Text preview for : MAC Mode Imaging of Biological Molecules with 7500 AFM - Application Note 5991-3672EN c20141020 [2]. part of
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Keysight Technologies
MAC Mode Imaging of Biological
Molecules with Keysight 7500 AFM
Application Brief
Introduction examples of high-resolution MAC mode
imaging of biological samples in liquid
One of the most appealing features of using Keysight 7500 AFM will
atomic force microscopy (AFM) for the be demonstrated.
field of biology is that it can operate
in aqueous environments, making this Instrumentation
technique capable of monitoring or
measuring biological molecules under The Keysight 7500 AFM/SPM
physiological conditions. In terms of microscope is a high-performance
AFM imaging, the complexity of probe instrument that delivers high resolution
oscillations and tip/sample force imaging with integrated environmental
interactions in solutions is much higher control functions. Keysight's patented
than that under ambient conditions. For magnetic AC mode (MAC Mode) is Figure 1. MAC mode imaging of lambda phage
instance, ordering of the liquid at a solid offered as a system option. Switching DNA in a buffer solution.
interface is a ubiquitous phenomenon imaging modes with the Keysight
and such liquid structuring could have a
profound effect on the operation of an
7500 AFM/SPM microscope is quick Imaging of DNA in
and convenient, a result from the
AFM, causing the tip to hop between scanner's interchangeable, easy- a Buffer Solution
different stable configurations in the to-load nose cones. Every aspect of
ordered region of the fluid. A Keysight the Keysight 7500 AFM's design and Lambda phase DNA is used as the first
Technologies, Inc. patented magnetic construction are optimized to reduce example to demonstrate the effective
AC (MAC) mode is developed as a better mechanical noise, and deliver industry AFM imaging in liquid. When DNA in a
approach to AFM in liquid, in which only leading performance. The compact buffer solution containing Ni++ or Mg++
the cantilever part of an AFM probe is and completely encapsulated scanner is added to a mica substrate, these
coated with a thin magnetic coating provides easy cantilever exchange, a divalent cations can form a charge
and the introduction of an alternating slot for (optional) preamps for STM bridge between the negatively charged
magnetic field will induce a direct tip and CSAFM operation, as well as an phosphate groups on the DNA backbone
oscillation. This method eliminates integrated, high-reliability connector to and the negatively charged siloxy groups
the spurious resonances seen in fluid interface with the control electronics. on the mica. This will lead to stable
tapping mode. In addition, a substantial All 7500 AFM's come with the lowest immobilization of DNA molecules onto
signal-to-noise advantage is obtained noise closed loop position detectors the substrate. The amount of DNA that
for A/C mode AFM in fluids if the tip to provide the ultimate convenience can be adsorbed to bare mica strongly
is oscillated by direct application of a and performance in imaging, without depends on the DNA concentration in
magnetic force, as opposed to indirect sacrificing resolution and the solution, soaking/reaction time,
mechanical excitation with an acoustic image quality. as well as the buffer composition
transducer. In this application brief, and buffer pH. Figure 1 is an AFM
Figure 2 (right). Careful examination also
shows the donut like trimer structures
that are likely connected by fibrous arms.
This demonstrates that MAC mode
AFM is a favorable method in studying
the topography of soft and weakly
attached biological samples, capable of
revealing single- or even sub-molecular
structures with high resolution under
physiological conditions.
Summary
Using imaging of DNA and purple
Figure 2. MAC mode imaging of purple membrane proteins in a buffer solution. membrane proteins in well-controlled
buffer solutions as two examples, it
is demonstrated that high-resolution
topographic image showing a case of force microscopy (DFM) methods like imaging of biological samples at single
high surface coverage of DNA molecule. tapping mode and MAC mode are molecular level can be achieved readily
Individual DNA molecules with various designed to reduce the lateral force with Keysight 7500 AFM.
configurations are clearly observed. exerted on the sample surface, thus
better suited for imaging soft and weakly
Molecular-level attached biological samples. However, AFM Instrumentation
tapping mode in solution is difficult to
Visualization of Purple operate. Also for delicate samples like
from Keysight
PM, the force for imaging has to be very
Membrane Proteins via extremely low; otherwise the lattice
Keysight Technologies offers high-precision
modular AFM solutions for research
MAC Mode Imaging structure of the PM would be easily industry, and education. Exceptional
destroyed. Fortunately with MAC mode, worldwide support is provided by
Bacteriorhodopsin is a light-driven the oscillation of the cantilever is directly experienced application scientists and
proton pump in the cell membrane driven by an oscillating electromagnetic technical service personnel. Keysight's
of Halobacterium salinarium. The field, thus the interaction force between leading-edge R&D laboratories are
bacteriorhodopsin molecules adsorb the tip and the sample can be more dedicated to the timely introduction and
on freshly cleaved mica substrate to accurately controlled. Consequently, optimization of innovative and easy-to-use
form a densely packed 2-dimensional high resolution images of the lattice AFM technologies.
membrane. The membrane has a structure of PM can be achieved using www.keysight.com/ind/afm
distinguished purple color, thus often MAC mode in solution.
called purple membrane (PM) in the
literature. PM is stable on mica under The densely-packed bacteriorhodopsin
a pH ranging from 4 to 9, and they can molecules form highly ordered 2-D
form patches of several hundred nm to trigonal lattices in the purple membrane.
several microns in size. The thickness of A topography image of a patch of PM
the membrane is about 5.5nm in average of about 200nm in size, obtained by
and changes slightly with pH. Contact MAC mode, is presented in Figure 2
mode AFM has been used to image PM (left). The hexagonal arrangement of
in KCL solutions of different pH level. the bacteriorhodopsin molecules with
Till today, most of the high resolution a repeating unit of about 6nm and a
images of PM seen are achieved by corrugation of less than 0.8nm is clearly
contact mode AFM. However, contact- revealed by the MAC mode image. At
mode imaging is less suitable for weakly high magnification, the arrangement of
attached bio-sample, because bio- bacteriorhodopsin trimers in each of the
molecules are often disrupted by the repeating unit in the membrane revealed
AFM stylus during scanning. Dynamic a distinct donut-like shape, as seen in
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