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X-Ray Fluorescence and Spectro-Microscopy
for Biomedical Applications
Barry Lai
Experimental Facilities Division, Advanced Photon Source
Supported by Dept. of Energy, Basic Energy Office of Science, Contract No. W-31-109-Eng-38.
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1. X-ray fluorescence microprobe→→ Motivation: Trace metal detection→→ Instrumentation
2. Exogenous Metals→→ Cellular transformation of chromium carcinogen (Cr)→→ Cisplatin and derivatives in cancer cells (Pt)• Microbe-metal interaction in planktonic bacteria & biofilms (Cr)• Transfection of TiO2 nanoparticle-oligonucleotide composites (Ti)• Metal complexes of anti-inflammatory drugs (Cu, Ni, Co)
3. Endogenous Metals→→ Host-parasite interaction in mycobacterial infection (Fe)→→ Cell differentiation mediated by metalloproteins (Zn)• Quantifying trace elements in marine protists (Fe, Cu, Zn)
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2
3 4 5 6 7 8 9 10
11 12 13 14 15 18 17 18
19 20 31 32 33 34 35 3621 22 23 24 25 26 27 28 29 30
37 38 49 50 51 52 53 5439 40 41 42 43 44 45 46 47 48
55 56 81 82 83 84 85 8671 72 73 74 75 76 77 78 79 80
87 88 113 114 115 116 117 118103 104 105 106 107 108 109 110 111 112
61 62 63 64 65 6657 58 59 60 67 68 69 70
91 92 93 94 95 9689 90 97 98 99 100 101 102
H He
Li
Na
Ne
Ar
Kr
Xe
2
3 4 5 6 7 8 9 10
11 12 13 14 15 18 17 18
19 20 31 32 33 34 35 3621 22 23 24 25 26 27 28 29 30
37 38 49 50 51 52 53 5439 40 41 42 43 44 45 46 47 48
55 56 81 82 83 84 85 8671 72 73 74 75 76 77 78 79 80
87 88 113 114 115 116 117 118103 104 105 106 107 108 109 110 111 112
61 62 63 64 65 6657 58 59 60 67 68 69 70
91 92 93 94 95 9689 90 97 98 99 100 101 102
H He
Li
Na
Ne
Ar
Kr
Xe
Rn
F
Cl
Br
I
At
O
S
Se
Te
Po
N
P
As
Sb
Bi
C
Ge
Sn
Pb
B
Al
Ga
In
Tl
Zn
Cd
Hg
Cu
Ag
Ag
Ni
Pd
Pt
Co
Rh
Ir
Mt
Fe
Ru
Os
Hs
Mn
Tc
Re
Bh
Cr
Mo
W
Sg
V
Nb
Ta
Db
Ti
Zr
Hf
Rf
Sc
Y
Lu
Lr
Be
Mg
Sr
Ba
Ra
Yb
No
Tm
Md
Er
Fm
Ho
Es
Dy
Cf
Tb
Bk
Gd
Cm
Eu
Am
Sm
Pu
Pm
Np
Nd
U
Pr
Pa
Ce
Th
La
Ac
Si
Fluorescence Mapping
Fluorescence Mapping and Spectroscopywith hard x-rays excitation (Einc = 5 – 30 keV)
K excitation
L excitation
Fluorescence Spectroscopy
D. Legnini, Oct/01
CaK
Constituents of Earth’ crustElements intrinsic to biological systemsElements with Biological activity
Rb
Cs
Fr
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Why study metal concentrations in biol. cells?
• Some metals are integral components of numerous classes of proteins. These proteins often have regulatory functions– Ca in Calcium-binding proteins: second messenger pathways, e.g.
Troponin C in muscle– Zn in Zinc finger proteins:
transcription factors • By studying the metal
concentrations conclusions can be drawn about regulatory functions of these proteins. Zn
left: zinc finger motif from yeast transcription factor SWI5 [from Encylopedia of Molecular Biology]
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Why use x-ray-excited fluorescence to study trace metals?
• Higher fluorescence cross sections• Better signal/background ratio⇒ sub-ppm (part-per-million) sensitivity⇒ quantitative
• Less radiation damage• Better resolution for sample >1 µm thick• Selectively excite one particular element• Map chemical states by XANES• SIMPLE SAMPLE PREPARATION !!⇒ no fluorescence markers needed⇒ no staining nor thinning⇒ can study hydrated “natural” samples
* C.J. Sparks, Jr., in Synchrotron Radiation Research(Plenum Press) 1980.
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Detection Limit for Transition Elementsfor 1 sec. acquisition time, 0.2 x 0.2 µm2 spot, E=10 keV
Attogram
(10-18gm
)
10 4
10 5
10 6
2 4 6 8 10
10
100D
etec
tion
Lim
it (a
tom
s)
Fluorescence Energy (keV)
Zn
Fe
Ti
K
Cu
Co
Mn
V
Ca
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Schematic of Scanning X-Ray Microprobe
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Performance of 2-ID-D X-Ray Microprobe
145
µm
0.9 µm
0.1 µm
0
1000
2000
3000
4000
5000
6000
7000
-0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3
Inte
nsity
(a.u
.)
Y (µm)
FWHM spot size: 1st order = 150 nm 3rd order < 90 nm
0.4
Zone Plate Resolution Test
Spatial Resolution = 150 nm FWHM
Efficiency = 20-25% (with two Au ZPs)
Flux density = 2 x 1011 photons/sec/µm2/0.01%BW
Flux density gain = 3 x 104
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Hard X-ray Microprobe Facility, APS sector 2
beam splitting crystal
first mirror
"white" SR beamStorage ring with ID
pink beam
Energy DispersiveDetector
mono beam
double crystal monochromator
zone plate
sample chamber
slitsOSA
spectraelemental mapelemental map spectra electronics
Main branch 2-ID-D: E = 5 – 30 keV, δ = 150 nm ↔ 2·109 phot/s
Side branch 2-ID-E: E = 7 – 15 keV, δ = 250 nm ↔ 5·108 phot/s
Integrated epi-fluorescence microscope
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2-ID-D/E Hard X-ray Microprobe Facility
2-ID-D
2-ID-EEpifluorescenceMicroscope
sample
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Cellular Uptake and Cellular Metabolism of Chromium Carcinogens
C.T. Dillon, P.A. Lay, B.J. KennedyUniversity of Sydney
M. CholewaUniversity of Melbourne
G. Shea-McCarthyBrookhaven National Laboratory
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Chromium Carcinogenesis• Cr(VI) is a known
carcinogen causing cancers in the respiratory tract.
• It is predominantly encountered in the workplace.
• Recent attention has focused on environmental exposure to Cr(VI), resulting from the poor disposal practices of Cr(VI) into unlined ponds.
ErinBrockovich
others26%
nickel20%
PAH4%
chromium25%
arsenic3%benzene
4%
cadmium3%
Commonly Encountered Carcinogens in the Workplace
asbestos15%
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Preparation of Whole Cell Samples• Cells were treated for
4 hr in cell growth medium.
• The medium was removed and the cells were washed to remove extracellular material.
• The cells were harvested, deposited on a formvarsupport in ammonium acetate solution and freeze-dried using liquid nitrogen cooled isopentane.
Distribution of V79 cells on a 3 mm diameter gold grid
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Preparation of Thin Sectioned Cells
• Cells are treated and harvested as for whole cell preparation.
• Cells are centrifuged to a pellet, washed, dehydrated, set in Spurr’s resin, and microtomed to 1-µm thin sections
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Effect of Oxidation State on Cr Genotoxicity
0 2 4 6 8 100
40
80
120In
cide
nce
of M
N/1
000
BN c
ells
[Cr] (µmol/dish)
Cr(VI) Cr(V) phen Cr(III) phen
Dillon, C. T.; et al. Chem. Res. Toxicol. 1998, 11, 119-129; Dillon, C. T.; et al. Chem. Res. Toxicol. 2000, 13, 742-748.
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Cr Uptake Analysis of V79 Whole Cells
Cr(III)-Treated CellP K Cr Zn
Cr(VI)-Treated CellP K Cr Zn
30 µm
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control Cr(III)phen Cr(V)phen Cr(VI)0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Rel
ativ
e Pe
ak A
rea
Cell Treatment
Micro-SRIXE and GFAAS Determination of Cr Uptake into V79 Cells
GFAAS of 106 cellsMicro-SRIXE of individual cells, n=5
Control Cr(III)phen Cr(V)phen Cr(VI)0
50
100
150
200
250
300
350
Cr C
onte
nt
Cell Treatment
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K-Edge XANES Spectra of Cr Standards
10.0 10.5 11.0 11.5 12.0
[CrO(mampa)]−Rel
ativ
e In
tens
ity
Monochromator Distance (mm)10.0 10.5 11.0 11.5 12.0
[CrO(ehba)2]−
10.0 10.5 11.0 11.5 12.0
Cr(VI)
10.0 10.5 11.0 11.5 12.0
[Cr(glygly)2]−
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K-edge XAS Spectra of Cr-Treated Cells
* Dillon, C. T., et al. Chem. Res. Toxicol. 1997, 10, 533-535.
Cr(VI)
Inte
nsity
(Arb
itrar
y U
nits
)
Monochromator Distance (mm)
[CrO(mampa)]− [CrO(ehba)2]
−
10.0 10.4 10.8 11.2 11.6
Cr(III) standard
Cr Treated Cells
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Thin-Section of V79 Hamster Lung Cell Following Exposure to Cr(VI)
P CrK Cu Zn
NucleolusMin Max
Optical Micrograph Nucleus
Scan dimensions = 11 × 11 µm; Beam size = 300 nm diameter.
Dillon, C.T. et al., J. Biol. Inorg. Chem., 2002, 7, 640-645.
.
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Cellular Metabolism of Chromium
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May 14-15, 2001
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X-Ray Fluorescence Microscopy
Pro: • No fluorescence dyes or markers are needed• Minimal sample prep, allow hydrated/in situ studies• Parallel acquisition of ~ 10 elemental images• Quantification to ppm level for most metals• 200 nm spatial resolution (≈ optical, better than EM for thick specimens)• Substantially less radiation damage than EM• Reveal oxidation state by micro-XANES
Con: • Long integration time (1-2 hrs)• Non-specific to binding partners
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Future Enhancements
• Install multilayers monochromator – gain of 20x in flux – wider energy bandwidth
• Design a second generation fluorescence microprobe– higher resolution scanning stages – monitor ZP and sample position with interferometer – increase stiffness, reduce thermal drift
• Option to run in vacuum– facilitate detection of low-Z elements (Na, Mg, P, S…)– reduce scattering from air or helium– increase acoustic and thermal isolation
• Implement plunge freeze and vacuum dried (no fixation)• Develop a cryo sample holder (frozen hydrated samples)• Future, full-field imaging fluorescence microscope….
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Contributors
MicroprobeZ. Cai, J. Maser, W. Yun, D. Legnini, S. Vogt, P. Ilinski, A. Stampfl, W. Rodrigues
BeamlineI. McNulty, S. Xu, C. Roehrig, E. Trakhtenberg, D. Shu, A. Khounsary, J. Barraza, B. Tieman, G. Wiemerslage
Zone Plate OpticsA. Krasnoperova, Z. Chen, Y. Vladimirsky, F. Cerrina (UW-Madison)E. Di Fabrizio, M. Gentili (Inst. of Solid State Technology, Italy)R. Divan, D. Mancini (XFD/APS)