Lecture 14.
X-Ray Fluorescence Microscopy
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.
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.
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.
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)
X rays
• Absorption dominates
• Inelastic scattering is weak
• No multiple scattering
Electrons
• Inelastic scattering dominates
(energy filters)
• Multiple scattering often
present
• High contrast from small things
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)
Phase contrast: even thicker!
Fibroblast reconstruction: Z
slices
Frozen-hydrated
(ethane plunge)
3T3 fibroblast:
Y. Wang et al., J.
Microscopy 197,
80 (2000)
•
Analyzing full fluorescence
Peaks from trace elements
spectra
can be on shoulders of strong
peaks from common
elements.
• Setting simple energy
windows can give poor
quantitation. Record full
spectrum and do curve-fitting!
• 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.
•
X-ray focusing: Fresnel zone
Diameter d, outermost zone width
plates
Diffractive optics: radially
dr , focal length f, wavelength :
varied grating spacing
• 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)
N
d drN  f 
Centra stop and order
sorting aperture (OSA)
to isoate first order
focus
Fresnel zone plate images
R. W. Wood (1898): zone
plate figure drawn with a
pen and a compass!
Photographically reduced
•
•
•
•
Zone plates by electron beam
Electron beam lithography: produces the finest possible structures (other than
lithography
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
Zone plate microscopes
STXM
TXM
• Incoherent illumination; works
well with a bending magnet;
exposure time of seconds
• More pixels (e.g., 20482)
• Moderate spectral resolution
in most cases: E/(E)3001000
• Coherent illumination; works best
with an undulator; exposure time
seconds to minutes
• Less dose to sample (~10%
efficient ZP)
• Better suited to conventional grating
monochromator: E/(E)3000-5000
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)
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?
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  I0 exp- (E ) t   I0 exp[ -D(E )]
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
•
Near-edge absorption fine structure
(NEXAFS) or
X-ray absorption near-edge structure
Fine-tuning of the x-ray energy
near an atom’s edge gives sensitivity
(XANES)
to the chemical bonding state of atoms of that type
• First use in microscopy: Ade et al., Science 258, 972 (1992)
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
•
Spectromicroscopy by image
Acquire sequence of images
over XANES spectral region;
stacks
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.
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
“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.”
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?
Cluster analysis of sperm
Air-dried specien; 140 separate iages
Comparison with mitochondrial DNA
spectrum
Mitochondrial DNA spectrum: K. Kaznacheyev
Mitochondria
NA
Purpe region
Purple region: DNA packed with protamines
N, O edges
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
• Makes it hard to view
living cells!
Experient by V.
Oeher, J. Fu, S.
Wiias, and C.
Jacobsen, Stony
Brook using specien
hoder deveoped by
Jerry Pine and John
Gibert, CaTech
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
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
Radiation damage
resistance in cryo
Left: frozen
hydrated iage
after exposing
severa
regions to ~1010
Gray
Right: after
warup in
icroscope
(eventuay
freeze-dried):
hoes indicate
irradiated
regions!
Maser et a., J.
Microscopy 197,
68 (2000)
Lignocellulosics
• Radicals are formed by
the interaction of
peroxide and metal that
can damage cellulose
• Damage results in
carboxylic acid groups
• Visualize the damage
physical and chemical
testing show
H2O2 Mg
H2O2 Mg
•
•
•
•
•
•
•
•
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
Locating Carboxylic Acids
(unbleached)
285
TEM grid hole = ~125 μm
289
Locating Carboxylic Acids (2)
(bleached)
285
TEM grid hole = ~125 μm
289
Locating Carboxylic Acids (3)
(bleached)
285
TEM grid hole = ~125 μm
289
High Resolution
(bleached)
• Stepper scan (.5 μm)
vs. piezo scan (30
nm).
• High resolution
images of damaged
regions.
• Perhaps evidence of
hollow center.
Top = 20 μm
Bottom = 8 μm
285
289
285
289
High Resolution (2)
(unbleached)
Left = 72 μm
Right = ~36 μm
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.
Acknowledgements
• Chris Jacobsen and Janos Kirz (BNL)
• Doug Mancosky (Hydro Dynamics)
• Alan Rudie (Forest Products
Laboratory)
• Hiroki Nanko (Georgia Institute of
Technology)