Download Atomic Force Microscopy (AFM) for Biological Imaging

Practical exercise no. 7
Atomic Force Microscopy (AFM) for Biological Imaging
and Mechanical Testing
Block Course HS 2013 – Structural Biology and Biophysics
Prof. Roderick Lim
Dr. med. PhD Marko Loparic
Dr. Marija Plodinec
Practical exercise no. 7
1. Introduction
The atomic force microscope (AFM) offers a unique opportunity to visualize, manipulate,
and quantitatively assess structural and mechanical aspects of native biological samples with
nanometer (nm) resolution. An unparalleled advantage of AFM over other high-resolution
microscopes is that biological specimens ranging from tissues to cells and molecules can be
investigated in a physiological liquid environment. The AFM can be operated at 37° C, which
makes it ideal for in situ cell/tissue studies. Combined with optical microscopy, the AFM has
the
added
power
to
directly
correlate
structural/nanomechanical
changes
with
optical/fluorescence images. This ability is unprecedented in biology where the simultaneous
acquisition of structural and functional information is invaluable. After introducing the basics
of AFM, we will focus our attention on investigating the inner limiting membrane (ILM), a
basement membrane found in the eye.
1.2 AFM Operating Principle
At the heart of the AFM lies a mechanical
probe that is composed of a flexible cantilever
with an ultra-sharp tip at its free end (Fig.1).
A standard mode of AFM operation in biology
is referred to as ‘contact mode’. Here, a laser
that reflects off the back of the AFM cantilever
is used to monitor any bending in the cantilever
as it is moving across a sample surface. A
photo diode then monitors the fluctuations in
the reflected laser while the force on the sample
is held constant by a piezoelectric scanner. The
force impinging the sample surface can be
Fig. 1: Operating principle of an AFM
calculated by invoking Hooke‘s Law ( F = kc·d, F = force, kc = cantilever spring constant, d
= cantilever deflection). The signals generated by either the cantilever deflection or vertical
piezoelectric scanner displacement can be used to generate a 3D image of the sample surface.
Another widely used scanning mode is the ‘tapping’ or ‘intermittent’ mode. In this case, the
AFM tip is oscillated vertically while scanning the sample. By applying this method,
frictional forces are reduced. Furthermore, deformation and displacement of the sample is
Practical exercise no. 7
minimized. This method is thus very useful in imaging the surface topography of weakly
immobilized molecules.
1.3 Mechanical Testing of Biological Samples
Beyond imaging, the AFM is ideally suited to measure nanomechanical properties such as
stiffness in tension or compression, plasticity and viscoelasticity of biological samples in
physiological conditions. Instead of scanning the probe during imaging, the AFM is engaged
in ‘force curve’ or ‘force measurement’ mode. This involves the tip approaching and
indenting the sample surface vertically, and then retracts away from the surface.The
cantilever displacement is continuously monitored as the tip-sample separation changes to
produce “force curves/profiles” (Fig. 2).
Fig. 2: Force measurement mode
The slope (gradient) of each curve (∆force/∆displacement) represents a measure of the
specimen’s stiffness. The resulting stiffness measurements can be interpreted as the elastic
modulus (also known as Young’s modulus).
Practical exercise no. 7
2. The Inner Limiting Membrane
The inner limiting membrane is a basement membrane which acts as a boundary between the
vitreous and the retina of the eye. This thin (3-4 µm), transparent layer is derived from
footplates of Mueller cells and mainly composed of nidogen, laminin, collagen IV and
perecan. Due to its mechanical properties, the ILM is an ideal sample for atomic force
microscopy.
2.2 Medical Relevance
Excessive contact between the ILM and the vitreous is a common cause of ocular pathology:
In a process called vitreoretinal traction, the vitreous adheres to the ILM and shearing forces
are conveyed to the retina during eye movements. This is a normal phenomenon with
advancing age. Vitreoretinal traction concentrates around the Macula lutea, the small area in
the center of the retina which is responsible for central vision. These forces ultimately result
in macular holes, which in turn may lead to a rapid loss of sight.
Surgical removal of the ILM leads to a complete relief of traction. During surgery, the
surgeon grasps the ILM with a fine forceps and carefully peels it off the underlying retinal
layers. This procedure is extremely delicate, as the ILM is transparent, extremely thin and in
direct contact with highly damageable retinal structures. Numerous dyes have been employed
to make the ILM more visible and because some dyes have been described to improve “grip”
of the ILM during its extraction, most likely due to photochemical interference with ILM
material properties.
Existing dyes are unrewarding however. Some substances have only weak staining capacities,
others display retinal toxicity or are not approved for intravitreal use. Little is known on their
effect on ILM material properties. Yet with an AFM, the direct structural and mechanical
effects of these dyes can be quickly assessed and thus could reveal important information on
their toxicity. These facts thus make the ILM an interesting candidate for atomic force
microscopy measurements.
3. Objectives
In this session, we will focus our attention on investigating the biomechanical and
topographical properties of the inner limiting membrane. The main aim is that every student
understands the principles of atomic force microscopy and the advantages of such a method.
Practical exercise no. 7
4. Material and Methods
4.1 Samples
The samples acquired were removed by pars plana vitrectomy and preserved in PBS and
sodium azide. After applying fluorescent antibodies (primary antibody: L3993-Anti-Laminin,
secondary antibody: Alexa-FLO 488), Polylysine was then applied to fix the membranes to
glass slides. For fixation, the glass holders were centrifuged for 3 minutes at 3000 RPM.
4.2 Microscope
The experiment in this course will be carried out on commercial microscopes (JPK
NanoWizard I). The AFM contains the optics to detect the cantilever, a cantilever holder, a
scanner, and electronics to approach the sample. The cantilever is mounted on the holder and
held in position by an S-shaped spring. For imaging basement membranes, the AFM is
mounted on an optical microscope (Zeiss Axiovert 135 TV).
4.3 Cantilever Mounting
Put the cantilever holder upside down on the pedestal under the stereomicroscope. Take a
cantilever out of the box and put it on the frosted/matte plane of the holder that is under an
angle. Take the S-shaped spring with a pair of tweezers as shown by the instructor. By
squeezing one side of the S-spring, the other side opens up and can be shifted over the
cantilever. Check whether the cantilever chip and spring are mounted straight. Correct if
necessary.
Warning for mounting the holder: Prevent eye damage when working with lasers!!
The AFM uses laser light that is hardly visible by eye (λ~830 nm) to detect the cantilever
deflection. It is automatically switched off when the AFM is tilted. Nevertheless: Do not look
into the opening where the laser emits when the warning LED is on or put any shining objects
into the laser trajectory to avoid reflection of the laser into your eyes or those of the people
around you.
4.4 Determining the Cantilever Properties
In the following experiment you will characterize the cantilever. First, a detection property of
the cantilever must be determined: The deflection sensitivity, which is the distance over
which the cantilever must be pushed up for the diode signal to change by ΔV=1V. This is
accomplished by using the piezo motor for z-displacement of the cantilever. To measure a
cantilever deflection, the cantilever needs to be in contact with a sample or support. To
Practical exercise no. 7
approach the sample the microscope makes a ‘saw-tooth’ movement towards the sample:
First the cantilever is moved down maximally by the piezo (~15µm at maximum range). If no
contact is established (no large cantilever bending), the cantilever is pulled up again, and the
AFM is moved down by the same distance (or slightly less) using the three step-motors, with
which the microscope stands on the base. This procedure is repeated until contact is
established. The deflection signal stays more or less constant until the cantilever makes
contact with the sample and the cantilever is pushed up. The microscope finally makes a fine
adjustment with the three step-motors, until the preset voltage difference of the photodiode is
reached in the middle of the z-piezo motor. Using the force spectroscopy utility of the
software, the deflection sensitivity can be measured.
a) Place the sample you want to image on the base.
b) Select “Force Spectroscopy” (icon on the upper right). This opens a new window and
changes the parameter bar on the left side.
c) Put the Setpoint to ΔV = 0.6V and start an approach of the cantilever to the surface.
d) Press ‘run’. Force curves will now be shown.
e) Open the calibration manager (Setup → Calibration Manager) to measure the vertical
deflection signal (bottom left on screen) and press the ‘run’ button.
f) Press ‘Select fit range’ below the curve and draw with the mouse an x-range where
the force curve has a constant slope. Press ‘Accept value’ several times and write
down the values.
g) Withdraw from the surface.
h) In the calibration manager, activate the accepted value by selecting ‘use it’.
To establish the mechanical properties of the cantilever, its vibration caused by Brownian
motion of the surround air or liquid is measured. The motion of the cantilever is not random,
but has a preferred vibration frequency – the resonance frequency of the cantilever. The
shown frequency spectrum is created using Fourier transformation of the cantilever noise in
time. For the cantilever to be able to move freely, it should NOT be in contact with the
sample. From the noise spectrum of the cantilever, the spring constant can be calculated.
The next step shows how to calculate the spring constant:
a) Withdraw the cantilever once more (button with red arrow up).
b) Next, go to the ‘spring constant’ tab to measure a noise spectrum. To start the
measurement, press the ‘∞’ button. A spectrum will be shown with much noise in it at
the start, but upon averaging this noise is reduced and a clear peak is observed. After
Practical exercise no. 7
stopping the recording (‘∞’ button again), the peak can be fitted (‘select fit range’),
yielding resonance frequency and spring constant.
4.5 Sample Imaging (Contact Mode only)
a) Place the sample you want to image on the base. If you are concerned that the cantilever
will touch the sample, move out of the step-motors and then carefully place the microscope
on the base: Check from the side whether the cantilever is going to touch the surface and
remove the microscope if necessary to move the step-motors further out (i.e. stepper motor
‘up’).
b) Search for a nice spot for scanning. Put the Setpoint to ΔV = 1V and start an approach of
the cantilever to the surface (button with blue arrow down).
c) After contact: Enter -10V as the Setpoint in order to lose contact between tip and sample:
The cantilever is moved up, either the vertical deflection becomes -10V or the piezo has
reached its highest position.
d) Observe the vertical deflection value. Add 0.2V to this value and enter it as a Setpoint.
e) Adjust the scan parameters. You can start with a scan size of 100x100µm (fast and slow
scan axes), 512x512 pixels, a scan velocity of 40µm/s and a scan rate of 0.7 Hz. Later,
depending on your observations while scanning, these parameters will be adjusted
accordingly.
f) To start (and stop) scanning press ‘run’.
g) Adjust IGain (speed) and PGain (magnitude) for optimizing the servo-system to maintain
constant force and look how the images change. You will see later that the Gains can be
increased when using a smaller range of the z-piezo as result of electronic noise reduction.
h) Change the scan parameters according to your observations. The supervisor will show how
to use the mouse for zooming in ect..
4.6 Force Mapping
a) Start an approach of the cantilever to the surface
b) Change to force spectroscopy mode by selecting the icon on the upper right.
c) Set 20nN as a Rel. Setpoint in the left window. After this is achieved, you may start the
scan by pressing ‘run’.
Note: This kind of measurement is very susceptible to noise. Try to be as quiet as possible.
Practical exercise no. 7
References
Springer Handbook of Nanotechnology, Bharat Bhushan, Springer-Verlag Berlin Heidelberg
New York, 2003
Live Cell Imaging – A Laboratory Manual, Robert D. Goldman, Jason R. Swedlow, David L.
Spector, Cold Spring Laboratory Harbor Press, 2010
Tutorial Questions
1. Briefly explain/draw how an atomic force microscope works.
2. Name the advantages and disadvantages that the atomic force microscope has over
light and electron microscopes.
3. What is the difference between contact mode and tapping mode? In what case would
tapping mode make more sense than contact mode?
4.
How does the AFM acquire information on biomechanical properties of a sample?