1. AFM Imaging of Biological Samples
in Dry and Liquid Conditions
L. Clark1
and M. Goedert2
Phase Imaging using Intermittent Contact Mode in “Just Dry”
Conditions with High Stiffness Probe
Figure 4. (a) 20 µm topography image of unidentified structures in a HeLa cell.
This topography image was generated by feedback of resonant frequency
oscillation displacements. (b) The same structures were imaged
simultaneously by phase lag feedback, where higher resolution was achieved.
The probe used was the PPP-NCHR high spring constant, high resonant
frequency probe with 125 µm cantilever. The sample was hydrated briefly in
de-ionized water and dried by careful blotting of the slide. (c) No sample
damage was observed or measured after repeated imaging.
(a) (b)
Conclusions
• Biological samples were successfully imaged by AFM in dry and
liquid conditions
• Liquid imaging resulted in increased resolution and an 82% increase
in sample height
• Intermittent and contact modes were performed with a low and high
stiffness probe with no apparent sample damage
Acknowledgments
L. Clark and Dr. Michel Goedert were partially supported by the Defense
Microelectronics Activity Cooperative Agreement # H94003-08-2-0806.
References
[1] H.G. Hansma, E. Oroudjev, S. Baudrey, and L . Jaeger, “TectoRNA and ‘Kissing-Loop’ RNA: Atomic Force
Microscopy of Self-Assembling RNA Structures,” Journal of Microscopy, 212 ( 3), pp. 273–279. (2003).
[2] Agilent Technologies, ”Agilent Technologies 5500 Scanning Probe Microscope.” User’s Guide.
Rev. B, 2008.
[3] B. Alberts, Molecular Biology of the Cell (Garland Publishing, Inc. New York, USA, 1994) as appears in A.
Lima-de-Faria, Praise of Chromosome “Folly,” Confessions of an Untamed Molecular Structure, (World
Scientific, 2008), pg 46.
[4] Swiss National Science Foundation, Frontiers in Genetics, “Protein Synthesis [Online],” Available at:
http://www.frontiers-in-genetics.org/page.php?id=protein-synthesis_en (accessed 5 April 2009).
[5] J. Widom, “Chromosome Structure and Gene Regulation.” Physica A, 244, pp. 497-509 (1997).
Methods
Agilent 5500 equipped with a 100 µm scanner (Agilent Technologies,
Santa Clara, CA) and PPP-NCHR and PNP-DB probes (Nanosensors and
NanoWorld, Neuchâtel, Switzerland). Abbott/Vysis Normal Male
Metaphase CGH Target slides from Abbott Molecular (DesPlaines, IL,
#30-806010) and HeLa whole cell samples from ATCC (Manassas, VA,
#CCL-2).
Liquid Conditions: imaged dry, removed plate, added ultra-pure
de-ionized water 15 minutes and imaged in liquid with PNP-DB probe:
pyrex nitride probe with 100 µm with gold-reflex-coated silicon nitride
cantilever, 0.48 N/m spring constant, 67 kHz resonant frequency, and
tip radius of curvature <10 nm.
Just Dry Conditions: sample held in ambient conditions for 30 minutes,
hydrated for 15 minutes in ultra-pure de-ionized water, tilted and
carefully blotted the edge of slide before imaging with PPP-NCHR probe:
silicon probe, 125 µm silicon aluminum-reflex-coated cantilever,
42 N/m spring constant, 330 kHz resonant frequency, and tip radius of
curvature <7 nm.
Figure 3. Using the same probe, Abbott/Vysis male metaphase chromosomes
from normal lymphocytes were imaged in (a) dry and (b) liquid conditions.
With a de-ionized water buffer, higher resolution was observed. Hydration of
the chromosomes resulted in separation of the chromatids and a height
increase of 82% from approximately ~30 to ~170 nm. A gain setting between
1-5% resulted in optimal height and resolution.
Dry vs. Liquid Conditions in Contact Mode with Low Stiffness Probe
Figure 2. (a) 50 µm image of cervical tumor cell (“HeLa”) metaphase
chromosomes. (b) 1 µm 3D image of HeLa chromatin. The probe used was the
PNP-DB low spring constant, low resonant frequency probe with silicon nitride
100 µm cantilever. In dry conditions, high resolution to the level of chromatin
was achieved.
(a) (b)
(a) (b)
Abstract
The purpose of this study was to
image biological samples by atomic
force microscopy (AFM) in dry and
liquid conditions and to optimize the
images by varying the probe type
and AFM mode. The samples were
chromosomes in the metaphase stage of
mitosis from normal male lymphocytes
and cervical tumor cells.
Chromosomes were chosen due to the potential of materials
characterization studies of the self-assembly mechanisms of
chromosomes and potential applications of self-assembling,
programmable bio-materials in the micro- and nano-packaging
industries. While contact mode in liquid increased the resolution,
high resolution images were also achieved using a form of dry imaging
in intermittent contact mode. Where low stiffness probes are often
considered optimal for imaging biological samples, we demonstrate
that a high stiffness probe in intermittent contact mode can be used
to produce high resolution images with no apparent sample damage.
This statement is evidenced by consecutive height measurements that
showed a percent difference of nominal statistical significance. Our
method shows potential for future studies in biological micro- and
nano-materials characterization.
Figure 1. Normal male metaphase
chromosomes in de-ionized water
using contact mode with PNP-DB
probe and 100µm cantilever.
Objectives
• Image biological samples by AFM in dry and liquid conditions
• Evaluate imaging effects by varying the mode and probe type
Background
Motivation to image biological samples by AFM
• AFM requires minimal sample preparation and reduces risk of
sample damage
• Imaging can be performed in liquid to mimic or preserve the
native environment
• Investigation of biological mechanisms has applications in the
development of “self-assembling, programmable biomaterials [1].”
A simplified AFM
probe operation [2].
Stages of chromosome folding [4].
With approximately 1.8 m of DNA in
a cell [3], the chromosome is an
efficient self-assembly mechanism
that is thought to undergo a
“10,000-fold linear compaction [5].”
Formation of a
chromosome [4].
1st stage “11-nm
fiber” chromatin
2nd stage “30-nm fiber”
Results
Contact Mode in Dry Conditions with Low Stiffness Probe
Presented at Materials Research Society Spring Meeting, San Francisco, California, April 6, 2010 1 Department of General Engineering, Bio-Engineering Concentration 2 Department of Materials Engineering
Table 1. Height Measurement in "Just Dry" Conditions with
High Spring Constant and High Resonant Frequency Probe
Test
#
Image#
Image Size
(µm)
Height 1
(nm)
% Diff from
Max Result
1 900702-25 10 166.5 Max
The percent
difference
decreased from
the initial to
the final test.
This result
supports our
claim of no
apparent
sample
damage
2 900702-26 10 162.5 2.4%
3 900702-27 10 151.6 8.9%
4 900702-28 10 156.1 6.2%
5 090702-29 5 163.9 1.6%
6 090702-30 5 160.8 3.4%
7 090702-31 5 162.0 2.7%
8 090702-33 5 162.5 2.4%
9 090702-34 5 162.8 2.2%
(c)