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Quantitative study of mammalian cells by scanningtransmission soft X-ray microscopy- Application of soft X-ray microscopy toenvironmental microbiology ofhydrosphereK Takemoto, M Yoshimura, T Ohigashi etal.

To cite this article: K Shinohara et al 2017 J. Phys.: Conf. Ser. 849 012003- The history and future of X-ray microscopy
J Kirz and C Jacobsen- Making the most of microscopy

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X-Ray Microscopy Conference 2016 (XRM 2016)
IOP Conf. Series: Journal of Physics: Conf. Series 1234567890
849 (2017) 012003

IOP Publishingdoi:10.1088/1742-6596/849/1/012003

Quantitative study of mammalian cells by scanningtransmission soft X-ray microscopy
K Shinohara1, T Ohigashi2, S Toné3,5, M Kado4 and A Ito1
1

School of Engineering,Tokai Univ, Hiratsuka, Kanagawa 259-1292, JapanUVSOR Synchrotron, Inst Molecular Science, Okazaki, Aichi 444-8585, Japan3
Dept Biochem, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan
4
Kansai Photon Science Institute, QST, Kizugawa, Kyoto 619-0215, Japan
5
Present address: Tokyo Denki Univ, Hatoyama, Saitama 350-0394, Japan
2E-mail: kshino-tky@umin.net
Abstract. Molecular distribution in mammalian cells was studied by soft X-ray scanningtransmission microscopy with respect to the quantitative aspect of analysis. NEXAFS profiles
at the C, N and O K-absorption edges were combined and used for the analysis. For theestimation of quantity for nucleic acids and proteins, NEXAFS profiles of DNA and bovine
serum albumin (BSA) at the N K-absorption edge were applied assuming that those were theirrepresentatives. The method has a potential to explore the other molecular components than
nucleic acids and proteins.

1. Introduction
Scanning transmission X-ray microscope (STXM) has been established and used in a wide area ofscience. In a biological field, distribution of DNA in a bean chromosome [1] and a bull sperm [2] hasbeen imaged by the spectromicroscopy. In addition, quantitative approach has been developed for theanalysis of molecules and elements in biological specimens such as microbial biofilms [3], quinoa [4]and bacterial endospores [5] using STXM. For the quantitative analysis of molecular distribution of
DNA and proteins, near edge X-ray absorption fine structure (NEXAFS) at carbon K-absorption edge
has been applied. We have proposed that NEXAFS at the N K-absorption edge has a merit of clear
separation between DNA and proteins in addition to the exclusion of biomolecules containing no N [6].
2. Materials and methods
Cultured CHO cells at interphase were collected, fixed with ethanol-acetic acid (3:1) solution or
glutaraldehyde (2.5% in 0.075M KCl) and dropped on the commercially available formvar membrane(#10-1008, Okenshoji. Japan). The cells and molecules such as DNA and bovine serum albumin(BSA; protein) were observed with scanning transmission X-ray microscope (STXM) installed at
BL4U, UVSOR-III, Okazaki, Japan. The results were analyzed using the aXis2000 program. For
quantitative analysis, we use NEXAFS spectra of DNA and BSA at the N K-absorption edge, sincenucleic acids and proteins are major components of cells containing nitrogen and were clearly
separated at 1s-π* transition.
Calculation has been made as follows: At the first step, each spectrum of image, DNA or BSA at
the C, N and O K-absorption edges was combined to form one absorption spectrum covering theContent 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 Ltd1X-Ray Microscopy Conference 2016 (XRM 2016)
IOP Conf. Series: Journal of Physics: Conf. Series 1234567890
849 (2017) 012003

IOP Publishingdoi:10.1088/1742-6596/849/1/012003

energy rrange from thhe C K-abso
orption edge to O K-abso
orption edgee. Combined image specttrum wasnamed aas spect1. DNA
Dspectrumm was adjuusted to specct1 so that it fitted the optical denssity (OD)
differencce between ODs
O at 399.6
6 eV and 3988 eV (the peaak and the vaalley in DNA
A NEXAFS) of spect1and nam
med as DNA
Aspect. DNAAspect was suubtracted fro
om spect1 reesulting in a spectrum namednasspect2. B
BSA spectruum was adjussted to spect22 so that it fited
fthe OD difference bbetween ODss at 401.4
eV and 3398 eV (the peak and thhe valley in B
BSA NEXAAFS) of spectt2 and nameed as Prspectt. Prspect
was subttracted fromm spect2 resu
ulting in a sppectrum nam
med as spect3
3. ODs in DN
DNAspect andd Prspect
were connverted to massmabsorptio
on coefficiennt. For compparison of thee profile of eeach spectrum, it wasrescaled so that the difference
doff maximum llevel and minnimum levell was adjusteed to be the samesOD
differencce and set to be the same at the left ennd of the eneergy.
3. Results and Disccussion
3.1. NEXXAFS of DN
NA and BSA
Figure 1 shows the NEXAFS prrofiles of DN
NA andBSA at the C, N annd O K-absorption edgess. Peaks
were refferred to the papers
pfor DN
NA [7] and pproteins[8]. NEXXAFS of DN
NA at the N K-absorptioon edge
shows a peak of 1s tot π* transition of N=C bond at399.6 eVV, which is clearly
cseparrated from a peak of
1s to π* transition ofo amide at 401.4
4eV in BSA. Itshould bbe noted thaat DNA andd RNA incluude thesame typpe of N=C bond,bhence the present aanalysisof DNA
A correspondds to nucleicc acids. In aaddition,
amide iss common inn proteins, soo that the anaalysis ofBSA is ffor proteins.
3.2. Imaages of CHO
O cells
Figure 2 shows the opticalodensitty images at 285 eVand RGB
B maps for interphase ceells fixed witth either
ethanol-aacetic acid oro glutaraldeehyde obtainned withSTXM. The spect3 wasw estimateed from the averagem (spect1) of a whole iimaging
absorptioon spectrum
area by the method shown in seection 2. RGGB maps
abcd

Figure 22. Optical densitydimag
ges (a,b) annd their
RGB maaps (c,d) of CHO cells fixed with eethanolacetic accid (a,c) andd glutaraldeh
hyde (b,d). IIn RGBmaps (c,,d), red, greeen and blue area for nucleeic acid,protein, and spect3, respectively.
rBar, 2 μm.

2Fiigure 1. Masss absorptionn coefficient of DNA(ssolid line) annd BSA (dottted line) at thet C, N
an
nd O K-absorption edgess.

X-Ray Microscopy Conference 2016 (XRM 2016)
IOP Conf. Series: Journal of Physics: Conf. Series 1234567890
849 (2017) 012003

IOP Publishingdoi:10.1088/1742-6596/849/1/012003

were obtained by thhe aXis2000 program wiith the masss absorption coefficient spectra of DNA
DandBSA, annd OD of speect3.
The rresults in figgure 2 show differentdRG
GB maps duee to the differrence in the fixatives of the cells,ethanol-aacetic acid anda glutaralddehyde. In thhe center of the cells (nuucleus) fixedd with glutarraldehyde(figure 22d), color is blueb meaningg that the maain componeent was spectt3 (residual ccomponent otherothan
nucleic aacids and prooteins). The result is not reasonable tot our knowledge that nuucleus mainly
y consistsof DNA
A and proteinns. In contraast, the resullt for the ceells fixed with ethanol-aacetic acid shhows thereasonabble feature. TheT reason foor this discreepancy is attributable to the glutaralddehyde. The observeddata for the cells fixxed with glu
utaraldehydee may not bee sensitive enougheto seeparate the spectra ofDNA annd proteins at the presentt condition. TThe results suggestedsthaat fixatives oof the cells shoulds
beselected for the purpose of the annalysis, thouggh tomograp
phic sliced-im
mage will cleear the probleem.
XAFS of locaal areas in CHO
Ccells3.3. NEX
The NEXXAFS profilles for the loocal areas in CHO cells werew
studiedd as the next step. The arreas werechosen ffor nucleus (1, green), nuucleus containning nucleollus (2, blue) and cytoplassm (3, pink) as shownin figuree 3. It shouldd be noted th
hat the areas for region 1 and 2 in th
he cells fixedd with glutarraldehyde
was not adopted beccause of the reasonrmentiioned above.. Figure 4 shhows the specct1 of each local
larea
a1
200b

33Figure 3. Local areeas in CHO cells
cfixedwith ethanol-acettic acid (a), andglutaralldehyde (b) for the analy
ysis. Partsof 0 (red) for outside thetcells(backgrround), 1 (ggreen) for nucleus,n2(blue) for nucleu
us including
g mainly
nucleollus, and 3 (pink)(for cytoplasm.
cThe sizzes of the arreas were a, 2.25 μm2
each annd b, 0.73μm
m2.
for compparison. In figure 4, each part infigure 3 correspondds to the samme colour
mber, i.e., part1
pfor greeen (solidand num
pect1 of threee local areass (figure 3a parts1-3)
pline), parrt2 for blue (broken(line)), part3 for Figure 4. Sp
and oneinaCHO
cellfixedwithw
ethanol-acetic
acidpink (brooken at longg distance) ofo the cellsarea(figureCHOcellfixe3bpart3;g-part3)
inaC
xedwithfixed wiith ethanol-aacetic acid (ffigure 3a),glutaraldehy
ydeforthespectrasattheeC(top),N(middle)
and g-paart3 for purpple (dotted liine) of thecells fixeed with glutaaraldehyde (ffigure 3b). and O (botttom) K-abssorption edgges. Left hallf showsparison of observed specct1 and righht half is
It is iinteresting too note that th
he profiles direct compof each aarea are quitte similar at the N and for the rescaled spectra.
O K-abssorption edgees (right half in figure4). However, the proofiles of the spectrasfromm different poosition are noot the same aat the C K-abbsorption
edge whhich may be applicableato
o identify thee molecules other than nu
ucleic acids and proteinss, or evenamong tthe nucleic acids
aand prroteins. Leftt half in figu
ure 4 reflectts the compaarison of the density
differencce of the moolecules. Thee density is low at cytop
plasm and high at nucleuus for the ceells fixedwith ethhanol-acetic acid.
aIn conntrast, the deensity in cyto
oplasm (figuure3b, 3) forr the cells fiixed withglutaralddehyde was much
mhigher compared too the cells fix
xed with ethaanol-acetic aacid.

3

X-Ray Microscopy Conference 2016 (XRM 2016)
IOP Conf. Series: Journal of Physics: Conf. Series 1234567890
849 (2017) 012003

IOP Publishingdoi:10.1088/1742-6596/849/1/012003

3.4. NEXXAFS of spect3 for locall areas in CH
HO cells
Figure 5 shows the spect3
sof each local area for comparisson. The spect3 indicatedd the spectruum for themoleculees other thann nucleic aciids and proteeins. Therefoore, it shouldd be useful tto find the molecularmcomponeents in the cells.
cFigure 5 suggests tthat there is not much difference
dat all the locall areas incells for the distributtion of moleccules containning nitrogenn and oxygenn, but carbonn, at which siignificant
differencce in the NE
EXAFS was observed. A sharp decreease in the sp
pectra may bbe caused byy either asmall shhift of the peak
pfor proteins or theepresencee of compoonent(s) which have apeak cloose to the C=O amidee absorptionnpeak.
With the presentt results forr DNAspectt
and Prsppect, the amoount of nucleeic acids andd
proteins at the locaal areas werre estimatedd(Table1)). The ratio ofo nucleic accids/proteins
were alm
most the samme regardless of the locall
areas in the cells fiixed with ethanol-acetic
acid in aaddition to thhe cytoplasm
m of the cells
fixed wiith glutaraldeehyde, thoug
gh molecularr
species should be different, i.e.,
imainlyy
DNA forr nucleus andd RNA for cyytoplasm.
Table1. Ratio of nuucleic acids per
p proteins
estimatted from DN
NAspect and Prspect for
local arreas in CHO cells shown in figure 3.
part123g-partt3

nucleicProteins(PP)acids(N))
N/P
2μμg/cm
6.771
33
3.1 0.20
10..348
8.4 0.21
5..523
3.5 0.23
11..157
7.7 0.19

Figure 5.
5 Spect3 of three locaal areas (figure 3aparts1-3)) in a CHO cell fixed withh ethanol-acetic acidand one area (figure 3b part3; g-ppart3) in a CHO
Ccellfixed wiith glutaraldehyde for thhe spectra ata the C(top), N (middle) and O (boottom) K-ab
bsorptionedges. LeftL
half shhows direct comparisonn of thespectra anda right halff is for the reescaled spectrra.

In conclusion, quuantitative annalysis using combined NEXAFS
N
pro
ofiles at C, N
N, and O K-ab
bsorptionedges shhowed that thhe ratios of nucleic acids per proteins were the samme level in aall the cell reg
gions.
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