Atomic force microscopy (AFM) is well
known for its ability to image and measure
surface properties. Whether these properties
are electrical, magnetic, physical, or
chemical characterization, the images of
these properties are typically represented
as 3-D images. A device called the NTegra
NanoTome™ (NT-MDT, Zelenograd,
Russia) (Figure 1) integrates an AFM into
an ultramicrotome to extend these surface
investigations into the real world of 3-D,
opening new possibilities for imaging biological
ultrastructure as well as nanostructures
and polymer domains, inclusions,
and voids.1
Figure 1 - NTegra NanoTome.
System operation
The basic approach underlying the new
technology is conventional: The microtome
then slices the AFM images. As with
any tomographic approach, the process is
repeated until the necessary number of
individual images is collected to adequately
represent the 3-D structures of interest.
Figure 2 - Nanotomography diagram of
AFM/knife interface (image courtesy of NT-MDT).
1) Sample, 2) sample holder, 3) ultramicrotome
arm, 4) ultramicrotome knife, 5)
AFM/SPM scanner, 6) holder for AFM/SPM
cantilever or probe, 7) AFM/SPM probe.
What makes the NanoTome
distinctive is its integration.
As shown in Figure 2, the
AFM images directly from the
block face after each slice,
eliminating typical artifacts
such as stretching, tearing, and
wrinkling inherent in imaging
individual slices. Additionally,
imaging from the block face
ensures accurate slice-to-slice
alignment in the 3-D reconstruction,
minimizing the
tedious and complex alignment
processing involved
when working from individual
slices instead of the block face.
The NanoTome is based on the
NTegra microscope (NTMDT),
a next-generation
design whose open architecture
permits both extensive modularity
and flexibility in the base
instrument and facile integration
with other devices such as
the ultramicrotome and Raman spectrometers. A slight
modification of the standard
scanner allows use of the routine
stereo viewing system.
Coupled with the use of a 35°
diamond knife, it makes installation
quick and easy. The
manufacturer recently entered
into a strategic agreement with
Leica (Bannockburn, IL) to
retrofit its UC6.
Looking beyond
topography for ultra-structure
Figure 3 - Cross-section of C. elegans imaged using a)
conventional tomography mode, and b) feedback mode,
measuring local variation in elasticity. Scan size: 25 μm ×
25 μm. (Sample courtesy of Dr. Martin Mueller and Dr.
Nadejda Matsko, ETH, Zurich, Switzerland.2 All sample
images courtesy of Dr. Anton Efimov, NT-MDT.)
Because the diamond knife leaves an ultrasmooth
surface, the commonly used AFM
topography mode reveals little information
(Figure 3a). However, the use of techniques
that elicit other physical responses, such as
local differences in elasticity, uncovers hidden
details (Figure 3b). Animations illustrating
various imaging modes are available
at www.nt-america.com.
Standard sample preparation/multiple microscopies
Figure 4 - The nematode, C. elegans, is a well-known biological model. a) and b) Sequential single
sections, phase imaging (each image is 10 μm × 20 μm). c) 3-D reconstruction made from a stack of
seven sequential block face images, sectioning interval of 200 nm each. (Sample courtesy of Dr. Martin
Mueller and Dr. Nadejda Matsko, ETH.)
Because the AFM does not limit the
ultramicrotome, this technology presents
an opportunity for multiple imaging
modes that present complementary
information. Biological samples are prepared
using standard freeze substitution
protocols. As a result, the AFM can
image from the block face while the slices can be further processed and observed
using transmission electron microscopy (TEM). For example, the C. elegans used
in Figure 4 was prepared by standard
freeze substitution. Figure 4a and b illustrate
sequential atomic force images
taken from the block face, with contrast
generated using phase imaging. Figure 4b
shows the 3-D reconstruction developed
from seven such images. The individually
cut sections can also be mounted for further
TEM investigation.
Figure 5 - a) Fifteen sequential images of
polystyrene/high-impact polystyrene (HIPS) blend with
silica (hard inclusions). Image size: 40 × 20 μm with
200 nm between sections. b) 3-D reconstruction made
from those 15 sections: 40 × 20 × 3.0 μm. (Sample
courtesy of Dr. Aliza Tzur, Technion, Israel.)
Sample preparation for polymers,
advanced materials, and new nanomaterials
is even easier. In many cases, a block of
material can be cut and mounted directly
on the microtome. Because the AFM has a
variety of imaging modes, 3-D information
can be acquired regarding domains, inclusions,
voids, distribution of magnetic fields,
etc. For instance, Figure 5 clearly illustrates
the volumetric distribution of hard silicon
clusters embedded in a polystyrene/high-impact
polystyrene matrix. For the first
time, material scientists can image structures
such as spherulites in true 3-D,
gaining important information to
relate the material’s internal structure
to its function and behavior.
New questions
The new technology has
prompted expected questions, the
most fundamental of which is,
“How thin a slice can be imaged
and what is the XY limit of resolution?”
As with any microtomy
process, the depth of the cut and
therefore the Z resolution is determined
by the nature of the material
and the features of interest.
Typically, slices are on the order of
tens of nanometers in thickness.
Because the AFM uses nonoptical
imaging modalities, the XY resolution
is limited only by the AFM
technique. For local elasticity, for
instance, that distance is on the
order of 10 nm.
A second key question centers on the
availability of the microtome for other
routine activities. Because of the ease
of installation, the AFM can be readily
attached or detached as needed,
leaving the microtome free for other
routine uses in the laboratory.
A third question arises from the 3-D
reconstructions themselves. Specifically,
how are they viewed, and are measurements
such as distances in 3-D or volumes
available? Using the integrated
NOVA™ software, the system presents
“aquarium” constructions that can be
rotated, cut, and viewed from any angle.
Data can also be exported into existing
software such as 3-D Constructor® for
Image-Pro Plus (Media Cybernetics,
Silver Spring, MD) for a variety of measurements
including distance in 3-D
space, volume, and branch length.
Conclusion
The NanoTome is the latest in a growing
arsenal of 3-D imaging tools. Because it is
derived from conventional AFM and
microtomy techniques, the learning curve
is short. However, the resulting benefits are
greater than the sum of its parts, opening
intriguing new opportunities for 3-D imaging
of ultrastructure and the understanding
of structure-to-function relationships.
References
- Efimov AE, Matsko N, Mueller M,
Saunin S. 3D reconstruction of internal
cell structures with ultramicrotome
SPM. Poster presented at the American
Society of Cell Biology, Washington,
DC, Dec 4–8, 2004.
- Matsko N, Mueller M. AFM of biological
material embedded in epoxy resin. J
Struc Biol 2004; 146(3):334.
Ms. Foster is President of Microscopy/Marketing
& Education, 313 S. Jupiter Rd., Ste. 100,
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fax: 972-954-8018; e-mail: [email protected].