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Soft X-ray microscopy with a cryo scanning transmissionX-ray microscope: I. Instrumentation, imaging andspectroscopy
J. MASER* 1 , A. OSANNA*, Y. WANG*, C. JACOBSEN*, J. KIRZ*, S. SPECTOR* 2 , B. WINN* &
D. TENNANT²*Department of Physics and Astronomy, State University of New York at Stony Brook,
Stony Brook, New York 11794-3800, U.S.A.
²Lucent Technologies Bell Laboratories, Holmdel, New Jersey 07733-1988, U.S.A.
Key words. Cryo microscopy, frozen hydrated, soft X-ray microscopy,
spectromicroscopy, synchrotron radiation, zone plate.
Summary
We have developed a cryo scanning transmission X-ray
microscope which uses soft X-rays from the National
Synchrotron Light Source. The system is capable of imaging
frozen hydrated specimens with a thickness of up to 10 mm
at temperatures of around 100 K. We show images and
spectra from frozen hydrated eukaryotic cells, and a
demonstration that biological specimens do not suffer mass
loss or morphological changes at radiation doses up to about
1010 Gray. This makes possible studies where multiple images
of the same specimen area are needed, such as tomography
(Wang et al. (2000) Soft X-ray microscopy with a cryo
scanning transmission X-ray microscope: II. Tomography.
J. Microsc. 197, 80±93) or spectroscopic analysis.
Introduction
Soft X-rays have unique characteristics which make them
well suited for studies of organic specimens. Their short
wavelength (l < 2±5 nm) makes high resolution imaging
possible, and presently available X-ray optics produce the
®nest focus (20±50 nm) of electromagnetic radiation of any
wavelength (Schneider et al., 1995; Spector et al., 1997). By
operating in the `water window' spectral region between
the carbon K-edge at 284 eV and the oxygen K-edge at
540 eV, one can image samples in up to 10 mm of water or
ice, whereas electron microscopes require ice layers under
1 mm thick (Grim et al., 1998; Jacobsen et al., 1998) (see
Fig. 1). One also has access to spectroscopic signatures
which re¯ect the chemical bonding state of major low-Z
constituents, without the plural inelastic scattering back-
ground which is present in electron energy loss spectro-
scopy. The depth of ®eld of today's optics is well matched to
the typical dimensions of cell-sized specimens, allowing
for the acquisition of tomographic projections in the
microscope.
These characteristics have been known for some time
(Wolter, 1952; Sayre et al., 1997), and a large number of
laboratories are carrying out studies using X-ray micro-
scopes (see e.g. Kirz et al., 1995; Thieme et al., 1998).
However, as with other techniques using ionizing radiation
for probing the specimen, radiation damage limits studies of
hydrated specimens at room temperature (Foster et al.,
1992; Williams et al., 1993). Soft X-ray imaging at 50 nm
resolution typically involves a dose to the specimen of , 106
Gray. This dose must be compared with a dose level of
, 102 Gray where cell cultures die, , 104 Gray where
immediate changes are seen on initially living specimens
(Foster et al., 1992; Kirz et al., 1995), and 106ÿ107 Gray
where mass loss and shrinkage is seen even on chemically
®xed specimens (Williams et al., 1993). Radiation damage is
especially signi®cant for studies in which multiple images
must be taken of the same specimen, such as in tomography
(Haddad et al., 1994; Lehr, 1997; Wang et al., 2000) and
for spectroscopic mapping of chemical states (Ade et al.,
1992; Zhang et al., 1996). In addition, the dose required to
achieve suf®cient image contrast increases signi®cantly
with the resolution as more powerful X-ray optics become
available. To reduce structural changes caused by radiation
damage, most previous X-ray microscopy studies have been
done on dehydrated or chemically ®xed specimens. Even
though ®xation allows single images of organic specimens
to be made at moderate resolution levels, it causes changes
Journal of Microscopy, Vol. 197, Pt 1, January 2000, pp. 68±79.
Received 10 February 1999; accepted 30 May 1999
q 2000 The Royal Microscopical Society68
Correspondence to: Chris Jacobsen.
Present addresses: 1 Advanced Photon Source, Argonne National Laboratory,
Argonne, Illinois 60439, U.S.A and 2 Lucent Technologies Bell Laboratories,
Holmdel, New Jersey 07733-1988, U.S.A.
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to the specimen morphology, and does not preserve lipid-
rich structures (Coetzee & van der Merwe, 1984; Stead et al.,
1992).
To overcome these limitations, we have built a cryo
scanning transmission X-ray microscope (cryo STXM)
(Maser et al., 1998). This system allows imaging and
spectroscopy of fully hydrated specimens at temperatures of
around 100 K. At these temperatures, specimens several
micrometres in thickness can be preserved in an amorphous
ice matrix. Structural damage caused by the ionizing
radiation is reduced owing to reduced quantum yield for
ionization of chemical bonds at these temperatures, and by
immobilizing free radicals which are created by ionizing
radiation, thus preventing secondary damage. This has
been exploited in electron microscopy for a long time (see
e.g. Dubochet et al., 1988), and ®rst was demonstrated in an
X-ray microscope by Schneider & Niemann (1994) and
Schneider (1998). Our ®rst measurements (see Fig. 7)
indicate a structural stability of the specimen for doses up to
1010 Gray, which represents a 104-fold increase in radiation
stability for hydrated biological specimens relative to ®xed
specimens at room temperature. This observation agrees
with X-ray microscopy results for a specimen in cold
nitrogen gas, as described by Schneider et al. (1995). This
dose level is at least an order of magnitude higher than the
one at which sample `bubbling' is observed in cryo electron
microscopy (Grimm et al., 1998)
The Stony Brook cryo STXM
The design of our cryo STXM draws considerably from the
experience of the cryo transmission electron microscope
(TEM) community. In cryo TEMs, one is able to mechani-
cally position cryo specimen holders at high precision and
with acceptable drift. The use of an airlock allows for rapid
specimen interchange, and good vacuum conditions (10ÿ6
to 10ÿ7 Torr) minimize specimen contamination buildup.
For our cryo STXM, we therefore chose to use a TEM airlock
(JEOL 1000 type) and cryo specimen holders (E.A. Fischione
and Oxford Instruments). However, a STXM has some
important differences from a TEM which have been
addressed in the following ways:
X Synchrotron X-ray beams propagate horizontally, rather
than vertically, and the working distance of soft X-ray zone
plates is small; this necessitates modi®cations to the usual
TEM cryo holder design.
X Focusing is accomplished by changing the distance from
the zone plate to the specimen, and an order sorting
aperture (OSA) must be placed between the zone plate and
the specimen to allow only X-rays in the desired focus to
reach the specimen.
X Moving the X-ray beam for scanning the specimen
involves mechanical motion of complex beamline compo-
nents or the X-ray optics, which poses signi®cant challenges
in the mechanical design of these components. Instead, we
chose to hold the optic ®xed and scan the specimen for
image acquisition. This requires motions of several milli-
metres for coarse positioning and large ®eld imaging at
micrometre resolution, and over a small range for high
resolution imaging.
We describe below how our cryo STXM has been designed
to meet these requirements. We also note that other
approaches have been used for cryo X-ray microscopy,
whereby the specimen is immersed in a cold nitrogen gas
environment (Schneider, 1998), but for our applications
which include nitrogen edge spectroscopy we preferred to
avoid the need to normalize signals carefully to correct for
absorption in such a cryogenic gas.
Layout
The cryo STXM operates at the X1A undulator beamline
(Winn et al., 1996) at the National Synchrotron Light
Source at Brookhaven National Laboratory (Fig. 2). The
beamline provides soft X-rays of high spectral brightness in
the energy range 250±700 eV with an energy resolution of
0´1±0´5 eV. The incident coherent X-rays are focused into a
small spot using a Fresnel zone plate, yielding a ¯ux of
2 ´ 107 photons sÿ1 in a diffraction limited spot. The
specimen is positioned into the focal spot, and raster
scanned for image acquisition. Placing the focusing optics
upstream of the specimen has the advantage of minimizing
q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 68±79
Fig. 1. X-ray/electron dose comparison. The required dose for ima-
ging 20 nm protein structures in different ice thicknesses is shown
for soft X-ray and 100 keV electron microscopy. Electrons are best
suited to observing thin structures (e.g. viruses in thin ice layers),
whereas X-rays offer exponentially increasing advantages for ice
thicknesses corresponding to whole cell dimensions. (Samples in
1±2 mm thick ice layers may become accessible to transmission
electron microscopy if 400 keV energy ®lters become available.)
For both X-rays and electrons, a detective quantum ef®ciency of
100% was assumed. The X-ray calculations are based on the
work of Sayre et al. (1977) and Schmahl & Rudolph (1987),
whereas the electron calculations of Jacobsen et al. (1998) used
Crewe & Groves (1974) and Langmore and Smith (1992).
SOFT X -RAY MICROSCOPY WITH A CRYO STXM 69
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the radiation dose to the specimen. It also enables us to use
secondary X-ray induced processes such as X-ray induced
luminesence (Jacobsen et al., 1993; Moronne, 1999) as
contrast forming mechanisms in addition to absorption or
phase contrast. Spectra from small areas are obtained by
changing the X-ray wavelength and refocusing the zone
plate in a coordinated manner. The latter is necessary since
the focal length of a zone plate changes with the photon
energy.
For the development of 3D imaging, the cryo STXM has
been designed to allow a one-axis rotation of the specimen.
This allows us to record tilt series of specimens and obtain
tomographic reconstructions with high spatial resolution. A
demonstration of this capability on a frozen hydrated
specimen using the cryo STXM is described by Wang et al.
(2000).
Our cryo STXM operates at a pressure of 10ÿ6 to
10ÿ7 Torr. This has several advantages. First, no convective
heat exchange between the cooled specimen and the
environment takes place, and thermal drifts of the specimen
stage and nearby optics are minimized. Second, the full
spectral range of X-rays provided by the X1A beamline can
be exploited for imaging and spectroscopy, without addi-
tional X-ray absorption by a gaseous specimen environ-
ment. This results in a reduction of image noise, which is of
particular importance for spectroscopic applications. Third,
contamination of the cold specimen is kept at a minimum
when good vacuum conditions are maintained. In fact, we
were able to image the same specimen for time periods on
the order of 40 h without any noticeable build-up of
contamination.
The zone plate and the order sorting aperture (OSA) are
mounted inside the vacuum chamber (Fig. 4), where they
can be positioned with respect to the X-ray beam using ®ne
adjustment stages with micrometre replacement actuators
(New Focus piezomikes). The OSA is mounted at the end of
a thin (0´5 mm wide) strip, which is aligned parallel to the
rotation axis of the specimen. This allows us to rotate the
specimen around the OSA and achieve tilting angles of
6 608. The entire assembly of zone plate and OSA is
mounted on a linear stage inside the vacuum chamber to
allow focusing.
The vacuum chamber with the X-ray optics and specimen
stage is mounted on an optical table with vibration isolation
supports. The vibration isolation supports were specially
chosen to have better than 30 mm position reproducibility so
as to maintain stable alignment of the system to the 200±
300 mm wide X-ray beam from the beamline. The vacuum
chamber has a number of viewports to allow the microscope
operator to verify positioning of components. A turbomo-
lecular pump is used to pump the chamber to a pressure of
10ÿ6 Torr. To avoid mechanical vibrations and improve the
vacuum further, the turbo pump can be valved off, and the
system can be pumped with an ion getter pump which is
mounted directly to the chamber with an isolating gate
valve.
Focusing optics
Fresnel zone plates combine high spatial resolution, broad
wavelength coverage, ease of alignment and good stability.
Zone plates (ZPs) are circular diffraction gratings with
radially increasing line density (see e.g. Michette, 1986).
Incident X-rays are focused into a series of foci of positive
and negative diffraction orders. For imaging, the ®rst
positive diffraction order is usually chosen. Other diffraction
orders as well as undiffracted radiation contribute to
background signal in the image, and have to be blocked
from reaching the specimen by placing an OSA next to the
specimen (Fig. 2). The distance from the OSA to the
specimen is typically 30% of the focal length, and
determines the working distance.
The focusing ef®ciency of ZPs depends on the choice of
materials and on some geometrical factors (Kirz, 1974).
Typical soft X-ray ZPs are made of germanium or nickel,
and achieve a focusing ef®ciency on the order of 10±20%
(see e.g. Thieme et al., 1998). The smallest achievable spot
size is determined by the size drN of the outermost grating
lines. Using electron beam lithography and successive
pattern transfer, nickel ZPs with a thickness of 60 nm
with an outermost zone width of drN�18 nm and a
corresponding theoretical Rayleigh resolution of 22 nm
have been manufactured (Spector et al., 1997). Our highest-
resolution ZPs were previously fabricated with a maximum
outer diameter of d�80 mm, corresponding to a focal length
f � ddrN/l and working distance , f/3 on the order of
0´5 mm or less at an X-ray energy of 516 eV. However, the
Fig. 2. Optical set-up of the cryo STXM at the X1A beamline at the
NSLS. The X1A undulator delivers tuneable soft X-rays of high
spectral brightness from the storage ring. A monochromator
selects the desired energy and bandpass. A ZP is used to focus
the monochromatized beam to a small X-ray spot. An OSA blocks
background radiation not contained in the ®rst order zone plate
focus. The specimen is placed in the focal spot and raster scanned
for image acquisition. Focusing is achieved by moving ZP and OSA
along the X-ray beam direction.
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construction of the cryo specimen holder and the need for
tilting the specimen by angles in excess of 6 608 for
tomographic experiments required longer working distances
for cryo STXM. Therefore, we have developed ZPs with
diameters of 160 mm, and outermost zone widths drN of
60 nm and 45 nm with corresponding Rayleigh resolution
of 73 nm and 55 nm, respectively (Spector et al., 1998).
These provided us with working distances of a millimetre or
more at E > 500 eV, which considerably eased microscope
commissioning and initial tomographic experiments. We
believe that it will be possible to improve on this in the
future; for example, a ZP with 240 mm diameter and 45 nm
outermost zone width would have a usable working
distance of 0´8 mm when used at the carbon K edge.
Cryo specimen holder
Cryo specimens are prepared on electron microscope grids,
which are mounted in turn on the cryo specimen holder in
its workstation (E. A. Fischione Inc., Oxford Instruments).
The tip of the cryo holder with the specimen grid is cooled to
about 100 K by thermal conduction to liquid nitrogen in a
small dewar. The specimen temperature is continuously
monitored, and can be raised in a controlled way using a
built-in heater circuit. The heater can be used to freeze-dry
the specimen in-situ by slowly warming the specimen to
room temperature, or to investigate ice crystal formation in
the specimen by raising the temperature above the
recrystallization temperature of solid water. A shutter can
be placed over the specimen to protect it from contamina-
tion during transfer into the main vacuum chamber. The tip
of the specimen holder ends in a small ball of borosilicate
glass, which forms the interface between the sample and the
scanning stage. In comparison with a standard TEM cryo
holder, the dewar has been rotated by 908 to accommodate
the horizontal X-ray beam. The tip area has been stiffened
to better accommodate the forces caused by scanning the
specimen holder. The geometry of the tip has been modi®ed
to minimize interference with ZP and OSA during tilt, and to
reduce shadowing effects at large tilt angles.
Scanning mechanism
As mentioned previously, we scan the specimen through the
®xed X-ray focus to acquire images. In order to achieve
highest resolution, the mechanical precision and stability of
the scanning mechanism has to exceed the diffraction-
limited resolution provided by the X-ray optics. This requires
high precision in the positioning of the specimen, small
vibration amplitudes of all components relating to the
q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 68±79
Fig. 3. Scanning mechanism of the cryo STXM as seen from the
direction of the incident X-ray beam. The cryo specimen holder is
inserted into the vacuum chamber through an airlock, which in
turn is connected to the vacuum chamber through bellows. A pre-
cision lever mechanism allows horizontal and vertical motion of
airlock and specimen holder. The tip of the cryo holder is placed
in a receptacle on the scanning stage, and held in place by an
adjustable preload. For high resolution scans, an in-vacuum ¯ex-
ure stage moves the specimen holder horizontally and vertically.
Coarse scans are performed by moving the whole ®ne stage and
the specimen holder using out-of-vacuum linear stages driven by
stepping motors.
Fig. 4. Side view of the cryo STXM. Monochromatized X-rays enter
the vacuum chamber from the left. The ZP and the OSA are
mounted on separate three-axis positioning stages for alignment.
Both stages are mounted to an in-vacuum stepping motor to
allow focusing. The coarse and ®ne scanning specimen stages are
placed on the upstream side of the vacuum chamber (see also
Fig. 3). The X-ray detector is mounted in a separate vacuum can
on a platform, which can be positioned using out-of-vacuum man-
ual positioners. Using an in-vacuum slide, the X-ray detector can
be replaced with a phosphor screen, a silicon photodiode, or an
in®nity-corrected visible light microscope objective. The micro-
scope head is located outside the vacuum chamber, along with
an alignment telescope. The vacuum chamber is supported on
kinematic mounts. This allows alignment of the chamber to the
X-ray beam without moving the scanning stages and the detector
set-up.
SOFT X -RAY MICROSCOPY WITH A CRYO STXM 71
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scanning mechanism or the X-ray optics, and small thermal
drifts between X-ray optics and specimen. Here we describe
the overall layout of the scanning mechanism and the
interface between specimen holder and vacuum chamber
(see Figs 3 and 4).
The scanning motions are provided by a combination of
coarse motion for covering large ®elds, and a ®ne stage for
high resolution. The coarse stage is formed by stepping
motors which are mounted outside the vacuum chamber,
and provide a motion of 12 mm ´ 9 mm with a resolution
of 0´1 mm. They are used to take overview scans of the
whole specimen grid, or to position into the X-ray beam
alignment features, which are mounted next to the
specimen on the cryo holder. For high-resolution scans,
we use a piezo-driven ¯exure stage with closed-loop
capacitance feedback, which covers a ®eld of 60 mm. Our
design is based on one used in an atomic force microscope,
which demonstrated 3 nm resolution and 1 : 104 linearity
in scans of a holographic grating (Lindaas, 1994; Lindaas
et al., 1996). In the cryo STXM, noticeable image
distortions are observed if the vacuum force of the airlock
bellows is not properly compensated for by springs
installed for this purpose (an area to be addressed in
future development of the instrument). This ¯exure stage is
positioned on a platform inside the vacuum chamber, and
this platform is coupled to the out-of-vacuum stepping
motors by three posts passing through welded bellows.
Coarse scans using the stepping motors move the airlock,
¯exure stage and cryo holder with specimen inside the
vacuum chamber.
The specimen holder is loaded into the cryo STXM using
a TEM-type side entry airlock (JEOL). The airlock is
connected to the vacuum chamber using welded bellows,
and suspended in a lever mechanism which allows vertical
and horizontal translation of the cryo holder tip about a
distant, well de®ned point. After transfer of the specimen
holder into the vacuum chamber, a capture nut provides a
tight connection between the cryo holder and the airlock,
so both are moved as one unit when moving and scanning
the specimen. This avoids any relative stick±slip motion
which would compromise the spatial resolution during
scanning. The ball at the tip of the specimen holder is
placed into a cone-shaped receptacle on the scanning
stage, thereby providing a precise kinematic link between
the two. The receptacle is made of invar, which, owing to
its low coef®cient of thermal expansion, reduces drifts
caused by the large temperature gradient between the cold
specimen holder and the scanning stage. A preload is
provided by adjustable springs on the airlock to keep the
specimen holder ®rmly placed in the receptacle on the
scanning stage during operation. The inner part of the
airlock can be rotated using a small actuator. This allows
rotation of the specimen holder inside the vacuum
chamber for acquisition of tomograpic data.
Detector and alignment optics
Our detector for transmitted X-rays is a screen coated with
P47 phosphor, and coupled through a light pipe to a
photomultiplier tube. A 100-nm thick silicon nitride
window coated on both sides with 40 nm of chromium is
placed at the entrance to the detector to make this system
relatively insensitive to visible light. The photomultiplier is
run in pulse counting mode, and water cooling pipes are
routed to its enclosure to minimize thermal noise in
operation. The overall ef®ciency of this system is only about
10%. We plan to replace this system with a more ef®cient
silicon drift detector and/or an avalanche photodiode in the
near future.
The X-ray detector is mounted to a platform inside the
vacuum chamber, which is connected through bellows-
sealed tripod posts to an out-of-vacuum manual translation
stage (Fig. 4). This allows positioning of the detector on the
X-ray axis. A sliding stage on top of the detector platform
can be externally manipulated to move the X-ray detector
aside and place other detectors in the beam. We use a large
area silicon photodiode in current mode for ¯ux monitoring,
and a phosphor screen for alignment purposes. Also, an
in®nity-corrected visible light microscope lens, which
operates in connection with a microscope head outside
the vacuum chamber, allows in-situ previewing of the cold
specimen. The microscope head and an alignment micro-
scope are installed at the downstream side of the vacuum
chamber. A mirror allows switching between the two
instruments. The telescope is used to view ZP, OSA and
specimen at adequate magni®cation for alignment pur-
poses. We achieve better than 100 mm pre-positioning of
these components, thereby signi®cantly speeding up the
®nal alignment step which uses X-rays.
Controls
The microscope is controlled by a Unix workstation. The
electronics and software system is similar to that used in a
scanning force microscope (Lindaas et al., 1996), where the
computer is involved in motion instruction and data
transfer once per scanline rather than at every pixel. Up
to eight channels of 16-bit analogue data can be collected,
and up to six channels of pulse rate data can be collected
with 16-bit dynamic range and rate prescaling. This allows
data collection from different detector channels (for example
from con®gured detectors), and can be used for recording
single measurements per pixel (for example temperature of
the sample). The graphical user interface is written in IDL
(Research Systems, Inc. Boulder, CO), which allows easy
control of the different operation modes of cryo STXM such
as 2D imaging, focus scans and acquisition of sequences of
energy-tuned images for spectromicroscopy (Jacobsen et al.,
2000), as well as display of ongoing data collection and
image processing.
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Early operation and experimental results
Preparation of frozen hydrated biological specimens
Specimens are either grown or deposited on formvar-coated
gold grids. When cooling the specimen, the formation of ice
crystals has to be reduced as far as possible in order to avoid
damage to the morphology of the specimen. This can be
achieved by cooling at a high rate. We use plunge freezing, a
method where the specimen is plunged at high speed
(several m sÿ1) into a liquid cryogen (this freezing technique
is used by electron microscopists to prepare vitri®ed
specimens with little or no ice crystal artefacts; see e.g.
Dubochet et al. (1988)). For specimens several micrometres
in thickness, cooling rates on the order of 103ÿ104 K sÿ1
can be achieved, and ice crystal formation suppressed. As
cryogen, we use liquid ethane, which is cooled by liquid
nitrogen to a temperature of around 90 K, just above its
melting point. The use of liquid ethane greatly improves
specimen freezing, because gas bubbles would form around
the specimen and reduce thermal contact with the cryogen
if liquid nitrogen was used directly.
After plunging the grid with the specimen into the
cryogen, it is transferred to the cryo holder. This is done in a
workstation, where the specimen is kept under cold nitrogen
vapour above a liquid nitrogen reservoir to maintain low
specimen temperature and to avoid contamination. After
mounting, a small shutter on the cryo holder is closed over
the specimen for transfer into the airlock and successively
into the microscope's vacuum chamber. The shutter can
now be opened to begin operation.
Instrumental performance
We performed a number of tests to evaluate the perfor-
mance of the microscope. We tested the focusing properties
of the optics and the stability of the stage against vibrations
by imaging a resolution test pattern mounted on the
specimen holder. With a 160 mm diameter ZP with an outer
zone width of 45 nm we obtained the image shown in Fig. 5.
Periodic lines and spaces of 35 nm are visible, even though
the ZP was partially coherently illuminated. With a 45-nm
outermost zone width and an ideal object with 100%
contrast, one would expect to see periodic lines and spaces
as small as 23 nm if the ZP were fully coherently
illuminated. We conclude that the system is able to operate
near the X-ray optical resolution limit. We use data from
this and similar images to verify that the stage is linear,
orthogonal and correctly calibrated at the level required to
make quantitative measurements. We measured thermal
drifts by mounting a resolution test pattern on the specimen
holder and imaging it repeatedly at a temperature of 110 K.
From the apparent shift of the image as a function of time
we deduced an upper limit of the thermal drift of 4 AÊ sÿ1.
We veri®ed the thermal stability of the specimen by
q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 68±79
Fig. 5. Images of a germanium test pattern demonstrate the ability of the cryo STXM to image high resolution structures. The test pattern
consists of 58 spokes, with rings located at radii where the spoke widths are 40, 60, 80, 140 nm, and so on as indicated. As can be seen at
left, the germanium test pattern is located within a 5-mm pinhole of nickel. The image at right was taken at 24 nm step size using a ZP with
drN �45 nm outermost zone width and diameter d�160 mm, showing spokes of width below 35 nm. The resolution was limited in this case
by partial spatial coherence of the X-ray beam.
SOFT X -RAY MICROSCOPY WITH A CRYO STXM 73
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monitoring the thermocouple built into the tip of the holder.
We looked for the build-up of contamination on the frozen
hydrated specimen by imaging the same area repeatedly
over a period of 40 h. We observed no change in mass
thickness over this period of time when the pressure in the
chamber was 0´5±2 ´ 10ÿ6 Torr.
Imaging of frozen hydrated 3T3 ®broblasts and study ofstructural damage of frozen hydrated specimens
EM grids containing live 3T3 ®broblast cells were taken
from the culture medium, brie¯y rinsed in phosphate-
buffered saline prior to plunge freezing, and transferred into
the microscope. The cells were imaged at a temperature
around 110 K. In practice, we end up with a layer of
vitreous ice that is not uniform. To ®nd areas where cells are
in ice of optimum thickness, we ®rst performed a coarse
exploratory scan that covered the entire grid. On this overall
map we recorded the coordinates of interest for higher
resolution imaging. We then recentred the image ®eld on
one of these coordinates. To focus, we collected a series of
repeated line scans near the cell of interest at decreasing
focal distances and selected the line with the sharpest
features (Wang et al., 2000). We then shifted to the cell for
high resolution imaging. Figure 6 shows an image of a
frozen hydrated 3T3 cell obtained in this way.
To study radiation damage to the specimen at cryogenic
temperature, we exposed a frozen hydrated ®broblast to
high radiation doses and looked for morphological changes
during and after exposure at low temperature, as well as
after warming the specimen to room temperature. After
initial imaging of a ®broblast, we increased the size of
various beamline apertures to greatly increase the X-ray
¯ux at a loss of spatial and spectral resolution. We let the
beam dwell on several specimen regions in and around the
nucleus for as long as 45 min per area for accumulated
Fig. 6. Image of an un®xed, frozen hydrated 3T3 ®broblast obtained in the early operation of our cryo STXM. The image is taken at an energy
of 516 eV, close to the oxygen absorption edge where transmission of water is high. The ®broblast was grown in culture medium on a form-
var-coated gold grid, brie¯y washed in a buffer solution, and plunge-frozen at high speed into liquid ethane at a temperature of 90 K. The
cell nucleus with several nucleoli can be clearly seen. Dense lipid vesicles can be seen surrounding the nucleus. The second image shows a
detail at the lower edge of the nucleus. Fine structure surrounding the carbon-dense lipid vesicles can be observed, and the nuclear mem-
brane can be seen. In the water surrounding the cell, as well as in the cytoplasm, groups of lamellar structures are visible. We believe these
are an indication of ice crystal formation in the layer of frozen water. Ice crystals could have formed if the water layer around the cell was
relatively thick prior to plunge-freezing. A typical plunge-frozen grid contains specimen areas of large water thickness which are virtually
opaque to X-rays, areas of moderate thickness where lamellar structures such as the above are observed, as well as areas where the
water layer is very thin and no artefacts are visible (see e.g. Fig. 8).
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q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 68±79
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protein doses of more than 1010 Gray at a photon energy of
516 eV. Figure 7 shows an image of the ®broblast taken at
low temperature after radiation exposure to 1010 Gray. No
structural damage is obvious from this image, and no
structural changes are seen in comparison to an image
taken prior to the exposure. We then warmed the specimen
slowly to room temperature by heating the cryo holder. This
had the effect of freeze-drying of the specimen in the
vacuum chamber, allowing all specimen constituents of low
molecular weight to escape. The inset in Fig. 7 shows the
cell after warm-up, revealing the full extent of the radiation
damage inherent in the specimen areas that received high
doses. Most of the mass in these areas is lost, leading to a
series of holes in the cell structure. Even though radiation
damage on a molecular level was present in the cold
specimen after exposure to high radiation doses, it did not
manifest itself in mass loss or morphological changes at the
sub-100 nm resolution limit we worked at during these
measurements. Figure 8 shows another cell which was
selectively exposed to a dose of around 5 ´ 1011 Gray. At this
dose level, the exposed area shows slight mass loss even
without warming the specimen to room temperature,
indicating a limit of structural stability of frozen hydrated
specimens in a cryo STXM at doses exceeding 1011 Gray. We
note that we have yet to see signs of specimen bubbling even
at these selectively high doses, whereas in cyro electron
microscopy such bubbling is seen at electron exposures of
, 104 eÿ nmÿ2 at 100 keV or about 4 ´ 108 Gray. It is unclear
whether this might be due to the lower dose rate delivered
here, to the lower ratio of ice to organic absorption with
`water window' soft X-ray illumination, or some other reason.
Spectromicroscopy
Soft X-rays are much more likely to be absorbed than
elastically scattered, and the cross-section for inelastic
q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 68±79
Fig. 7. Demonstration of the radiation dose tolerance of a frozen hydrated 3T3 ®broblast which was rapidly plunged into liquid ethane. Fol-
lowing acquisition of a 100-nm resolution image at a temperature of 110 K, an intense, , 0´2 mm beam spot was left stationary on several
locations (noted with arrows) for several minutes (in particular, several spots on the nucleolus were given a dose of about 1010 Gray, in con-
trast to the , 106 Gray delivered to the cell over the course of many images). The image at left was acquired after these exposures, with no
discernible difference from an initial image which is not shown here. The specimen was then allowed to slowly warm up to room tempera-
ture. The smaller insert shows an image of the cell taken after warm-up. The heavily dosed regions lost signi®cant mass, presumably due to
the reaction of radiation-produced free radicals as the temperature approached room temperature, while the rest of the cell shows less pro-
nounced artefacts from radiation damage and freeze-drying. No particular freeze-drying protocol was followed when warming the specimen,
which may have led to artefacts in the image.
SOFT X -RAY MICROSCOPY WITH A CRYO STXM 75
Page 9
scattering is negligibly small by comparison. As a result,
images are largely free from multiple scattering effects and
provide easily quanti®able maps of organic mass. Further-
more, one can exploit X-ray absorption near edge spectro-
scopy (XANES) resonances, which are caused by excitations
of inner shell electrons into available molecular orbital
states, to obtain selective contrast of certain chemical states
of atoms which are present at high concentration. The
combination of high resolution imaging with this sensitive
spectral information is termed XANES spectromicroscopy. In
the soft X-ray range, this approach has been exploited in
applications including the characterization of polymers
(Ade et al., 1992) and the mapping of protein/DNA
concentrations in biological specimens (Zhang et al.,
1996), in addition to other applications cited in a recent
review (Ade, 1998). Because there is no background of
plural inelastic scattering, the signal-to-noise ratio is much
higher than in electron energy loss spectroscopy (Isaacson
& Utlaut, 1978; Rightor et al., 1997).
The natural energy width of soft X-ray XANES reso-
nances is about 0´3 eV, so for quantitative studies one
requires correspondingly high energy resolution. This
requirement is met using a spherical grating monochro-
mator at the X1A beamline (Winn et al., 1996). Studies to
date have been conducted mostly at the carbon absorption
edge. Work at the nitrogen and oxygen edges has been
dif®cult owing to the residual air present in the helium-
¯ushed sample environment in our room temperature
STXM (Kirz et al., 1995). With the specimen placed in the
vacuum of our cryo STXM, we are now able to pursue
spectromicroscopy through the whole spectral range
provided by the X1A beamline.
For biological applications, it may be possible to predict
the dominant features of the near-edge absorption spectra of
proteins from previously acquired amino acid absorption
spectra (Kirtley et al., 1992; Boese et al., 1997), and
therefore estimate from knowledge of amino acid sequences
the likely contrast for chemical state mapping experiments
of proteins. These applications require that several images
be taken of the same object at different photon energies, so
that the radiation dose stability of the cryo method is needed
for high resolution studies of hydrated specimens. In Fig. 9,
Fig. 8. An example of the effects of a dose in excess of 1011 Gray on a frozen hydrated specimen. The insets show a detail at the right edge of
the nucleus. The image in the ®rst inset is taken at the resolution limit of the 60 nm ZP (total accumulated dose roughly 106 Gray). A small
area in the centre was exposed to an accumulated dose of approximately 5 ´ 1011 Gray after this image was taken. The second inset shows the
same detail as before, with the specimen still at cryogenic temperature. An area of reduced density can now be seen in the area where the
focused X-ray beam dwelled, which we attribute to structural damage of the cryogenic specimen. This is an indication of the dose level at
which morphological changes will be encountered in frozen hydrated specimens in a cryo STXM.
76 J. M AS ER ET AL .
q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 68±79
Page 10
we show the absorption spectrum of glutamic acid versus
that of ice taken in cryo STXM. It illustrates that the
resonance near 531 eV due to the C�O bond in glutamic
acid is well separated in energy from the oxygen edge in ice.
This should make it possible to perform XANES imaging of
the organic components of hydrated specimens at the
oxygen absorption edge in the presence of several micro-
metres of ice. Studies of this type are now in progress. An
important consideration for these studies will be the
radiation dose sensitivity of near-edge absorption reso-
nances in organic materials at cryo temperatures. It is
known that ®lms of polymethyl methacrylate at room
temperature suffer a 15% decline in C�O bond strength
following absorbed doses of 107 Gray at 290 eV (Zhang et al.,
1995), but this polymer is especially sensitive to radiation
exposure, which is why it is commonly used as a
photoresist. We are now beginning studies of the radiation
dose sensitivity of XANES resonances at liquid nitrogen
temperatures.
Discussion
We have presented early results from operation of the cryo
STXM. The system has unique characteristics which are
advantageous for studies where 2D and 3D imaging of
labelled or unlabelled structures is desired of un®xed,
hydrated specimens with the 1±10 mm thickness typical of
eukaryotic cells. In addition, resonances in absorption
spectra can be used for mapping of chemical states of low-
Z atoms at high spatial resolution.
Images of unmodi®ed samples of the sort shown in Figs 6±
8 are dif®cult to acquire by other means. Optical microscopy
of unlabelled regions of cells tends to have a point-to-point
resolution $ 200 nm; , 100 nm resolution can only be
obtained using interferometric and deconvolution techni-
ques on ¯uorescently labelled structures (Carrington et al.,
1995; Hell et al., 1997). Near-®eld (Betzig et al., 1991) and
related (Zenhausern et al., 1995) microscopes deliver higher
resolution images, but only of near-surface features. Using
1 MeV (O'Toole et al., 1993) or energy ®ltered 120 keV
(Grimm et al., 1998) electron microscopes, one can study
samples at higher resolution in ice as thick as 0´5±1 mm,
but this thickness limit is not suf®cient for most intact
eukaryotic cells. Cryo X-ray microscopy offers a way to
study the 2D and 3D organization of larger scale aggregates
in unmodi®ed frozen specimens at a resolution intermediate
between the capabilities of optical and electron microscopes.
In addition, efforts are underway to develop labelling
methods appropriate for X-ray microscopes (Jacobsen et al.,
1993; Moronne et al., 1994; Chapman et al., 1996;
Moronne, 1999).
Our ®rst studies with the cryo STXM were taken using
ZPs of lower resolution and correspondingly larger focal
length than the present state of the art. We expect to
improve the spatial resolution as methods for fabrication of
ZPs with large diameters advance, and as additional
operational experience allows us to use high resolution
ZPs with shorter focal length. We also expect to improve the
ef®ciency and the dynamic range of our X-ray detector to
speed data acquisition and improve image statistics. We
®nally plan to adopt other contrast mechanisms such as X-
ray-induced luminescence and phase contrast methods for
use in our cryo STXM. The experimental program pursued
with this system will involve signi®cant efforts in micro-
spectroscopy, spectromicroscopy and nanotomography, and
could be extended to specimens such as polymers and other
radiation-sensitive soft matter.
Acknowledgements
We thank Raymond Fliller, Konstantin Kaznacheyev, Jan
Warnking, Matthias Weigel and Sue Wirick for their
contributions to cryo STXM, Azeddine Ibrahimi for his
help with cell culture, and Marc Adrian, Wolfgang
q 2000 The Royal Microscopical Society, Journal of Microscopy, 197, 68±79
Fig. 9. Soft X-ray absorption spectroscopy provides information on
the chemical state of organic compounds. The absorption spectrum
of , 100 nm thick ice (which we believe is in the amorphous
rather than the crystalline state) is shown, along with the absorp-
tion spectrum of a , 200 nm thick ®lm of glutamic acid. Both spec-
tra were normalized to tabulated absorption coef®cients Henke et al.
(1993) at 510 and 570 eV using a procedure described for example
by Boese et al. (1997), and assumptions of thin ®lm densities of
0´95 g cmÿ3 and 1´35 g cmÿ3 for ice and glutamic acid, respec-
tively. The absorption resonance at , 531 eV is thought to be
caused by C�O bonds in the amino acid, and the exact energy at
which it appears may differ among amino acids. Absorption edge
spectra of amino acids can be used to predict the absorption spectra
of organic complexes Boese et al. (1997), and images acquired
using near-edge absorption resonances can be used to form maps
of chemical bonding states (Ade et al., 1992), including quantita-
tive maps of protein and DNA distribution (Zhang et al., 1996).
SOFT X -RAY MICROSCOPY WITH A CRYO STXM 77
Page 11
Baumeister, Frank Booy, Jacques Dubochet, Robert Glaeser,
Rainer Hegerl, Richard Henderson, Richard Leapman,
Jonathan Sedat and Dieter Typke for many helpful
discussions. We gratefully acknowledge support from the
Of®ce of Biological and Environmental Research, US DoE
under contract DE-FG02-89ER60858, the National Science
Foundation under grants DBI-9605045 and ECS-9510499,
and the Alexander von Humboldt Foundation (Feodor-
Lynen Fellowship, JM). This work was carried out at the
National Synchrotron Light Source at Brookhaven National
Laboratory, which is supported by the US Department of
Energy.
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