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.

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)

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

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]

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.

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

Schematic of Scanning X-Ray Microprobe

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

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

2-ID-D/E Hard X-ray Microprobe Facility

2-ID-D

2-ID-EEpifluorescenceMicroscope

sample

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

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%

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

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

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.

Cr Uptake Analysis of V79 Whole Cells

Cr(III)-Treated CellP K Cr Zn

Cr(VI)-Treated CellP K Cr Zn

30 µm

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

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]−

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

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.

.

Cellular Metabolism of Chromium

May 14-15, 2001

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

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….

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)