging, the cell suspension was injected into a glass capillary tube with a fine tip that was
created by using a puller, PC-10 (Narishige). For acquisition of whole data set, 75 X-ray projection
images were acquired with rotating the sample 4 degree each (180 degree rotation). I0 images wereacquired every 5 projections to compensate decay of beam current. The cross sectional images werereconstructed with convolution back projection algorithm. The reconstructed volumetric data wasvisualized using VGStudio MAX 2.0 (Volume Graphics GmbH). The spatial resolution of the crosssectional image was approximately 160 nm based on the edge response (20 - 80%) of the glass capillary
at 517 eV.
3. Results and discussion
Figure 1 shows XM images of air-dried P. foetida. Although filamentous bacteria are often surroundedwith sheaths, P. foetida cells were not surrounded with sheaths. Any metabolite-like agar or gelatin were
not also observed. Several granules were observed in the cells 4 weeks after inoculation, log growthphase (Fig.1 a). As reported previously, the granules disappear at 532 eV. This result indicates that the
granules are oxygen-enriched structures such as polyphosphate granules. A cell 1 week after inoculation,
early log growth phase, showed interesting structures which have been not observed under previous XM
observation (Fig.1 b). In the middle of the cell, a boundary line is seen. From the boundary line, thestructures extend to both ends of a cell. The structures are almost linearly symmetrical with the boundaryline. It is noteworthy that the structures appear not only in 620 eV image but also in 532 eV image. This
result indicates that a dividing cell has been observed and the structures are carbon-enriched structures
such as nucleoid or prokaryotic cytoskeleton [9].

a 620 eV532 eVb 620 eV532 eV

Figure 1. XM images of air-dried P. foetida. Images of cells 4 weeks after inoculation, log growthphase, were taken at 620 eV and at 532 eV (a). Images of a cell 1 week after inoculation, early log
growth phase, were taken at 620 eV and at 532 eV (b). Exposure time was 120 s. Scale bar is 2 µm.

2X-Ray Microscopy Conference 2016 (XRM 2016)
IOP Conf. Series: Journal of Physics: Conf. Series 1234567890
849 (2017) 012010

IOP Publishingdoi:10.1088/1742-6596/849/1/012010

The carbon-enriched structures were observed throughout log growth phase. However, few oxygenenriched granules were observed in early log growth phase. In log growth phase, 2-MIB productivity ofeach cell is increased depending on the period of its culture. The result suggests that the oxygen-enriched
structures such as polyphosphate granule relate to 2-MIB productivity and the carbon-enriched
structures such as nucleoid or prokaryotic cytoskeleton are not related to it.
To verify the oxygen-enriched structures, distribution of oxygen and oxygen K-edge XANES spectra
were collected by the scanning transmission X-ray microscope (STXM) at UVSOR BL4U [10, 11].
Figure 2 shows STXM images and a distribution of oxygen in P. foetida grown to early stationary phase.
Four oxygen-accumulated granules consisting of large and small sizes were observed. Althoughabsorption spectra were noisy, a spectrum from the granule obviously exhibited a pre-peak which isdifferent from that of organic carbonyl group (C=O) at about 532 eV. To identify the granules, we intend
to promote a more detailed analysis.

530 eV538 eV

OxygenFigure 2. STXM images and oxygen K-edge XANES spectra. STXM images and distribution ofoxygen in the air-dried P. foetida cells grown to log growth phase. Scale bar is 1 µm. Oxygen K-edge
XANES spectra of cytoplasm (black line) and granule (grey line).
Figure 3 shows 3D reconstructed tomograms and 2D slice images of P. foetida cells grown to late
log growth phase. Many rod-shaped bacteria without sheath were observed. In the cells, heterogeneous
X-ray absorption was seen, whereas carbon-enriched granules were not seen. These results are the samethat in air-dried 2D observation at 532 eV (Fig.1 a). However, P. foetida cells grown to death phase
contained several carbon-enriched granules (Fig.4). The cells shrank because of radiation damage during
repeated X-ray exposure. However three different granules were inside different cells. Although thisresult indicates that the granules are carbon-enriched structures, candidate prokaryotic organelles
corresponding to the granules have not been assigned yet.
Side view

Bottom view

White arrow

Black arrow

Figure 3. Cryo-XM images of P. foetida grown to late log growth phase in a hydrated state at -110 °C.
Three-dimensional rendering images (side and bottom views) and longitudinal section images of cells(white and black arrows). Images were taken at 532 eV and exposure time was 60 s.
4. Conclusions
To investigate how the microstructure is related to the 2-MIB productivity, unprocessed P. foetida cells
were observed by using soft XM. The P. foetida cells were not surrounded with sheath. Carbon-enriched3

X-Ray Microscopy Conference 2016 (XRM 2016)
IOP Conf. Series: Journal of Physics: Conf. Series 1234567890
849 (2017) 012010

IOP Publishingdoi:10.1088/1742-6596/849/1/012010

structures such as nucleoid or prokaryotic cytoskeleton were observed throughout log growth phasealthough few oxygen-enriched granules such as polyphosphate granule were observed in early log
growth phase. In addition, carbon-enriched granules were observed in cells grown to death phasealthough any candidate prokaryotic organelles corresponding to the granules have not been assigned yet.
During log growth phase, 2-MIB productivity of each cell is increased depending on the period of its
culture. The result suggests that the oxygen-enriched structures relate to 2-MIB productivity and thecarbon-enriched structures do not relate to it.
0°

79.2°

120°

3 µm

Figure 4. XM images of P. foetida grown to death phase in a hydrated state at room temperature.
Image was taken at 517 eV and each exposure time was 120 s. Three different granules (black, whiteand yellow arrows) were inside different cells.
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Proc. 8th Int. Conf. on X-ray Microscopy 7 82-84[8] Yoshimura M, Ishii T, Murakami S, Takemoto K, Kihara H , Namba H and Ohta T 2015 Mem.
SR Center Ritsumeikan Univ. 17 149–151[9] Shih YL, Rothfield L 2006 Microbiol. Mol. Biol. Rev. 70 729–54[10] Ohigashi T, Arai H, Araki T, Kondo N, Shigemasa E, Ito A, Kosugi N and Katoh M 2013 J. Phys.
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Acknowledgments
Samples were provided by Dr. Ichise (Lake Biwa Environmental Research Institute). The soft X-ray
imaging work was performed at the SR Center of Ritsumeikan University and the STXM imaging work
was performed at UVSOR under the MEXT Project for Creation of Research Platforms and Sharing ofAdvanced Research Infrastructure. A part of this study was supported by JSPS KAKENHI Grant
Numbers 24510039 and 15K00572, and the Strategic Research Foundation at Private Universities(S1101034).

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