X-Ray Fluorescence Microscopy:
Powerful Biomaterials
Characterization Tool
1
X-Ray Synchrotron
• Giant x-ray ring located at
Brookhaven National
Laboratory in Long Island
New York.
• Utilized soft x-ray
microscopy to visualize
chemical groups in paper.
• ESCA (XPS) with pictures.
2
The Microscope
• Scanning Transmission Xray Microscope (STXM)
• Operates between the K
edges carbon and oxygen
with good penetration in
samples slightly less than
1μm, therefore well suited
for the study of specimens
like single biological cells.
• Can operate under
standard conditions or cryo
conditions.
3
The Microscope (2)
• Soft x-ray microscopy
uses X rays with an
energy of 100-1000 eV,
or a wavelength of
about 1-10 nm. X-ray
energy (eV).
• 30nm resolution
• Only about 1 dozen
sychrotron STXMs
available worldwide.
4
Biological imaging
• Consider
penetration
distance: 1/e
absorption length
for x rays,
scattering mean
free paths for
electrons
• Water window:
Wolter, Ann. Phys.
10, 94 (1952)
5
X rays
Electrons
• Absorption dominates
• Inelastic scattering is weak
• No multiple scattering
• Inelastic scattering dominates
(energy filters)
• Multiple scattering often
present
• High contrast from small things
6
Thick samples:
photons come out ahead
X-rays: better for thicker specimens. Sayre et al.,
Science 196, 1339 (1977) Schmahl & Rudolph (1990)
These plots: Jacobsen, Medenwaldt, and Williams, in X-ray
Microscopy & Spectromicroscopy (Springer, 1998)
7
Phase contrast: even thicker!
8
Fibroblast reconstruction: Z slices
Frozen-hydrated
(ethane plunge)
3T3 fibroblast:
Y. Wang et al., J.
Microscopy 197,
80 (2000)
9
Analyzing full fluorescence spectra
• Peaks from trace elements
can be on shoulders of
strong peaks from common
elements.
• Setting simple energy
windows can give poor
quantitation. Record full
spectrum and do curvefitting!
• Wavelength dispersive
detectors can help – but
often with lower collection
solid angle
• This example: Twining et al.,
Anal. Chem. 75, 3806
(2003). Also ESRF,
elsewhere.
10
11
X-ray focusing: Fresnel zone plates
• Diffractive optics:
radially varied grating
spacing
Diameter d, outermost zone width
drN, focal length f, wavelength :
d drN  f 
• Largest diffraction angle
is given by outermost
(finest) zone width drN as
=/(2drN)
• Rayleigh resolution is 0.61
/()=1.22drN
• Zones must be positioned
to ~1/3 width over
diameter (10 nm in 100
m, or 1:104)
Central stop and order
sorting aperture (OSA)
to isolate first order
focus
12
Fresnel zone plate images
R. W. Wood (1898): zone
plate figure drawn with a
pen and a compass!
Photographically reduced
13
Zone plates by electron beam lithography
•
Electron beam lithography: produces the finest possible structures
(other than what nature can be persuaded to make by itself). Many
efforts worldwide!
•
M. Lu, A. Stein (PhD 2002; now BNL), S. Spector (PhD 1998; now Lincoln
Lab), C. Jacobsen (Stony Brook)
•
D. Tennant (Lucent/New Jersey Nanotech Consortium)
•
JEOL JBX-9300FS: 1 nA into 4 nm spot, 1.2 nm over 500 m, 100 keV
A. Stein and JBX-9300FS
14
Zone plate microscopes
TXM
STXM
• Incoherent illumination;
works well with a bending
magnet; exposure time of
seconds
• Coherent illumination; works best
with an undulator; exposure time
seconds to minutes
• More pixels (e.g.,
20482)
• Moderate spectral
resolution in most cases:
E/(E)300-1000
• Less dose to sample (~10%
efficient ZP)
• Better suited to conventional
grating monochromator:
E/(E)3000-5000
15
Soft x-ray imaging
NIL 8 fibroblast
(glutaraldehyde fixed):
V. Oehler, J. Fu, C.
Jacobsen
Human sperm (unfixed):
S. Wirick, C. Jacobsen,
Y. Shenkin
Test pattern: see Jacobsen et al., Opt. Comm. 86, 351 (1991)
16
Immunogold labeling
•
H. Chapman, C. Jacobsen, and
S. Williams, Ultramicroscopy
62, 191 (1996).
•
Fibroblast, antibody labeled
for tubulin.
•
More recent work:
– C. Larabell et al., LBL/UCSF
– S. Vogt et al., then at
Göttingen
•
Labels must be comparable in
size to optical resolution.
Vogt and Jacobsen,
Ultramicroscopy 87, 25
(2001)
•
Challenge: how to label
without altering cell?
17
Absorption edges
Lambert-Beer law: linear absorption
coefficient µ
This coefficient makes a jump at
specific elemental absorption edges!
This example: 0.1 µm protein, silica
I  I 0 exp  E   t   I 0 exp[  DE ]
18
X-ray microscopy of colloids
• U. Neuhäusler (Stony Brook/Göttingen), S. Abend (Kiel), G. Lagaly
(Kiel), C. Jacobsen (Stony Brook), Colloid and Polymer Science 277,
719 (1999)
• Emulsion: water, oil droplets, clay, and layered double hydroxides
(LDH)
• “Caged” part of oil droplet remains fixed; “uncaged” part can
disperse
346 eV: calcium
weakly absorbing.
Clays and LDHs
absorb equally
352.3 eV: calcium
strongly absorbing.
Calcium-rich LDHs
are highlighted.
290 eV: carbon
284 eV: carbon
strongly absorbing (oil drop) weakly
absorbing
19
Near-edge absorption fine structure (NEXAFS) or
X-ray absorption near-edge structure (XANES)
• Fine-tuning of the x-ray energy near an atom’s edge gives
sensitivity to the chemical bonding state of atoms of that type
• First use in microscopy: Ade et al., Science 258, 972 (1992)
20
C-XANES of amino acids
• K. Kaznacheyev et al., J. Phys. Chem. A 106, 3153 (2002)
• Experiment: K. Kaznacheyev et al., Stony Brook (now CLS)
• Theory: O. Plashkevych, H. Ågren et al., KTH Stockholm; A.
Hitchcock, McMaster
21
Spectromicroscopy by image stacks
• Acquire sequence of images over XANES spectral region;
automatically align using Fourier cross-correlations or laser
interferometer; extract spectra.
• C. Jacobsen et al., J. Microscopy 197, 173 (2000).
Images at N=150
energies are
common.
22
DNA packing in sperm
• X. Zhang, R. Balhorn, J.
Mazrimas, and J. Kirz, J.
Structural Biology 116, 335
(1996)
• DNA packing in sperm
mediated by protamine I and
protamine II; fraction of
protamine II can vary from
0% to 67% among several
species
• Bulk measurements:
compromised by immature or
arrested spermatids
• Images at six XANES
resonance energies for each
specimen
23
“Sperm morphology, motility, and concentration
in fertile and infertile men”
Guzick et al., New England Journal of Medicine 345, 1388 (2001)
“Although semen analysis is routinely used to evaluate the male partner
in infertile couples, sperm measurements that discriminate between
fertile and infertile men are not well defined… Threshold values for
sperm concentration, motility, and morphology can be used to classify
men as subfertile, of indeterminate fertility, or fertile. None of the
measures, however, are diagnostic of infertility.”
24
What are the predictors of fertility?
• Use chemical state mapping
of x-ray microscopy to
investigate sperm types from
different patients (Holger
Fleckenstein, Physics; Dr.
Yefim Sheynkin, Dept.
Urology)
• Preparation: compare room
temp wet (at right), frozen
hydrated, freeze-dried
• One in-vitro fertilization
method: single sperm are
selected for injection into
egg. What’s the basis for
choosing one sperm over
another?
25
Cluster analysis of sperm
Airdried specien; 140 separate iages
26
Comparison with mitochondrial DNA spectrum
Mitochondrial DNA spectrum: K. Kaznacheyev
Mitochondria
NA
Purple region
Purple region: DNA packed with protamines
27
N, O edges
28
Radiation damage on
(initially) living cells
• X-rays are ionizing
radiation. The dose
per high resolution
image is about 100,000
times what is required
to kill a person
Experiment by V.
Oehler, J. Fu, S.
Williams, and C.
Jacobsen, Stony
Brook using specimen
holder developed by
Jerry Pine and John
Gilbert, CalTech
• Makes it hard to view
living cells!
29
Wet, fixed samples: one image is OK
• Chromosomes are
among the most
sensitive specimens.
• V. faba chromosomes
fixed in 2%
glutaraldehyde. S.
Williams et al., J.
Microscopy 170, 155
(1993)
• Repeated imaging of
one chromosome shows
mass loss, shrinkage
30
Frozen hydrated specimens
Grids with live cells are
• Taken from culture medium
and blotted
• Plunged into liquid ethane
(cooled by liquid nitrogen)
• Loaded into cryo holder
31
Radiation damage resistance
in cryo
Left: frozen
hydrated image
after exposing
several regions
to ~1010 Gray
Maser et al., J.
Microscopy 197,
68 (2000)
Right: after
warmup in
microscope
(eventually
freeze-dried):
holes indicate
irradiated
regions!
32
Lignocellulosics
• Radicals are formed by
the interaction of
peroxide and metal that
can damage cellulose
H2O2 Mg
• Damage results in
carboxylic acid groups
• Visualize the damage
physical and chemical
testing show
H2O2 Mg
33
Sample Prep
•
Peroxide bleached and unbleached
handsheets
•
Cut ~1cm by 2cm samples
•
Soaked in water
•
Dehydrated in ethanol
•
Used 50/50 mixture of epoxy resin
(Epon 812) and propylene oxide
•
100% epoxy and vacuum
•
Cured in oven between plastic sheets
•
Sectioned to 200nm thick (transverse)
and placed on TEM grids
34
Locating Carboxylic Acids
(unbleached)
285
TEM grid hole = ~125 μm
289
35
Locating Carboxylic Acids (2)
(bleached)
285
TEM grid hole = ~125 μm
289
36
Locating Carboxylic Acids (3)
(bleached)
285
TEM grid hole = ~125 μm
289
37
High Resolution
(bleached)
• Stepper scan (.5 μm)
vs. piezo scan (30
nm).
• High resolution
images of damaged
regions.
285
289
285
289
• Perhaps evidence of
hollow center.
Top = 20 μm
Bottom = 8 μm
38
High Resolution (2)
(unbleached)
Left = 72 μm
Right = ~36 μm
39
Conclusions
• Resolution is 20-40 nm now; pushing towards 10 nm…
• Tomography lets you look at whole cells up to 10 µm
thick (thicker at higher energies?).
• Radiation damage is less than with electrons, but is
still a consideration
• STXM is a viable tool for the investigation of paper
chemistry.
• Peroxide bleached samples undergo a heterogeneous
enrichment of carboxylic acid groups due to radical
damage.
• Results confirm trends previously seen in TOF-SIMS
as well as other physical and chemical testing.
40
Acknowledgements
• Chris Jacobsen and Janos Kirz (BNL)
• Doug Mancosky (Hydro Dynamics)
• Alan Rudie (Forest Products
Laboratory)
• Hiroki Nanko (Georgia Institute of
Technology)
41