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USERS MANUAL
February 2015
CONTENT
A. AtomicJ License agreement ............................................. 5
B. Installation and running ........................................ 5
C. Structure of the Main window .............................. 7
D. Viewing AFM recordings .......................... 9
1. Working with the Open files assistant ..................... 9
2. Raw data dialog ......................................................................................... 11
3. Force curves dialog ................................... 12
E. Force curve processing ..................................16
1. Working with the Processing assistant ......................... 16
2. Which settings to use? ............................................................................... 22
3. Partitioning force maps into multiple batches ........................................... 24
4. Sensitivity calibration ................................... 27
5. Manual selection of contact point ........................... 29
F. Working with the results of curve processing ................................31
1. Results dialog ..........................................31
2. Customizing result format. .......................... 34
3. Creating histograms ............................................. 35
4. Histogram dialog ..................................... 36
5. Creating box plots ......................................................................................38
6. Running statistical tests .......................................... 40
7. Descriptive statistics .......................................... 41
8. Recalculation .................................................. 43
9. Graphical results dialog ............................................................................. 43
G. Mouse actions on charts ............................................... 47
1. Normal mode .................................................... 47
2. Measure distance mode ............................................ 48
3. Extract profile mode ............................................ 49
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g.
J.
Click on the AtomicJ.exe file (right in the main application directory, icon with yellow
triangle on black background. You may want to create a shortcut on your desktop).
AtomicJ will be opened with the default run parameters it will use up to 3 GB of the
RAM. AtomicJ.exe may not open if there is not enough memory. In such case follow
steps in B.
If Java 7 or later is not installed on your computer, AtomicJ will be run with the Java
Runtime Environment included in the distribution. Java can be downloaded free of
charge from Oracle website:
http://www.oracle.com/technetwork/java/javase/downloads.
B. For the Platform independent distribution and Windows distributions:
Click on the AtomicJ.jar file (icon with a steaming cup of coffee). AtomicJ will open with
the default run parameters, which depend on the version of Java installed on your
computer. You may want to override those default settings, especially the maximum
memory available for application. To do so, run AtomicJ.jar from the command shell (for
Windows, see appendix How to Open Windows Command Prompt).
On the shell, set the current working directory to the AtomicJ directory (in Windows,
just go to the directory using the cd command) and run the command:
java XmxmemoryMaximumm -jar AtomicJ.jar
The value memoryMaximum is the maximal amount of memory (in megabytes) that
AtomicJ can use. For example, if you work on a computer with 8GB RAM and you want
to process at once several large force maps, you may use 7GB of memory, using the
following command:
java Xmx7000m -jar AtomicJ.jar
The recordings used in this manual are included in the test files, available at
https://sourceforge.net/projects/jrobust/files/TestFiles/AFMRecordings.zip. You will find there
force curves (collected on prostate cancer cells), as well as a force map and a corresponding
AFM image of fibroblasts. Simulated curves for different tip shapes can be downloaded from
https://sourceforge.net/projects/jrobust/files/TestFiles/SimulatedCurves.rar. The curves are
accompanied by .properties files, which contain information about the recording conditions and
processing settings. Those files can be read-in to AtomicJ before processing (see section E.1.
Working with the processing assistant).
Fig. 1. Main window. The toolbar icons (a h) correspond to the commonly used options: a
process force curves and maps, b open curves or AFM images,
Menu options
The main menu bar contains three menus and can be navigated with either the mouse, or the
keyboard, using the mnemonics in the brackets:
o
o
o
Single force curves - the recordings of force acting between the sample and the
cantilever tip when it is pushed into the sample and then withdrawn.
Force volume recordings (or force maps)- multiple force curves recorded in a grid. The
grid is specified over the sample and force curves are recorded in each node of the grid.
Flexible force curves recordings - multiple force curves recorded on the sample at
arbitrary points.
Images obtained by scanning the sample. The images usually consist of different
channels, for example sample topography, friction or error signal.
All these types of AFM recordings can be viewed with the Open files assistant (Fig. 3). It can be
accessed from the Main Window with the shortcut Ctrl + 0, or through the toolbar icon
or the main menu (go to File (Alt + F) Open). It lets you read in and open multiple AFM
recordings, possibly of different types. The files that you read in to the Open files assistant are
displayed in the list in its center (Fig. 3).
You can read in or delete files to open with the buttons (Fig. 3 a) and the context menu (b):
to read in a new file, click the Add button (Alt + A). A standard file dialog will be
displayed. It is important to select the right type of AFM files (Fig. 4 a). You do not need
to add all files one by one. If you select a directory, all files of the right type in this
directory and its subdirectories will be automatically read in and added to the list.
You can open all read-in files or just the ones that you selected in the list.
to open all AFM recordings in the list, click the Open button (Alt + P).
to open some of the AFM recordings, select the corresponding file names in the list then
move the mouse over the selected files and click the right mouse button. The context
menu (Fig. 3 b) will appear, click Open.
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to add new files to the selection, hold down the Ctrl key and click the new files with the
left mouse button.
to select all files, use the shortcut Ctrl + A.
The force curves are plotted in the Force curve dialog (subsection D.3. Force curve dialog). All
individual curves from force volume recordings or flexible force curve recordings are plotted in
the Force curve dialog, too. If you wish to generate maps of mechanical properties from these
recordings, you need to process them see the section E Force curve processing. AFM images
are plotted in the Image dialog for details see the section J. Working with images.
The files can be also opened in the default application, associated with their extension by your
Operating System. This may be useful when you want to view the files in your AFM application
before processing in AtomicJ. To open files in the default application, select them in the list,
then click the selected files with the right mouse button. The context menu (Fig. 3 b) will
appear, click Open in default application.
The Open files assistant allows you to display the raw data that make up the AFM recordings.
This may be useful, for example, when the original recordings are saved in the binary form. To
display the raw data, select the files of interest in the list, then move the mouse over the
selected files and click the right mouse button. The context menu (Fig. 3 b) will appear. Click
Raw data. The raw data will be displayed in a separate dialog (Fig. 4).
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The Raw data dialogs are equipped with a main menu bar (Fig. 5 a) and a set of buttons (b) that
allow you to print, save to a file or customize the data in the tables:
to save the currently displayed table to a file, press Ctrl + S or go to File (Alt + F) Save
(Alt + S) or click the Save button (Fig. 5 b). A file dialog will be displayed, which allows you
to choose where the table should be saved and in which format (TSV or CSV).
to print the currently displayed table, press Ctrl + P, or go to File (Alt + F) Print (Alt + P),
or click the Print button (Fig. 5 b).
to change the number format, go to Customize (Alt + U) Format data (Alt + F). The
Customize number format dialog will be displayed (Fig. 22). It lets you specify the number
of decimal fraction digits and the decimal separator used in the statistics tables.
Anytime you open a force curve file, a chart is generated in the Force curves dialog. This is a
prototypical dialog for displaying charts, as more complicated dialogs in AtomicJ inherit its basic
structure. We will discuss it in greater detail and skip the redundant information in discussions
of more complex dialogs.
Force curves dialog consists of a main menu bar (Fig. 6 a), a set of buttons (b) for the most
commonly used actions, a central split pane, with the list of force curves on the left (c) and a
panel displaying the corresponding chart on the right (d).
The main menu bar contains of four menus:
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o
o
o
Open all in dialog (Ctrl + Shift + O) opens all the charts for the current sample in new
dialogs
Clear clears all the charts
Close closes the dialog
Measurement style opens a dialog for editing the style with which the
measurement lines are drawn.
o Measurements opens a dialog with the detailed
information about each measurement line you drew on
the currently displayed chart.
o Measure distance changes the way the mouse clicks are
interpreted by the chart. If this action is switched on
(when a tick-mark is visible), you can add a new
measurement line with mouse clicks.
Clear, Save all and Close options can be also accessed via buttons in panel b.
Preprocess menu provides many preprocessing procedures, that can be applied to all
force curves stored in the Force curve dialog, or just to the selected ones. The menu also
includes options for undoing and redoing the previous preprocessing operations.
o
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Gaussian lets you smooth the force curves using Gaussian filter, with user-specified
standard deviation.
Savitzky Golay lets you smooth or differentiate the force curves, using Savitzky Golay filter. The filter is usually applied to regularly spaced points. If the spaces
varies, AtomicJ will use linear interpolation to produce regularly spaced points before
applying Savitzky-Golay filter.
Local regression lets you smooth or differentiate the force curves, using robust local
regression procedure with tricube weights. More details can be found in the original
paper: Cleveland, W. S. (1979). Robust locally weighted regression and smoothing
scatterplots. Journal of the American statistical association, 74(368), 829-836.
Convolve lets you convolve the force curves with a kernel of arbitrary size and
elements. Convolution can be used for example to smooth or to differentiate the
curves. If the curve points are not regularly spaced, AtomicJ will use linear
interpolation to produce regularly spaced points before applying convolution.
The list of force curves is equipped with a context menu (Fig. 6 e), triggered by right mouse click.
The context menu contains eight actions:
Process allows you to calculate mechanical quantities from the currently selected force
curves.
Raw data (the same as in the main menu)
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Save selected opens the Batch save dialog, which will help you to save the charts for the
currently selected force curves.
Short names toggles between short names (usually filenames) and long names (usually
full paths) of force curves
Change font opens a font selection dialog. Click to change the font style or color of the
items in the list
Open all in dialogs (Ctrl + Shift + O) (the same as in the main menu)
Open in dialogs (Ctrl + O) (the same as in the main menu)
Delete deletes the selected force curves. Del key acts the same.
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c)
The buttons for selection of the data for analysis (Fig. 8 c). They allow you to add new
files with force curves or maps, clear the files that have already been added, as well as
to plot and preprocess the curves (Preprocess button) to verify and improve their
quality. The Patchwork button lets you choose curves from a force map that should
be included in the current batch based on an AFM image. It also lets you partition a
force map into multiple batches. For more details on partitioning curves from a single
force map into multiple batches, see section E.2. Partitioning force maps into multiple
batches.
d) The navigation buttons (<<Back, Next>> and Finish) (Fig. 8 d) for navigating between
different pages of the assistant.
e) The list of curves already added to the batch, equipped with a context menu,
triggered by the right mouse button (Fig. 8 e). The context menu includes options for
displaying the raw data for selected curves (see the section D.2. Raw data dialog),
previewing, opening and deleting selected curves from the batch.
The parameters of the analysis are specified in the second page of the processing assistant. The
settings specified in this page can be saved to a file, with the Export button. The saved settings
can be then read in with Import button, for example to speed up input processing settings for
subsequent batches.
The second page of the Processing assistant consists of three tabs General, Advanced
and Output. The General tab (Fig. 9) allows for input of the mandatory settings:
Batch name (subpanel a in Fig 9). The default name is the current batch index.
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The method of contact point estimation whether the search for contact point should
be manual or automatic, and if automatic, which estimator should be used (panel b)
The estimator used for fitting the contact mechanics model to force-indentation data
(panel b)
The shape and size of the AFM tip used to collect the force curves. Contact models for
different shapes are implemented :
1. Cone
2. Sphere (Hertz's approximation)
3. Sphere (Sneddon's solution)
4. Fungs (hyperelastic material, sphere)
5. Ogdens (hyperelastic material, sphere)
6. DMT (model of adhesive contact, sphere)
7. JKR (model of adhesive contact, sphere)
8. Maugis (model of adhesive contact, sphere)
9. Pyramid
10. Power-shaped (shape obtained by revolving a power function along the y axis)
11. Hyperboloid
12. Hyperboloid (Suns model of adhesive contact)
13. Blunt cone
14. Blunt pyramid
15. Truncated (i.e. flat - ended) cone
16. Truncated pyramid (Fig. 9, panel c)
Depending on the shape, you will have to specify tip radius (radius of curvature at the
apex), half angle and transition radius (see tip sections in the Appendix M.2.). For blunt
tips, the value of transition radius that ensures smooth merge between spherical cap
and the tip body can be calculated automatically click the
button.
Poissons ratio of the sample (panel d).
The method of calculating energy of adhesion (panel d). Energy of adhesion can be
calculated from the adhesion force directly measured on the chart (option Calculate
from fit) or treated as a fitting parameter (option From fit) This action is enabled only if
the selected model of indentation takes into account adhesive interactions.
The degree of the polynomial fitted to the off-contact region of a force curve (panel e)
The spring constant of the cantilever and the optical lever sensitivity (panel f).
Sensitivity has to be specified only if the deflection values are provided as raw
photodiode output. Otherwise this field will be greyed out. Sensitivity is used to
convert voltage into deflection expressed in microns. It can be calculated based on a
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force curve recorded on a clean, rigid substrate (i.e. mica), with the help of the
Calibration dialog (Fig. 14).
The Advanced tab (Fig. 10) allows for input of the optional settings:
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The extent of cropping how much of the extreme parts of the raw force curves should
be cropped (panel a in Fig. 10). You can either:
a)
b)
The maximal indentation or maximal load (panel c in Fig. 10). If these parameters are
set, parts of the force - indentation curve above the specified maximal values are
rejected.
Smoothing filter (panel b in Fig. 10). Two low-pass filters are available:
a)
Local regression.
Two parameters have to be specified:
Span determines what fraction of points closest to the current point is taken into
account for calculating local linear least-squares fit. Larger span ensures better
removal of noise, but smaller features may be lost or distorted.
Iterations determine how many robustness iterations should be performed.
Robustness iterations use residual from previous fits to calculate weights for new
least square fit. The greater the number of iterations, the better filters resistance
against outliers.
b)
Half width determines the number of points to the left and to the right of the
current point that are included in the moving filter window. Larger half width
ensures better removal of noise, but smaller features may be lost or distorted.
Degree determines the degree of the polynomial fitted to the points within the
moving window. Higher degrees ensure that higher moments of data are preserved.
Bottom effect correction (panel d in Fig. 10). Bottom effect is the effect of the rigid
substrate on the apparent stiffness of the sample. Thin samples on rigid substrates
appear stiffer than they really are. You can specify the thickness of the sample either as
an absolute value, or choose a topography image (discussed below). The correction
depends on whether the sample is bonded to the substrate or not (checkbox
Adherent). The corrections are available for two tip shapes: cone and sphere.
Using topography for bottom effect correction: If you recorded the topography image
of your sample, AtomicJ can use it to find the thickness of your sample in the positions
where the curves where recorded. You should:
1. select the Use topography checkbox, then
2. click the Browse button. The Sample topography assistant will be displayed. It
lets you select the image file, choose the topography channel (ex. trace or
retrace) and select the sample on the image (Fig. 12). Topography images are
often affected by various artifacts, so you will usually need to correct them
using the options accessible through the context menu (Fig. 12 c) Thickness of
the sample will be read directly from the corrected topography image.
If a force curve file does not contain information about the recording position, the
application will assume that the sample was of infinite thickness, unless you have
specified explicitly the thickness in the Thickness field (Fig. 10 panel d).
Fig. 12. Sample topography
assistant, displaying the Correct
topography page. This page
allows
you
correct
the
topography image, so that the
height of the sample can be read
from it for during force curve
processing. Image correction
actions can be accessed from the
context menu (c) and applied to
selected Regions of Interest
(ROIs). To select the sample on
the image (b) as ROI use the
buttons in panel a. For more
information on selecting 21
ROIs,
see section G.4. ROI modes.
The Output tab (Fig. 13) lets you choose what kind of processing output should be generated.
You can specify:
which curves to plot in the output (Fig. 13 a) and whether model fits should be also
drawn.
whether a force map should be generated. (Fig. 13 b, checkbox Include in maps). This
option is enabled only if it is possible to generate the force map, i.e. at least some of the
curves within the batch contain information about their position on the sample.
whether the image of the sample should be read-in alongside force curves (Fig. 13 b,
checkbox Plot map area scans). This option is enabled only if the file with curves contains
image data or specifies another accessible image file.
By default, three charts are generated for each curve. For larger sets of curves, this may result in
large consumption of memory. 4 GB of RAM is enough to draw around 30 000 - 60 000 charts,
depending on the size of curves.
sets of very poor force curves, it may be advisable to compare the results obtained with the
automatic and manual estimation, for a small subset of curves.
The quality of the automatic contact point estimates depends on how close is the modeled
force indentation relation to the true relationship. There are several sources of discrepancies
between model and reality in AFM indentation experiments. Analyzing force curves, we usually
assume that the sample is linear elastic, i.e. that the relationship between stress and strain is
linear. This assumption is usually satisfied if the indentations are small relative to the sample
thickness. If you suspect that at least some curves may exceed the limit of linear elasticity, you
may use robust estimators to fit model to data (LTS, LTA, HLTS or HLTA). Robust estimators will
reject the parts of force indentation data that grossly deviate from the model. Apart from the
material properties of the sample, force indentation relation depends on the tip shape and the
thickness of the sample (for equations, see Appendix M.2. Force - indentation relations.) If the
sample is thin, the cantilever feels underlying, rigid substrate, which leads to overestimation of
Youngs modulus. Fortunately, corrections for this effect are known for indentation with
spherical (Dimitriadis et. al 2002) and conical tips (Gavara and Chadwick 2012). These
corrections are implemented in AtomicJ.
The degree of a polynomial fitted to the off-contact may influence estimation of the contact
point. A straight line (i.e. degree 1) can be fitted if the off-contact parts of the force curves in
the batch are linear. However, the off-contact parts of force curves often exhibit some
curvature, so fitting a polynomial of degree higher degree usually works better. For single curves
polynomial of degree 2 is often optimal. Curves in force maps may exhibit more artifacts in the
off-contact part. In such cases you may consider fitting of a polynomial of degree 3.
The Poissons ratio is a property of the sample it tells how big lateral contraction (or
expansion) will accompany axial stretching (or compression). In principle, in order to obtain
accurate results of analysis, the correct value of Poisons ratio for our sample has to be
measured independently. However, biological samples are often assumed to be incompressible,
i.e. that their Poissons ratio is 0.5 (for example Radmacher 1996, Mathur 2001). Although
overestimation of Poissons ratio leads to underestimation of Youngs modulus, this error is
relatively small. In the extreme case, when you mistake a compressible sample (Poissons ratio
0) for an incompressible (0.5) one, Young's modulus is reduced by 25 %.
The spring constant of the cantilever has to be measured in order to convert cantilever
deflections into forces. Various methods of measuring cantilevers spring constant have been
described (Burnham et al 2003). Automatic measurement of spring constant usually can be
performed with programs operating AFMs. Overestimation of cantilver spring constant leads to
overestimation of Youngs modulus.
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Burnham NA, Chen X, Hodges CS, Matei GA, Thoreson EJ, Roberts CJ, Davies MC, Tendler SJB (2003) Comparison of
calibration methods for atomic- force microscopy cantilevers. Nanotechnology 14: 1 - 6.
Dimitriadis EK, Horkay F, Maresca J, Kachar B, Chadwick RS (2002) Determination of elastic moduli of thin layers of
soft material using the atomic force microscope. Biophys J 82:27982810.
Gavara N and Chadwick RS (2012) Determination of the elastic moduli of thin samples and adherent cells using
conical AFM tips. Nat Nanotechnol. 7: 733736.
Radmacher M., Fritz M., Kacher CM., Cleveland JP, Hansma PK (1996) Measuring the Viscoelastic Properties of
Human Platelets with Atomic Force Microscope. Biophys J 70 : 556 567.
Mathur AB, Collinsworth AM, Reichert WM, Kraus WE, Truskey GA (2001) Endothelial, cardiac muscle and skeletal
muscle exhibit different viscous and elastic properties as determined by atomic force microscopy. J Biomech 34
(12): 1545 1553.
Fig. 14. The first page of the Partition force map assistant,
which allows you to select the AFM image of the force map
area.
25
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Fig. 17. When some curves are not included in any of the selected patches, AtomicJ will ask you whether a new
batch should be created for these curves.
The third page of the Partition force map assistant also allows you change the style of ROIs for
the current patch, as well as to calculate the statistics, shape factors (i.e. area) and to draw
histograms for the selected ROIs. These actions can be accessed from the context menu. To
display the context menu, move the mouse over the image chart and click the right mouse
button.
E.4. Sensitivity calibration
Usually, cantilever deflection is measured with the optical lever technique. Force curves are
recorded as a relationship between voltage generated in the photodiode and the position of the
cantilever. Before processing, voltage should be multiplied by the optical lever sensitivity in
order to convert it into cantilever deflection. Optical lever sensitivity tells how much voltage
corresponds to a unit of deflection. It can be determined based on calibration curves, i.e. curves
recorded on a hard substrate (ex. mica) with the help of the Calibration dialog (Fig. 18).
There is no upper limit on the number of calibration curves you can use. If multiple curves are
used, the Calibration dialog will calculate average sensitivity. The Add button allows you to
choose new calibration curves from your computer file system. You have to choose at least one.
The newly chosen curves will appear in the list (Fig. 18 a). For each calibration curve, select the
range for calculation of the optical lever sensitivity (b). The range should encompass the linear,
in-contact part of the curve. The limits of this range can be dragged with mouse. The sensitivity
is the inverse slope of approach part of the curve.
Fig. 18. Dialog for calculation of the
optical lever sensitivity. Multiple
force curves can be used for
calibration (a), average sensitivity is
then calculated (c). The calibration
is done based on the part of the
approach curve within the blueshaded area (b), whose bounds can
be modified with the mouse. The
The navigation buttons (Fig. 18 d) allow you to commit you selections or to move to other
curves. Click Apply to commit sensitivity calculated from the current curve and range, Skip to
move on to the next curve, Finish to finish calibration (current average sensitivity will be used
as the final result) and Cancel to cancel calibration no result is returned. The Clear button (Fig.
18 a) discards all calculated values (you may choose new set of calibration curves and repeat
calibration).
TIPS!
You can choose all calibration curves from a single directory at once just select the
directory and AtomicJ will read in all force curves in this directory.
You can choose a force map and use the curves recorded on hard substrate as
calibration curves click Substrate button to select the rigid substrate on an image.
You can skip a low quality calibration curve click Skip without clicking Apply.
You can zoom-in the in-contact part of the calibration curve.
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Fig. 19. Dialog for manual determination of the contact point position.
select the contact point with a left mouse click on the chart a and adjust its position
dragging it with the mouse. The current coordinates of the contact point are displayed
in the fields d.
enter the coordinates in the fields d. The contact point appears on the chart, where it
can be repositioned.
You can choose whether to use both or just a single coordinate of the selected contact point,
using the radio buttons e. In the latter case, the missing coordinate is found automatically. For
example, if you specify the Y coordinate to be 100 nN, the application will find the point on the
curve closest to 100 nN. Please remember that the force values are calculated from raw
photodiode readings, so they are shifted by a constant corresponding to the voltage signal
generated by the laser bounced off the unengaged cantilever.
You can preview what will be the results of processing if you apply the current contact selection.
The Live preview dialog (Fig. 20, you can display it with the button c) shows Youngs modulus
and transition indentation corresponding to the current contact point position, together with
the force-indentation and pointwise modulus curves. It gets instantly updated when you move
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the contact point (ex. dragging it with mouse). This dialog can be used to demonstrate the
importance of proper selection of the contact point.
Once you have chosen the right point, you can apply it with the Apply button (Fig. 19 panel f).
The Apply to all button applies the current coordinates to all remaining curves. This is useful for
fast, cursory analysis, for example, of curves collected in a single location. The Skip button
allows you to skip the low quality curves that should not be included in the analysis.
Fig. 20.Live preview of the force indentation and pointwise modulus indentation curves, generated
based on the contact point selected manually.
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Batch histograms (Alt + B) lets you generate a separate histogram for each
batch.
Descriptive statistics (Alt + D) opens the Live results statistics dialog. Click to
view mean, median, variance and other statistics for all batches and pooled
results.
Show box plots opens the Box plots dialog, which stores all box plots generated
from processing results in the current session. It is initially disabled, as there are
no box plots to show.
Add box plots (Alt + X) opens the Box plots assistant. Click to generate box plots
for selected subsamples of processing results.
Inference (Alt + I) opens the Statistical inference assistant. Click to test
hypotheses about means of arbitrary subsets of results.
Recalculate opens the Recalculate assistant (see section F.8. Recalculation).
Mark on the maps marks the position corresponding to the selected entries on
maps of the mechanical quantities
Menu actions:
The menu bar consists of four menus and can be navigated with the mouse or using keyboard
mnemonics in brackets:
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o
o
o
o
o
Clear, Save, Print and Close actions can be also accessed through the respective buttons in the
button panel (Fig. 21 c).
The results are displayed in a table (Fig. 21 d). Each row corresponds to one force curve. The
table contains the calculated values of Young modulus, coordinates of the contact point,
transition indentation, transition force, adhesion force, the most important processing settings
spring constant, optical lever sensitivity, tip shape and the batch name. The format of the
data in the table can be customized via the customize menu (see above).
Individual or all rows of the result table can be selected. You can select multiple rows:
to select non-contiguous rows, right-click with the mouse while holding down the Ctrl
key
to select a range of contiguous rows, click the first row and then the last row, while
holding down the Shift key
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The results dialog is equipped with a context menu (Fig. 21 e). It contains options which are
performed on the currently selected rows:
Copy (Ctrl + C) copied the rows to the clipboard. The rows can be pasted to other
applications, including MS Excel.
Select all (Ctrl + A) selects all rows in the table
Jump to graphs shows the corresponding charts in the Graphical results dialog, if they
are available
Recalculate opens the Processing assistant. Click to recalculate the curves corresponding
to the selected rows. New results are added to the table, the old ones stays.
Recalculate and replace acts as Recalculate, but deletes the old results
Selection histogram opens the Histogram assistant. Click to generate a histogram for the
selected rows.
Selection statistics opens a dialog displaying statistics for the selected rows.
Delete deletes the selected rows.
F.2. Customizing results format
Format of the results can be modified with the Customize results table dialog (Fig. 22). It allows
you to change the number format, in particular:
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Each change to the format of results is saved as the applications default settings (in the registry
in the case of Windows), so you do not have to customize it again the next time you run
AtomicJ. The format is also preserved when results are saved to a text file.
F.3. Creating histograms
You can produce histograms of the results with the help of the Histogram assistant, which
gathers all the necessary settings. It consists of a variable number of pages that depends on the
number of samples to be plotted. If multiple samples are available, the first page (Fig. 23 left)
allows you to select which of them should be plotted. The next pages are the binning pages (Fig.
23 right), one page for each sample. If there is just one sample (ex. when you plot pooled results
for all batches), the assistant consist of a single binning page (Fig. 23 right).
In the binning page each histogram should be named (panel a). You can choose the binning
manually (filling either the bin width or bin count field), or use an automatic binning method
(panel b), one of the following:
Sturges k log2 n 1
Scotts h
Square root k n
the Freedman-Diaconis h
3.5
3
n
2 IQR
3
n
where n the sample size, k bin Count, h bin width, sample standard deviation, IQR
interquartile range, x is the smallest integer not less than x.
If the extreme values in your sample are dubious (ex. negative values of Youngs modulus), you
may want to discard a fixed percentage of largest/smallest values with the help of panel c.
Alternatively, you can change the range of data that are displayed in the histogram (panel e).
AtomicJ supports both ordinary and cumulative histograms. Bin height can be specified as the
count (number of values within the bin), its decimal logarithm, probability (i.e. the frequency of
values within the bin) or probability density (frequency divided by the width of the bin). A
theoretical distribution can be fitted to the sample and plotted together with the histogram.
AtomicJ supports the following distributions:
Normal
Log-normal
Laplace
35
Lorentz.
The log-normal distribution is not available when the sample contains negative values
Fig. 23.The first (left) and the second (right) page of the histogram assistant. The first page allows you to
specify the quantities for which you want to draw histograms, while the second page allows you to
specify the binning parameters
37
Fig. 24. Result histogram dialog. It stores all histograms that you have generated from the results of force
curve processing. The list of the samples (here named group of curves) (c) allows you to navigate
between histograms (d). For each sample several histograms can be generated for different quantities
for example for Youngs modulus and for transition indentation.
median
upper quartile
lower quartile
arithmetic mean
upper fence, i.e. the greatest value less than median + 1.5 times interquartile range
lower fence, i.e. the smallest value greater than median 1.5 times interquartile range
outliers, i.e. data greater than upper fence or less than lower fence
38
To create box plots from results of force curve processing, you click the Add box plot
toolbar item in the Results dialog (see section F.1). The Box plot assistant will popup. Its first
page contains checkbox with names of all quantities that were calculated from the force curves.
Select the quantities that you want to plot and click the Next button to move to the next page of
the assistant.
The Box plot assistant now displays its second page, which allows you to select the force curves
that will comprise the first sample. Click the Add button to choose the force curves from the
processing results. The number of samples visualized in a box plot is not limited to add new
samples, click the Next button. When you are done, click the Finish button in the Box plot
assistant. Box plots will be then created.
39
The Test results dialog is equipped with the main menu and a set of buttons. The following
actions can be performed:
Save (Ctrl + S) saves the test results to a text file (TSV or CSV)
Print (Ctrl + P) prints the test results
Close dismisses the dialog.
Fig.26. First (on the left) and second (right) page of the inference assistant.
40
Fig. 28. Live statistics dialog. The statistics are grouped in several tables (c), each for a different quantity,
under its own tab. Each table contains statistics for all batches taken together (i.e. all results in the
Results dialog) and for each batch separately.
Statistics dialogs can be displayed from the Results dialog (Fig. 21):
to display descriptive statistics for all results, click the toolbar icon
to Statistics (Alt + T) Descriptive statistics (Alt + D).
to display descriptive statistics for part of results, select the corresponding rows in the
results table, then move the mouse over the selected rows and click the right mouse
button. Click Selection statistics in the context menu (Fig. 21 e).
(Fig. 21 b) or go
The statistics dialogs are equipped with the main menu (Fig. 28 a) and a set of buttons (b), that
allow you to print, save to a file or customize the calculated statistics:
to save the currently displayed table to a file, use the shortcut Ctrl + S, or click the Save
button (Fig. 28 b), or go to File (Alt + F) Save (Alt + S). In each case, a file dialog will be
displayed, which lets you choose where the table should be saved and in which format
(TSV or CSV).
42
to print the currently displayed table, use the shortcut Ctrl + P, or click the Print button
(Fig. 28 b), or go to File (Alt + F) Print (Alt + P)
F. 8. Recalculation
You can recalculate all curves, the selected curves or only the curves from a particular batch. If
you want to delete old values, select the checkbox Delete old calculations, otherwise both old
and new results will be stored. Independently, you may specify whether the old curve charts
should be removed (select Delete old curve charts ) and whether the maps of mechanical
quantities (if any) should be modified (select Modify maps in place), or whether new maps
should be created and old ones kept intact.
F.9. Graphical results dialog
The Graphical results dialog stores charts generated during processing of force curves. Three
different types of charts can be generated for each curve (depending on the settings in the
Processing assistant):
43
The Graphical results dialog contains a main menu bar (Fig. 30 a), a toolbar (e), a set of buttons
(b) for the most commonly used actions, a central split pane, with the list of processed force
curves on the left (Fig. 30 c) and the tabbed panel displaying the corresponding charts on the
right, with each type of chart under its own tab (d). The menu bar contains four menus. The File
menu and Measure menu contain the options we discussed in the section D.3. Force curve
dialog.
Toolbar options:
Recalculate opens the Recalculate assistant (see section F.8. Recalculation).
Correct contact point - if pressed , you can drag the contact point with the
mouse. The force curve will be instantly recalculated, also the corresponding
maps and result dialog entries will be updates.
44
Correct transition point - if pressed , you can drag the transition point with the
mouse. The force curve will be instantly recalculated, also the corresponding
maps and result dialog entries will be updates.
Mark on the maps marks the position corresponding to the selected curves on
maps of the mechanical quantities, if they are available.
Measure lets you measure distances on the chart.
Crop lets you crop the end of all the curves or the selected ones.
Gaussian smoothing lets you smooth the curves (all or the selected ones) with
Gaussian filter.
Median smoothing lets you smooth the curves with the median filter.
Convolve
Raw data opens a raw data dialog (see the section D.2. Raw data dialog), containing
the coordinates of points for the selected force curves.
Overlay (Ctrl + Y) lets you plot the results for two force curves in a single chart. It opens
the Overlay assistant, which let you select the curves to overlay. This feature is useful
when you want to compare two different force curves or to compare the results of
processing obtained for the same curve, but with different settings, as in Fig. 31.
The Data menu (Alt + D) allows you fix contact point, i.e. to move it to the origin of the plot
coordinate system. All other points in the charts are translated accordingly.
Fix contact fixes contact point in the force curve plot that is
currently displayed.
45
Fix contact for all fixes contact point in all force curves
plots stored in the dialog.
The list of the processed curves has a context menu containing one additional action Jump to
results, which shows the numerical results for the selected force curves in the Results dialog.
46
Normal mode
Measure distance mode
Extract profile mode (or Extract slice for stacks)
ROI modes:
For line charts and histograms, only the normal and the measure distance mode can be used. All
mouse modes are available for maps, images and stacks. For slices you can use all the modes
except the extract profile mode.
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a) left click anywhere in the chart, then drag the mouse in any direction except in
the left down direction.
b) roll down the mouse wheel.
c) click Zoom-out action, accessible from the context menu. You can zoom out both
or just a single axis. The context menu is triggered by a right mouse click.
Panning to pan a chart (also when zoomed), hold down the Ctrl key, then left click
anywhere in the chart and drag the mouse
Adjusting title, legend and scale bars positions to move a title, legend or a scale bar
drawn inside the plot (i.e. within the data area), move the mouse over it, press the left
button and drag. The element you caught will follow the mouse movements.
to switch on/off this mode, click Measurements in the menu bar, then select the type of
the measurement in the drop-down menu. Three options are available: Line
measurement, Polyline measurement and Free hand measurement. In the Image dialog
and the Map dialog, you can click one of the toolbar icons with a pair of compasses
instead:
(line),
(polyline) or
(free hand measurement).
to add a new measurement, click the left mouse button on the start point, then move the
mouse towards the end point. To finish drawing a linear measurement, just click the left
48
button at the endpoint. For polyline measurements, you can add nodes with left mouse
click, while to finish drawing a polyline or free hand measurement, click the right mouse
button. When drawing is finished, a distance measurement dialog will pop up, containing
detailed information about the length and position of each measurement line.
to translate a measurement, move the mouse over the measurement line, press the left
mouse button and drag the mouse. The measurement line will follow the mouse.
to modify a measurement, move the mouse over the measurement and single click the
left mouse button. Moveable circles will appear at the ends of the line measurement, or in
the nodes of the polyline/free hand measurement. To reposition one of the endpoints or
nodes, move the mouse over the circle, press the left button and drag the mouse. To hide
the circles, single click the left button on the measurement line.
to remove a measurement, move the mouse over the measurement line and double click
the left mouse button.
The style of the measurement lines can be modified with the Measurement style dialog (go to
Measure Measurement style). For details, see the section H.2. Edit measurement style.
G.3. Extract profile mode
In this mode, you can extract height profiles from maps, images or stacks along an arbitrary
section line, polyline or free-hand path. For multi-channel images and maps, profiles are
automatically extracted from all the channels along the profile. This makes it easier to find
correlations between the imaged properties.
(line),
(polyline) or
(free hand
2. in a Stack dialog, go to Sections (Alt + T), then select either Line section, or Polyline
section, or Free hand section in the drop-down menu.
49
to add a new profile, click the left mouse button on the start point, then move the
mouse towards the end point. To finish drawing a linear profile, just click the left button
at the endpoint. For polyline profiles, you can add nodes with left mouse click, while to
finish drawing a polyline or free hand profile, click the right mouse button. The section
line will be drawn on all channels of the current image and the cross-section dialog will
pop up, containing chart with height profile extracted from all channels.
to translate a profile, move the mouse over it, press the left mouse button and drag the
mouse. The line will follow the mouse.
to modify a profile, move the mouse over it and single click the left mouse button.
Moveable circles will appear at the ends of the line profiles or the nodes of the
polyline/free hand profiles. To reposition one of the endpoints or nodes, move the
mouse over the circle, press the left button and drag the mouse. To hide the circles,
single click the left button on the line.
to remove a profile, move the mouse over the cross-section line and double click the left
mouse button.
Profiles are equipped with triangular, moveable markers. For each marker, another marker
is drawn in the cross-section chart in the same position. Both in the original image and the
cross-section chart markers can be moved, added or removed with the mouse.
to add a new marker, move the mouse over the section line and click the right mouse
button or double click the plotted curve in the cross-section chart.
to move a marker, move the mouse over, press the left mouse button and drag the
mouse. The marker will follow the mouse.
to remove a marker, move the mouse over and double click the left mouse button.
The style of section lines can be modified with the Profile style dialog (click Profiles in the main
menu bar, then Profile style). For details, see the section J.4. Editing style of profile lines.
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G.4.ROI modes
ROI modes allow you to select regions of interest of different shapes. Various actions can be
then applied to these areas. For multi-channel images and maps, ROIs are selected for all the
channels at once.
to switch on/off this mode, go to ROI (Alt + R), then select from the drop-down menu
the ROI mode of your choice, one of the following: Polygon ROI, Rectangular ROI,
Elliptic ROI or Free hand ROI. In the Image dialog and the Map dialog, you can select
the right shape from the toolbar.
to create a rectangular or elliptic ROI, click the left mouse button at the desired
position. A small ROI will be drawn, which can be then reshaped.
to create a polygon ROI, click the left mouse button, move the mouse to the next
vertex and click the left mouse button there. Repeat this until all vertices are added.
To finish, click the right mouse button.
to create a free-hand ROI, click the left mouse button and move the mouse. ROI
boundary will follow mouse movement. To finish, click the right mouse button.
to reshape a rectangular or elliptic ROI, move the mouse over its edge, press the left
mouse button and drag the mouse.
to reshape a polygon or free hand ROI, move the mouse over its edge and single click
the left mouse button. Moveable circles will appear at the vertices. To reposition one
of them, move the mouse over the circle, press the left button and drag the mouse.
To hide the circles, single click the left button on the line.
to translate a ROI, move the mouse over it, press the left mouse button and drag the
mouse. The ROI will follow the mouse.
to remove a ROI, move the mouse over its edge and double click the left mouse
button.
You can also draw ROIs which includes whole image, except for a hole (Fig. 33 B). The shape of
the hole depends on the current ROI mode. For example, when drawing holes is switched on
and the Rectangular ROI mode is on, you can select a ROI which includes the whole image
51
except for a rectangle. To draw ROIs with a hole, click ROI in the menu bar, then select in the
drop-down menu the Draw ROI hole item
Fig. 33. Regular Rectangular ROI (A) and a Rectangular ROI with a hole (B). The area inside ROI is
whitish.
The style of ROIs can be modified with the ROI style dialog (go to ROI ROI style). For details,
see the section J.9. Editing style of ROIs.
52
The dialog consists of the main menu bar (Fig. 34 a), a set of buttons (b) for the most commonly
used actions, and the central tabbed pane. Each tab contains an editor with controls for
modifying style of different chart elements (d).
The main menu bar contains the Style menu (Alt + T), with six options:
Reset to defaults (Ctrl + R) resets the style for the current chart element to the default
style (see above).
Undo (Ctrl + U) reverts the changes introduced to the current chart element.
Batch apply all (Ctrl +Shift + B) applies the style for all chart elements to all charts of the
same type in the same dialog. Charts of the same type are usually grouped under the
same tab in a chart dialog.
Save all as defaults (Ctrl + Shift + S) saves the style for all chart elements to an OSdependent backing store. From now on, this style will be used to as the default style to
draw the charts of the same type as the currently modified chart.
Reset all as defaults (Ctrl + Shift + R) resets the style for all chart elements to the default
style
Undo all (Ctrl + Shift + U) reverts the changes introduced to any of the chart elements
The chart is instantly updates when its style is edited. This feature helps you to find the best
style for the chart, as you can immediately see what the effects of the changes you are
introducing are.
The Live chart dialog consists of separate editors, each for different chart element. The number
and type of editors depend on the type of chart. Those editors are available for every chart:
a. Series editor
Series editor lets you modify the way the data are renderer. Different forms of this editor are
available for different types of charts. A series editor for a line chart is displayed in Fig. 34.
A more detailed description of series editors specific for histograms, images and maps can be
found in later sections.
b. Plot editor
Plot editor (Fig. 35) lets you modify the style of the plot, understood here as the inner area of
the chart, where data are actually draw. The editor allows you to set the plot background, to
specify the stroke of plots outline, hide or show range and domain gridlines, as well as to
change their stroke and color. The interval between gridlines is always equal to the interval
54
between the ticks on the corresponding axis. In addition, you can select the plot orientation.
There are two options:
1. Vertical orientation - the domain axis is horizontal, the range axis is vertical. This is the
usual orientation.
2. Horizontal orientation - the domain axis is vertical, the range axis is horizontal.
Fig. 35. The editor for plot - the inner area of the chart.
c. Axes editor
Axes editor (Fig. 36) contains separate subeditors for each axis. Most line charts contain two
axes domain and range axis, while stacks, as well as image and map channels contain also a
third axis depth axis, which is drawn next to the gradient bar. Those subeditors give you
control over every detail of the axis. Here, you can hide or show the axis, invert it or change its
range. You can also modify its main label, tick marks, ticks labels, axis line, position in the chart
or the number format (ex. the decimal separator).
You will usually want to keep the style of all axes the same. Axes editor allow you to choose
whether the axes are allowed to differ in their style:
to ensure that changes in style of one axis are also applied to other axes, select (press
in) the chain link button. When selected, its icon changes from
to . .
55
Fig. 36. The editor for axes. The style can be set separately for each axis. Press in the chain link
button if you want the same style to be used for all axes.
The Axes editor can be opened as a tab in the Live chart dialog, but it can be accessed even
faster:
to open the Axes editor, move the mouse cursor over one of the axes and double click
the left mouse button.
d. Title editor
The Title editor (Fig. 37) lets you add or modify the title of the chart. It consists of three tabs:
1. Text tab allows you to set text of the title, its font and color. Instead of simple color, you
can fill the letters with a gradient (check in the Use gradient box and then click the
Select button to choose the gradient).
2. Frame tab allows you to choose whether frame should be drawn around the title. You
can select the color and stroke of the frame, and the color of the title background.
3. Position tab allows you to choose whether the title should be drawn inside or outside
the area of the plot occupied by plotted data and in which position. Here you can also
set the title padding and margins. Note that the title can be simple dragged with the
mouse on the chart for details see the section G. Mouse actions on chart.
56
The Title editor can be opened as a tab in the Live chart dialog, but it can be accessed even
faster:
to open the Title editor, move the mouse cursor over the title and double click the left
mouse button.
to open the Scale bar editor, move the mouse cursor over the scale bar and double click
the left mouse button.
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f. Legend editor
Legend editor (Fig. 39) lets you change the style and position of a legend. If the chart has
multiple legends, for each of them a separate editor is included in the Live chart style dialog.
The Legend editor contains two or three (depending on the type of the legend) tabs:
1. Position tab lets you choose whether the legend should be drawn inside or outside the
area of the plot occupied by plotted data and in which position. Here you can also set
the legend padding and margins. Note that the legend can be simple dragged with the
mouse on the chart for details see the section G. Mouse actions on charts.
2. Style tab lets you set the font and color used for text in the legend. You can also choose
whether frame should be drawn around the title, select the color and stroke of the
frame, and the color of the title background. Note that legends for image and map
charts contain depth axes that can be edited from the Axes editor, but not from the
Legend editor.
3. Strip tab lets you set the outline and width of the color gradient strip that is the main
part of legends for image and map charts. This tab is included in the Legend editor only if
the edited legend contains a strip.
58
g. Chart editor
Chart editor (Fig. 40) lets you change the chart padding, background color and whether
antialiasing should be used. In addition, the lock aspect ratio action allows you to specify
whether natural aspect ratio of the chart (i.e. determined by unit lengths on the axes) should be
preserved when the dialog is resized.
59
The dialog (Fig. 41) lets you set different style for finished measurements and the measurement
lines that are being drawn. The measurement lines get instantly updated when you introduce
changes to their style, which makes it easier to find the optimal style parameters.
Batch apply (Alt + B) applies this measurement style to all charts of the same type in the
same dialog. Charts of the same type are usually grouped under the same tab in a chart
dialog.
60
61
H.3.Gradient dialog
The gradient dialog (Fig. 43) allows you to modify the color gradient used to find the color for
each z-axis value (ex. height in topographical images). The range and the colors that form the
gradient are important when you want to bring out interesting features of your image, map or
stack.
The Range panel is where you specify the range of the gradient. The minimal value will
be colored with the first color in the gradient, the maximal value with the last one. You can
specify the range in three ways:
1)
2)
The ROI masks panel allows you to specify a different color, independent of the gradient, which
will be used for the parts of the chart outside or inside regions of interest.
H.4. Editing color gradients
Color gradient editor (Fig. 45) allows you to modify the current color gradient or to choose a
new one. A gradient is determined by its color nodes, their position and the method of
interpolation between them. The control over the color gradient is provided by interactive
gradient bar (Fig. 45 a), a set of buttons (b) and widgets that control how colors are interpolated
(c).
63
to replace a color node, move the mouse over the droplet right of the gradient bar and
double click the left button. The Color chooser will appear. Choose the new color node
and click OK.
H.5. Gradient range dialog
The Gradient range dialog contains the data histogram plotted against the color gradient used
for their visualization (Fig. 46). The area painted with the color gradient is flanked by markers.
The gradient range can be specified in several ways:
to select the range manually, move the mouse over one of the markers flanking
colored area, press the left mouse button and drag the marker. Release the left
button when the marker is in the right position.
to choose the automatic range, which contains all the data except the distribution
tails, go to Range (Alt + G) Restore automatic (Ctrl + R) .
to choose the full range, which fits all the data, go to Range (Alt + G) Restore full
(Ctrl + Shift + R)
You can modify histogram binning to reveal all its features. Go to Data (Alt + D) Modify
binning (Ctrl + B)
Fig. 46. Gradient range dialog. To modify the range of values that the gradient is
applied to, drag with the mouse the lines on the edges of colored area.
64
65
AtomicJ gives you much flexibility in saving charts. It supports multiple image formats, both
raster (TIFF, JPEG, JPEG2000, Windows Enhanced Metafile, GIF, PPM, Bitmap and AVI for
movies) and vector formats (EPS, PS, PDF, SVG), as well as text formats (TSV and CSV). You can
save a single chart or multiple charts at once. The actions described in this section are applicable
to any type of charts, except the part on saving as a movie, which is applicable only to stacks.
66
vi. Save only data area. If this checkbox is selected, only the inner part of the chart, where
the image is actually drawn, is saved. Axes are left out, just are those legends or titles
that are draw on the margins of the chart.
Fig. 48. Dialog for setting options for the EPS file format. Dialogs for the PDF and PS formats are
identical.
Size of the image file. Enter the Width and Height of the image in printers points.
ii.
Keep aspect ratio. If this checkbox is selected, the aspect ratio of the chart is preserved
when you change the width, the height is updated accordingly, and the other way round.
iii.
Save only data area. If this checkbox is selected, only the inner part of the chart, where
the image is actually drawn, is saved. Axes are left out, just are those legends or titles
that are draw on the margins of the chart.
Fig. 49. Dialog for setting options for the PNG file format. Dialogs for the SVG, GIF, PPM and
bitmap formats are identical.
67
Compression. You can save the file Uncompressed, or use the LZW (lossless Lempel-Ziv
and Welch algorithm) or PackBits (lossless) compression.
ii.
Size of the image file. Enter the Width and Height of the image in printers points.
iii.
Keep aspect ratio. If this checkbox is selected, the aspect ratio of the chart is preserved
when you change the width, the height is updated accordingly, and the other way round.
iv.
Save only data area. If this checkbox is selected, only the inner part of the chart, where
the image is actually drawn, is saved. Axes are left out, just are those legends or titles
that are draw on the margins of the chart.
Fig. 50. Dialog for setting options for the TIFF file format.
Quality. The image quality can be specified as a number between 1 (highest quality) and
0 (lowest).
ii.
Size of the image file. Enter the Width and Height of the image in printers points.
iii.
Keep aspect ratio. If this checkbox is selected, the aspect ratio of the chart is preserved
when you change the width, the height is updated accordingly, and the other way round.
68
iv.
Save only data area. If this checkbox is selected, only the inner part of the chart, where
the image is actually drawn, is saved. Axes are left out, just are those legends or titles
that are draw on the margins of the chart.
Fig. 51. Dialog for setting options for the JPEG file format.
JPEG 2000.
i.
ii.
Quality. The image quality can be specified as a number between 1 (highest quality) and
0 (lowest). Quality cannot be set when lossless compression is used.
iii.
Size of the image file. Enter the Width and Height of the image in printers points.
iv.
Keep aspect ratio. If this checkbox is selected, the aspect ratio of the chart is preserved
when you change the width, the height is updated accordingly, and the other way round.
v.
Save only data area. If this checkbox is selected, only the inner part of the chart, where
the image is actually drawn, is saved. Axes are left out, just are those legends or titles
that are draw on the margins of the chart.
69
ii.
Trailing zeroes. The checkbox controls whether the trailing zeroes are shown.
iii.
iv.
Thousand separator (space, comma, dot or apostrophe. The Use separator checkbox
control whether the thousand separator is shown.
Fig. 52. Dialog for setting options for the CSV file format. The dialog for TSV format is identical.
AVI. Stack can be saved as a movie in this format. You can specify:
i.
Compression. You can save the movie Uncompressed, or use the PNG
compression (lossless, please note that you may need a codec to open the file!)
or JPEG compression (lossy).
ii.
Backwards. If this checkbox is selected, the movie is saved in reverse (as if played
backwards)
iii.
First frame. The first frame of the stack that will be included in the movie. You
can set this to a higher value to skip the initial frames of the stack, which often
appear noisy due to low signal-to noise ratio.
iv.
Last frame. The last frame of the stack that will be included in the movie. You can
set this to a smaller value to skip the last frames of the stack.
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v.
Rate. The rate of frames per second. Changing the frame rate results in the
changes of the movie length, because the number of frames is constant.
vi.
Length. The length of the movie. Changing the movie length results in the
changes of the frame rate, because the number of frames is constant.
vii.
Size of the frames. Enter the Width and Height of the frames in printers points.
viii.
Keep aspect ratio. If this checkbox is selected, the aspect ratio of the stack
frames is preserved when you change the width, the height is updated
accordingly, and the other way round.
ix.
Save only data area. If this checkbox is selected, only the inner part of the chart,
where the image is actually drawn, is saved. Axes are left out, just are those
legends or titles that are draw on the margins of the chart.
Fig. 53. Dialog for setting options for the AVI file format. The dialog allows you specify the
compression method, the range of the movie (i.e. which frames should be included), its speed
and the size of an individual frame.
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1.
2.
3.
click File (Alt + F) in the menu bar in the dialog where the chart is displayed, then click
Save (Alt + S) in the drop-down menu.
move the mouse over the chart, then click the right mouse button. Click the Save as
item in the context menu.
use the shortcut Ctrl + S.
In each case, the Save dialog will be
displayed (Fig. 54). It lets you specify
where the chart is to be saved (the
Browse button, Alt + B), select the
format and specify the saving options
(the Options button, Alt + P). The
saving options are format specific
and are described above.
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click File (Alt + F) in the menu bar in the dialog where the chart is displayed, then click
Save all in the drop-down menu.
click the Save all button at the bottom of the chart dialog
use the shortcut Ctrl + Shift + S.
The Batch save dialog will be then displayed. The dialog is where you specify which charts are to
be saved and where, in which format, and how to name the files. It consists of three tabs:
General tab (Fig. 55). Here, you
specify:
Fig. 56. Dialog for saving multiple charts at once. The tab with format settings is displayed.
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Prefix by default the name of the type of chart plus underscore (ex. Youngs
modulus_).
Root the central part of the name, by default the name of the file with the AFM
recording corresponding to the chart.
Suffix - by default a number incremented for each chart of a given type.
4.
Arbitrary string. The string can be typed into the appropriate field in the File
naming tab.
Name of the file with the AFM recording corresponding to the chart
Serial number, i.e. the file with the AFM recording corresponding to the chart.
The initial serial number, used for the first chart, can be set in the Initial serial #
field.
Type of the chart plus underscore (ex. Youngs modulus_ ).
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Format tab. Here you can specify the options for the AVI format (currently the only movie
format available) that are described in the subsection Options for file formats.
File naming tab. This tab is similar to the File naming tab in the Batch save dialog, described in
the previous section.
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Fig. 59 The Image dialog stores the AFM images opened in the current session. It contains a list of image
files (d) and a central panel (c), displaying each image channel under its own tab. Images can be
manipulated and analyzed through action accessible from the main menu bar (a), toolbar (b) and a set
of buttons (c).
The Image dialog is equipped with a menu bar (Fig. 59 a), a toolbar (b) and a set of buttons (c)
for the most commonly used actions and a list for navigating between the read-in image
recordings (d). The list is equipped with a context menu (d). The central panel (e) displays the
channels for the currently selected image recordings, each under its own tab.
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While working with images, you will use mainly the actions included in the menus from the
menu bar and the toolbar, which provide easy access to the most frequently used actions.
Toolbar options :
Draw histogram (Ctrl + D) lets you generate histograms for whole image
channels.
Draw ROI histogram (Ctrl + Shift + D) lets you generate histograms of values
inside or outside selected regions of interest (ROIs).
Show histograms (Ctrl + H) opens a histogram dialog with all the histograms
generated for images in the current session. It is initially disabled, as there are
no box plots to show.
Show box plots opens the Box plots dialog, which stores all box plots generated
for image ROIs in the current session. It is initially disabled, as there are no box
plots to show.
Draw ROI box plots. opens the Box plots assistant, which lets you generate box
plots for selected ROIs. See also section F.5. Creating box plots.
Statistics (Ctrl + T) shows statistics (including mean, median, variance) for each
image channel.
Statistics ROIs (Ctrl + Shift + T) shows statistics for the selected ROIs
Polygon ROI toggles polygonal Regions of Interest (ROIs), changing the way
mouse clicks are interpreted by the chart. If this action is switched on you can
add a new polygonal ROI with the mouse. See the section J.5. Creating and
modifying Regions of Interest.
Rectangular ROI toggles rectangular ROIs
Elliptic ROI toggles elliptic (this includes circular) ROI
Free hand ROI toggles free-hand ROIs.
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Wand ROI tool lets you build a ROI which includes a part of image with values
close to the value in an image position selected with the mouse.
Line measurement lets you draw a new straight line measurement on the image
with the mouse.
Polyline measurement lets you draw a polyline measurement (i.e. composed of
several connected line segments) with the mouse.
Free hand measurement lets you draw a polyline measurement with the
mouse. The measurement line will follow the movement of the mouse.
Line profile lets you draw profile lines on the image with the mouse. The crosssection dialog appears, displaying height profiles for all profile lines.
Polyline profile lets you draw a polyline profile (i.e. composed of several
connected line segments) with the mouse. Click left mouse button to add new
profile node (segment of the polyline), click right mouse button to finish
drawing of the profile.
Free hand profile lets you draw a polyline profile with the mouse. The profile
will follow the movement of the mouse. Click right mouse button to finish
drawing of the profile.
Convolve opens the Convolution dialog, which lets you convolve the image with
a kernel of arbitrary size.
Overlay (Ctrl + Y) lets you add channels from another image as new layers to the
charts generated for the currently selected image. It brings up the Overlay
assistant. Charts for the currently selected image are copied and new image is
created.
Resize lets you add a new layer to the currently selected image chart, containing
the same data, but with the number of pixels increased through bilinear or
bicubic spline interpolation. See: Smoothing dialog.
Lock aspect - when switched on (the button is pressed in), the natural aspect
ratio of the image (i.e. determined by its width and height in microns) is
preserved when the dialog is resized.
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Menu options:
The menu bar contains four menus and can be navigated with the mouse or using keyboard
mnemonics and accelerators in brackets:
File menu (Alt + F) (the same as in the Force curve dialog, see section D.3.)
Measure menu (the same as in the Force curve dialog, see section D.3.)
o
o
o
Live chart style (Ctrl + L) opens the Live chart style dialog.
Click to modify the way image is plotted.
Edit color gradient (Ctrl + G) opens the Color gradient
dialog. See Color gradient edition for details.
Gradient histogram (Ctrl + Shift + G) opens the Gradient
range dialog, where you can easily set the range of color
gradient against the image histogram. See Color gradient
edition for details.
Resize (Ctrl + M) (also in the toolbar)
Overlay (Ctrl + Y) (also in the toolbar
Lock aspect (also in the toolbar)
Data menu:
o
o
o
o
o
Process menu:
o Fix zero (Ctrl + F) subtracts the lowest value from the current image channel. For
example, if the lowest value on a topography image is 0.2 m, this value is
subtracted from each pixel in the image.
o Remove background (Ctrl + I) allows you to subtract a polynomial fitted to the
whole image, or the area that is inside or outside regions of interest.
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o Match lines
o Line fit correction
o Add function allows you to add a linear function (a plane) to the whole image
channel, or to the area inside or outside the selected ROIs.
o Threshold data allows you to replace with a constant value the parts of the image
that are either greater or lesser than a threshold. This can applied to the whole
image channel, or to the area that is inside or outside the ROIs.
o Replace allows you to replace the data inside or outside the ROIs with a linear
function. In particular, you can replace them with a constant.
o Transform is a submenu for basic image operations: flipping (i.e. reflection through
a vertical or horizontal line that goes through the center of the image), transposition
(reflection through diagonal ) and rotation.
o Filter is a submenu with several image processing actions for edge detection (Sobel,
Prewitt, Laplacian of Gaussian), enhancing (sharpening, unsharp mask), noise
removal (weighted median filter, Gaussian blur), derivative calculation and general
convolution.
ROI (Alt + R) menu, containing fifteen actions, most of which
are disabled unless at least one region of interest is selected:
o
o
ROI style opens a dialog for editing the style with which the
ROIs are drawn.
Apply mask (Ctrl + K) opens the ROI mask dialog, which lets
you apply a distinct color to the area inside or outside
regions of interest.
Draw histograms for ROIs (Ctrl + Shift + D) opens the
Histogram assistant, which lets you generate histograms for
the area inside ROIs. You can create at once a histogram for
each channel of the selected image.
Live ROI histogram (Ctrl + Shift + H) opens the Live histogram dialog, displaying
histograms of the data inside regions of interest for each channel of the currently
selected image. These histograms are instantly updated when you move, add or
delete a ROI.
Live ROI box plots opens the Box plot dialog, displaying box plots of the data inside
regions of interest for each channel of the currently selected image. These
histograms are instantly updated when you move, add or delete a ROI.
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o Raw data for ROIs (Ctrl + Shift + W) opens the Raw data assistant where you can
select the image channels for which you want to view raw data. The raw numerical
values for pixels inside regions of interest are then displayed in a raw data dialog.
o Statistics for ROIs (Ctrl + Shift + T) opens a statistics dialog, containing mean,
median, variance and other statistics for each image channel for data inside the
regions of interest
o Shape factors for ROIs (Ctrl + Shift + U) opens the ROI shape factors dialog with
detailed information about the geometry of each region of interest selected on the
current image. See the section J.9. ROI Shape factors dialog.
o Correct ROI plane (Ctrl + Shift + I) fits a plane the data inside regions of interest and
subtract it from the whole image. This action is useful for background removal.
The next four actions allows you to draw new regions of interest:
o
o
o
o
Polygon ROI switches on drawing Polygon ROIs (the same as in the toolbar)
Rectangular ROI switches on drawing Rectangular ROI (the same as in the toolbar)
Elliptic ROI switches on drawing Elliptic ROIs (the same as in the toolbar)
Free hand ROI switches on drawing Free hand ROIs (the same as in the toolbar)]
The last action controls the behavior of all ROI selection modes:
o Draw ROI hole controls whether in ROIs modes the shape that you draw with the
mouse is the inside or outside the ROI. If this action is selected, the shape is treated
as a hole in a larger ROI that encompasses all image
o
o
o
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to extract a new profile, click Profiles (Alt + P) in the menu, then select the type of the
profile: Line profile, Polyline profile or Free hand profile. This will turn on the Extract
profile mode, in which you can draw the profile line with the mouse. Please refer to the
section G. Mouse actions on charts for more details. The Profile dialog will be displayed
(Fig. 60).
to translate a profile, move the mouse over it, press the left mouse button and drag the
mouse. The line will follow the mouse and the cross-section will be instantly updated.
to modify a profile, move the mouse over it and single click the left mouse button.
Moveable circles will appear at its ends of line profiles and nodes of the polyline/free
hand profiles . To reposition one of the endpoints or nodes, move the mouse over the
circle, press the left button and drag the mouse. To hide the circles, single click the left
button on the line. The cross-section will be instantly updated.
to remove the profile, move the mouse over the cross-section line and double click the
left mouse button.
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to add a new marker, move the mouse over the section line and then click the right
mouse button. In the Image dialog, a new triangular marker will be drawn on the profile
line. In the Profile dialog, a new triangular marker will be drawn in the corresponding
position on the height profile.
to move a marker, move the mouse over the triangular marker, press the left mouse
button and drag the mouse. The marker will move along the section line.
to remove a marker move the mouse over the triangular marker, then double click the
left mouse button.
to add a new marker, move the mouse over the plotted profile. When the mouse cursor
changes to a cross, you can add markers by clicking the left mouse button. In the Image
dialog, a new marker will be drawn in the corresponding position on the profile line.
to move a marker, move the move over the triangular marker, press the left mouse
button and drag the mouse.
to remove a marker move the mouse over the triangular marker, then double click the
left mouse button.
to view the length, angle and position of the profile lines, go to Profiles (Alt + T)
Profile geometry in the Image dialog. A dialog with geometrical parameters of profile
lines will be displayed.
to edit the style of profile lines, click Profiles (Alt + T) Profile style in the Image
dialog. The Edit profile style dialog will be displayed. For more details on how to use this
dialog, see section H. Customizing style of charts.
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The Profile dialog (Fig. 60) stores charts of height profiles. The center of the dialog is occupied
by a list of image names and a tabbed panel, displaying profiles for different channels. Multiple
height profiles can be plotted on a single chart, if multiple profile lines were drawn on the
image. The Profile dialog is equipped with the main menu bar (Fig. 60 a) and a set of buttons (b)
for saving and clearing profiles. To show profile dialog from the Image dialog, use the shortcut
Ctrl + Shift + X, or go to Profiles (Alt + P) Show profiles.
Fig. 60 Profile dialog, containing height profiles for two different section lines for each channel
of an image. Topographical profiles are visible.
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You can also specify the number of points calculated for the cross-section, i.e. its resolution. For
image profiles, the default value is always 200. The choice of interpolation method influence the
visual appearance of the height profile, and the influence is greatest for low resolution images
(Fig. 62). To change the interpolation method, use the Profile interpolation dialog (Fig. 61). To
display it, use the shortcut Ctrl + I or click Interpolation (Alt + I) in the main menu bar of the
cross-section dialog, then click Change section interpolation (Alt + S).
Fig. 61. Dialog for specifying the method of interpolation and the number of points making up a height
profile. Nearest neighbor, bilinear and bicubic spline interpolation methods are supported.
The section interpolation dialog allows you to select the method of interpolation and the
number of points that make up the profile. The profiles are immediately updated to reflect the
introduced changes. The dialog is equipped with three buttons.
1.
2.
3.
The Apply to all button (Alt + A) applies the interpolation to profiles. This button is
disabled for horizontal and vertical cross-sections.
The Reset button (Alt + R) discards all the changes you have made.
The Close button (Alt + C)
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Fig. 62. Influence of interpolation method on the appearance of a height profile. The height profile was
cut along a diagonal of a 128 x 128 AFM topography image. A Nearest neighbor interpolation, B
Bilinear interpolation, C Bicubic spline interpolation.
The Profile style dialog allows you to set different style for finished profile lines and the profile
lines that are still being drawn. Profile lines get instantly updated when you introduce changes
86
to their style, which makes it easier to find the optimal style parameters. You can edit the stroke
of the profile lines, the size of the arrowhead that shows the direction of the profile, and the
color and font of the labels.
Actions accessible through buttons (Fig. 63 b):
Batch apply (Alt + B) applies this profile line style to all charts of the same type in the
same dialog. Charts of the same type are usually grouped under the same tab in a chart
dialog.
Save as defaults (Alt + S) saves this profile line style to a OS-dependent backing store. In
case of MS Windows, the backing store is simply the Windows Registry. From now on,
this style will be used to as the default style to draw profile lines on the charts of the
same type as the currently modified chart.
Reset to defaults (Alt + R) resets the style of profile lines to the default style (see above).
Undo (Alt + U) reverts the introduced changes.
or go to ROI (Alt + R)
or go to ROI (Alt +
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to select the free hand ROI mode, press in the toolbar icon
(Alt + R) Free hand ROI
or go to ROI (Alt + R)
or go to ROI
Once you have selected the type of ROI you need, you can draw and modify it with the mouse.
to create a rectangular or elliptic ROI, click the left mouse button at the desired
position. A small ROI will be drawn, which can be then reshaped.
to create a polygon ROI, click the left mouse button, move the mouse to the next vertex
and click the left mouse button there. Repeat this until all vertices are added. To finish,
click the right mouse button.
to create a free-hand ROI, click the left mouse button and move the mouse. ROI
boundary will follow mouse movement. To finish, click the right mouse button.
to reshape a rectangular or elliptic ROI, move the mouse over its edge, press the left
mouse button and drag the mouse.
to reshape a polygon or free hand ROI, move the mouse over its edge and single click
the left mouse button. Moveable circles will appear at the vertices. To reposition one of
the them, move the mouse over the circle, press the left button and drag the mouse. To
hide the circles, single click the left button on the line.
to translate a ROI, move the mouse over it, press the left mouse button and drag the
mouse. The ROI will follow the mouse.
to remove a ROI, move the mouse over its edge and double click the left mouse button.
to merge multiple ROIs, go to ROI Merge ROIs. A dialog will appear, which will let you
select which ROIs should be merged and whether individual ROIs should be deleted after
merging.
to subtract ROIs, go to ROI Subtract ROIs. A dialog will appear, which will let you
select the operands and whether individual ROIs should be deleted after subtraction.
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This action is unavailable if there are no overlapping ROIs drawn on the currently
displayed image.
to complements ROIs, go to ROI Complement ROIs.
You can also draw ROIs which includes whole image, except for a hole (Fig. 33 B). The shape of
the hole depends on the current ROI mode. For example, when drawing holes is switched on
and the Rectangular ROI mode is on, you can select a ROI which includes the whole image
except for a rectangle. To draw ROIs with a hole, go to ROI Draw ROI hole.
By default, ROIs are identified with consecutive natural numbers. You can also specify a name
for the ROI. Move the mouse over ROIs label, then double - click left mouse. A dialog will
appear, which will lets you specify a new name.
J.6. Descriptive statistics for ROIs.
Descriptive statistics (Fig. 64) are calculated for the area inside all ROIs taken together and for
each ROI separately. For the list of statistics calculated by AtomicJ, see the subsection
Descriptive statistics in Working with the results of curve processing.
The descriptive statistics are instantly updated when you move or reshape ROI, so you can use
ROI to probe the differences between parts of the sample.
Fig. 64. Descriptive statistics calculated for image ROIs. The statistics are grouped in several tables, each
for a different image channel, under its own tab. Each table contains statistics for all ROIs taken
together) and for ROI batch separately. The statistics are instantly updated when you reshape or
translate one of the ROIs.
to display descriptive statistics for ROIs, go to Data Statistics ROIs, or use the
shortcut Ctrl + Shift + T.
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to display histograms already generated in the current session, you click the toolbar
icon
, or go to Data Show histograms, or use the shortcut Ctrl + H.
, or go to Data
Histograms for ROIs are stored in the same dialog as the histograms for the whole image.
to display Live ROI histogram, you can use the shortcut Ctrl + Shift + H, or go to Data (Alt
+ R) Live ROI.
J.8. Box plots for ROIs
Image dialog lets you generate both static and live box-plots for the area inside ROIs. Static box
plots are not updated when you translate or modify ROIs. Live box plots are instantly updated in
response to every modification to ROIs. For more information about box plots, see section F.5.
Creating box plots.
to generate static box plots for ROIs, click the toolbar icon
ROI box plots.
to display histograms already generated in the current session, you click the toolbar
icon
, or go to Data Show box plots.
to display Live ROI box plots go to Data (Alt + R) Live ROI box plots.
or go to Data Draw
90
Fig. 65. Dialog containing shape factors for ROIs. The shape factors are instantly updated when you
reshape or translate one of the ROIs.
ROI the name of the ROI. The same name is used as a ROI label in the chart.
Area area in square microns.
Centroid X the first coordinate of the geometric center
Centroid Y the second coordinate of the geometric center
Perimeter
Box width the width of the smallest rectangle that completely encloses the roi and
whose sides are parallel to the axes
Box height the height of the smallest rectangle that completely encloses the roi ad
whose sides are parallel to the axes
Feret min the shortest feret (or caliper) length
Feret max the longest feret (or caliper) length
Circularity index a measure of circularity, calculated according to the equation:
f
4 A
P2
to display ROI style dialog, click ROI in the main menu bar, the click ROI style in the
drop-down menu.
The ROI style dialog allows you to set different style for finished ROIs and the ROIs that are
being drawn. The ROIs get instantly updated when you introduce changes to their style, which
makes it easier to find the optimal style parameters.
Fig. 67. The inside of the ROI can be left empty (A) or
filled with a color, preferably a semi-transparent one.
Batch apply (Alt + B) applies this ROI style to all charts of the same type in the same
dialog. Charts of the same type are usually grouped under the same tab in a chart dialog.
Save as defaults (Alt + S) saves this measurement style to a OS-dependent backing store.
In case of MS Windows, the backing store is simply the Windows Registry. From now on,
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this style will be used to as the default style to draw ROIs on the charts of the same type
as the currently modified chart.
Reset to defaults (Alt + R) resets the style of ROIs to the default style (see above).
Undo (Alt + U) reverts the introduced changes.
J. 11. Image processing
The Image dialog lets you modify the whole images and the selected Regions of Interest. Height
profiles, descriptive statistics and the Live Histogram are instantly updated whenever you
modify the images.
to ensure that the values in the image starts from 0, you can either:
1. go to Process Fix zero.
2. or move the mouse over the image, then click the right mouse button. Click Fix
zero in the context menu.
3. or use the shortcut Ctrl + F.
This option finds the lowest value in the image and subtracts it from all values in the
image.
to correct the substrate tilt, first draw a ROI (see subsection J.5. Creating and modifying
Regions of Interest (ROIs)) around the substrate in the image (or multiple ROIs), then
either:
1. go to ROI (Alt + R) Correct ROI plane.
2. or move the mouse over the image, then click the right mouse button. Click the
Correct ROI plane in the context menu.
3. or use the shortcut Ctrl + Shift + I.
This options fits a plane to the inside of all selected ROIs taken together and subtracts it
from the whole image.
to threshold the parts of image, go to Process Threshold . The Set data thresholds
dialog (Fig. 68) will be displayed. It lets you specify whether the thresholding should be
applied to the whole image, the area outside or the area inside of ROIs, set the lower
and upper thresholds, and specify the values that will replace the pixels smaller than the
lower threshold and greater than the upper threshold.
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Fig. 68. Dialog for thresholding images. Thresholding can be applied either to the whole image,
or to the area inside or outside the selected ROIs.
to add or subtract a plane, go to Process Add function. The Add function dialog (Fig.
69) will be displayed. It lets you specify whether the plane should be added to the whole
image, or to the area outside or inside the selected ROIs. Here you can also set the
parameters in plane equation.
Fig. 69. Dialog for adding (or subtracting) plane to images. The plane can be added to the whole
image, or the area inside or outsides the selected ROIs
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to replace parts of the image inside or outside ROIs with a place, go to Process
Replace. The Replace data dialog (Fig. 71) will be displayed. It lts you specify whether the
plane should replace the outsides or insides of ROIs and set the plane equation
parameters
Fig. 71. Dialog for replacing insides or outsides of ROIs with a plane.
to convolve the image with an arbitrary kernel, go to Process Convolve. The Convolve
dialog (Fig.) will popup. It lets you specify the components of the convolution matrix and
whether it should be normalized. You can apply the convolution to the whole image, or
only to its selected parts, outside or inside ROIs.
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Depending on the contact model used for force map processing, additional maps may be
generated:
The Map dialog resembles the Image Dialog. All actions that can be performed on images
can also be performed on maps. In addition, the map dialog allows recalculating of force
curves and for creating stacks.
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Fig. 72. The Map dialog stores the maps generated in the current session. It contains a list of processed
files with force-volume recordings (d) and a central panel (c), displaying each map under its own tab.
Maps can be manipulated and analyzed through action accessible from the main menu bar (a), toolbar
(b) and a set of buttons (c).
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to go to the processing results, the mouse over the position of interest, then click the
right mouse button. Click the Find results in the context menu. Unless the results have
been cleared, The Results dialog will be shown, with the rows corresponding to the
clicked position selected.
K.4. Generating stacks
Force volume recordings contain information about the changes in mechanical properties of
the sample across its depth. This information cannot be displayed in a single map, but you can
still extract this information in the form of stacks.
Stacks consist of series of frames and can be thought of as movies. Each frame is a map showing
the spatial distribution of some quantity, calculated either for a particular indentation depth
(Fig. 73 A) or tip displacement (Fig. 73 B). You can generate stacks for the following quantities:
pointwise modulus, calculated for the current indention depth or tip displacement
stiffening, which is the difference between Youngs modulus calculated from the fit
to force indentation curve and the pointwise modulus, calculated for the current
indentation depth or tip displacement.
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Often we are not interested in the whole area in the map, but only in a particular region. This is
the case when the map contains not only your sample, but surrounding substrate, ex. mica. The
map dialog allows you to create stack only for the selected ROIs.
All types of stacks can be generated through the main menu of the Map dialog:
In each case, a dialog similar to the dialog in Fig. 74 will appear. Here you can specify the
settings necessary for generating the stack:
o
o
Fig. 74. Dialog for specifying the parameters of
a stack.
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o
o
the Maximum field lets you specify the largest values of indentation/tip
displacement. It will be used for the last frame.
the Frame no field lets you specify the number of frames n. This number determines
the step between successive frames, which equals (Maximum Minimum)/(n - 1).
Once you specified all the parameters, click the OK button (Alt + O).
K.5. Force contour mapping
Force contour mapping resembles stacks
described above, but now each frame is
generated for a fixed value of force. Force
exerted on the sample is a function of the
position on the sample and the depth of
indentation (or, alternatively, tip displacement).
Each frame shows a contour of this function,
calculated for a particular value of force F, i.e. the
set of points for which force equals F. Fig. 75
explains how force contours are calculated.
Fig. 75. Force contour mapping involves generating
a series of 2D arrays or frames, each showing indentation
for a particular value of force.
Fig. 77. Series editor (see section H.1.a.) for maps. The Label tab is displayed. Here you can specify whether
labels should be displayed and what color and font should be used. You can also set the number format
used for labels. By default if the map is too large to display all labels clearly, the labels are hidden.
Fig. 78. A zoomed-in part of a Young's modulus map with labels visible. Labels are hidden by default if the
map is so large that the labels would overlap, but you can always switch them off for a smaller, zoomed-in
area, using the Live chart style dialog.
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102
to save all frames as separate image files, go to File (Alt + F) Save all frames (Ctrl +
Shift + S)
to save the whole stack as an AVI movie, go to File (Alt + F) Save as movie. The Save
dialog appear. See section I. Saving charts for details.
L.3. Playing stack as a movie
Stacks can be played as a movie both forward and in reverse. The user can modify the frame
rate and the movie length. The stack dialog is equipped with an easily accessible set of buttons
(Fig. 79 d) for playing the movie. Additional actions can be accessed from the Movie menu (Alt +
M in the main menu bar):
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to change frame rate or movie length, go to Movie (Alt + M) Edit parameters. The
movie parameters dialog (Fig. 80) will be displayed. Here you can specify the rate in
frames per seconds (fps) or the length in seconds. These parameters are interdependent
when you change one, the other gets updated automatically.
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to recalculate all force curves, go to Curves (Alt + U) Recalculate (Ctrl + R). This opens
The Processing assistant, allowing you to recalculate the force-volume recordings
corresponding to the stack. For instruction on how to process force curves, see section E.
Force curve processing. After processing is over, recalculated maps will be added to the
Map dialog. Now you can generate a new stack for details, see section K. Working with
maps.
to recalculate the curves within ROIs, go to Curves (Alt + U) Recalculate ROIs (Ctrl +
Shift + R)
to recalculate a single curve, move the mouse over the position in the stack that you
want to recalculate, then click the right mouse button. A context menu will appear. Click
the Recalculate curve item.
105
were not generated or they have been deleted), another dialog will pop up, asking
you whether you want to preview the force curve.
to go to the processing results, the mouse over the position of interest, then click the
right mouse button. A context menu will appear. Click the Find results item. Unless the
results have been cleared, The Results dialog (Fig. 21) will be shown, with the rows
corresponding to the clicked position selected.
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to draw a histogram for the current frame, go to Data Draw frame histogram
(Ctrl + D). The histogram assistant will appear, where you can specify how the histogram
should be drawn. For details about the Histogram assistant, see section F.3. Creating
histograms.
to view the histograms already generated for the frames, go to Data (Alt + C) Frame
histograms (Ctrl + H)
to draw a live histogram, go to Data Live histogram (Ctrl + Shift + L). If the live
histogram has not been already drawn, the Histogram assistant will be displayed to
collect drawing settings.
L.8. Displaying descriptive statistics for a stack
Descriptive statistics are calculated for each frame separately and are stored in a separate
dialog. Statistics for all frames are displayed in the same table, so you can easily compare them
(Fig. 81). See section F.7. Descriptive statistics for details about the descriptive statistics
calculated by AtomicJ. To display descriptive statistics for each frame, go to Data Statistics
(Ctrl + T).
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108
1. cross-section lines (white solid in Fig. 85) showing where the cross-sections were cut.
You can move these lines with the mouse - the cross-sections are then updated
accordingly.
2. triangular markers (white triangles in Fig. 85) that can be moved with the mouse along
the section lines. Their position is coupled to the position of the vertical lines in the
cross-section chart. You can delete the marker or add new markers with mouse.
Actions for manipulating markers, common to all stack cross-sections:
to add a new marker in the Stack dialog, move the mouse over the section line and then
click the right mouse button. In the Stack dialog, a new triangular marker will be added.
In the Cross-section dialog, a new vertical line will be drawn in the position
corresponding to the marker.
to add a new marker in the Cross-section dialog, go to Markers (Alt + K) Add marker
(Alt + A). If this item is selected, the mouse cursor appears as a cross over the crosssection plot and you can add markers by clicking the left mouse button. Once you added
markers, deselect the Add marker item.
to move a marker in the Stack dialog, move the mouse over the marker (small triangle),
press the left mouse button and drag the mouse. The marker will move along the section
line.
to move a marker in the Cross-section dialog, move the mouse over a vertical line, press
the left mouse button and drag the mouse.
to remove a marker in the Stack dialog move the mouse over the marker (small
triangle), then double click the left mouse button.
to display a stack cross-section, go to Sections (Alt + E) in the menu bar, then Horizontal
slices (Alt + H) or Vertical slices (Alt + V). A cross-section dialog will be displayed
(horizontal shown in Fig. 82, vertical in Fig. 83).
Fig. 82. The Horizontal section dialog. The central panel displays the current frame of the cross-section
movie (b). Cross-sections can be manipulated and analyzed through action accessible from the main
menu bar (a) or played as a movie with the help of dedicated controls (c, b).
The horizontal dashed line marks the Y-coordinate of the current frame in the sectioned stack.
110
to generate a new flexible cross-section, go to Sections (Alt + T) in the menu bar of the
Stack dialog, then select the Line section, Polyline section or Free hand section in the
drop-down menu. This will turn on the Extract profile mode, in which you can draw the
cross-section line with the mouse. Please refer to the section Mouse actions on charts
and stacks for more details. A cross-section dialog will be displayed (Fig. 84).
to translate the section profile in the Stack Dialog, move the mouse over it, press the
left mouse button and drag the mouse. The line will follow the mouse and the crosssection will be instantly updated.
to modify the section profile, move the mouse over it and single click the left mouse
button. Moveable circles will appear at its ends of line profiles and nodes of the
polyline/free hand profiles . To reposition one of the endpoints or nodes, move the
mouse over the circle, press the left button and drag the mouse. To hide the circles,
single click the left button on the line. The cross-section will be instantly updated.
to remove the cross-section, move the mouse over the cross-section line and double
click the left mouse button.
111
to view the length, angle and position of the section lines, go to Sections (Alt + T)
Section geometry (Alt + G) in the Stack dialog. A dialog with geometrical parameters of
section lines will be displayed.
112
nearest-neighbor interpolation
bilinear interpolation
bicubic spline interpolation
You can also specify the number of points calculated for the cross-section, i.e. its resolution. For
horizontal and vertical sections the default number of points is equal to the size of the grid. For
flexible cross-sections, the default value is always 200. The choice of interpolation method
influence the visual appearance of the cross-section.
to change the interpolation method, use the Section interpolation dialog (Fig. 86). To
display it, you can either:
1. click Interpolation (Alt + I) in the main menu bar of the cross-section dialog, then
click Change section interpolation (Alt + S) in the drop-down menu.
2. use shortcut Ctrl + I.
The section interpolation dialog (Fig. 86) allows you to select the method of interpolation and
the number of points that make up the cross-section. The images of cross-section are
immediately updated to reflect the introduced changes. The dialog is equipped with three
buttons.
1. The Apply to all button (Alt + A) applies the interpolation to all flexible crosssections. This button is disabled for horizontal and vertical cross-sections.
2. The Reset button (Alt + R) discards all the changes you have made.
3. The Close button (Alt + C)
113
114
M. Appendix
M.1. How to open Windows Command Prompt?
Vista, Windows 7:
1. Click Start (the Windows flag in left bottom corner)
2. Type cmd
3. Press enter.
Windows XP:
1. Click Start (left bottom corner)
2. Click Run
3. Type cmd
115
Fig. 89. Tip shapes: a. Paraboloid that approximates sphere in the Hertzs equation. R radius of
curvature at the apex. b. Hyperboloid. R radius of curvature at the apex, half angle
between the asymptotes.
e
Fig. 89 (cont.). Tip shapes. c. Cone.
half angle. d. Blunt cone (cone capped
by a sphere). R- radius of curvature at
the apex, b tip radius at the level of
transition between the capping sphere
and the cone, half angle. If the
transition from sphere to cone is
smooth,
then
b RCos .
e.
R.
116
4E
P
3 1 2
3
2
2. Sphere (Sneddon). Unlike Hertzs, Sneddons solution (Sneddon 1965) does not require that
R.
E R2 a2
Ra
P
Log
a R
2
1 2
Ra
a
Ra
Log
2
Ra
4E
3 1 2
3
2
4 2
8
4 2 3 16 3 3 2 4
R 2 1 2 2 3 3
4
15
where
R
h
Coefficients and depends on whether the sample is adherent to the substrate or not.
For adherent sample:
3 2
1
5 2
0.056
1
0.347
R Cot
1
Ea 3 2
2
ArcTan
where
2
2
a
1 R
2 2
2
117
1
a2
ArcTan
2R 2
2 2
2E Tan
8E Tan
3
2Tan
2
2
1
16 Tan 2
2h
h
2
E A
2
P
2 a 1
2
1 2 1
2
A 2 2
2 a
where is Eulers gamma function. Hertzs equation for sphere and Sneddons equation for
cone are special cases of the equation for the power-shaped tip, with = 2, A = 1/(2R) and = 1,
A = 1/Tan[], respectively. For example, the equation with = 2, A = 0.5 corresponds to the
Hertzs model with R = 1 m. In the AtomicJ interface, A is termed the factor and is termed the
exponent.
8. Blunt cone (Briscoe et al. 1994). The Hertz solution for the sphere is applied for small
indentations, for which the contact radius a is smaller than the transition radius b. This is the
case as long as
b2
. For larger indentations:
R
118
2E
a2
b a
a
ArcSin
a 2 b2
2
2 Tan 2
1
a 3R
a
b a 2 2
ArcSin a b
Tan 2
a R
1
2
1/2
b
a 2 b2
2Tan
3R
9. Truncated cone (Briscoe et al. 1994). Truncated cone can be treated as a special case of
blunted cone, for which the radius of curvature at the apex is infinite. Substituting R = into
the equations for blunt cone, we get:
2E
a2
b
a
ArcSin a 2 b2
2
2 Tan 2
1
a
1
2
2Tan
a
b
ArcSin
Tan 2
a
1.4906 ETan[ ] 2
2 1 2
11. Blunt pyramid, regular, four-sided (Rico et al. 2005). The Hertz solution for the sphere is
applied for small indentations, for which the contact radius a is smaller than the transition
radius b. This is the case as long as
2E
1 2
b2
. For larger indentations
R
21/2 a 2
b a
ArcSin
a 2 b2
a
Tan
2
a
3
R
23/2 a
b a 2 2
ArcSin a b
Tan 2
a R
1
2
21/2 b
a 2 b2
Tan
3
R
1/2
12. Truncated pyramid (Rico et al. 2005). Truncated pyramid can be treated as a special case of
blunted pyramid, for which the radius of curvature at the apex is infinite. Substituting R = into
the equations for blunt pyramid, we get:
119
2E
21/2 a 2
b
a
ArcSin a 2 b2
2
Tan 2
1
a
1
2
21/2 b
Tan
23/2 a
b
ArcSin
Tan 2
a
HYPERELASTIC MODELS
13. Sphere, Fungs hyperelastic model (Fung 1979) .The equation for force indentation depth
relation was derived by Lin et al (2009), who found that the equation for spheres contact radius
a
from Hertzz model (used here) holds for hyperelastic materials as long as 0.4 .
R
a3 15Ra 2
20 E0 a5 15Ra 4 75R 2 a3
P
exp b
2
2
3
2
3
9 1 2 5Ra 50 R a 125R
25R a 125R
a R
14. Sphere, Ogdens hyperelastic model (Ogden 1972).The equation for force indentation
depth for single-term Ogdens model relation was derived by Lin et al (2009):
/2 1
1
40 E0 a 2
a
a
P
1 0.2
1 0.2
R
R
9 1 2
a R
ADHESIVE CONTACT
15. Derjaguin-Muller-Toporov (DMT) (Derjaguin et al. 1975). DMT model was derived for
indentation with a sphere (approximated by a paraboloid) in the presence of adhesion forces.
We implemented the most commonly referenced form of the DMT model, in which the
additional load due to adhesive forces equals 2R and is independent of indentation. This
formulation is due to Maugis (1992). DMT model gives good approximation of the load
indentation relationship only for stiff samples, small values of tip radius R and small surface
energy .
P
4E R
3/2 2 R
2
3 1
120
16. Johnson-Kendall-Roberts (JKR) (Johnson et al. 1971). JKR model was derived for indentation
with a sphere (approximated by a paraboloid) in the presence of adhesion forces. JKR model is
a good approximation of the load indentation relationship for soft samples, large value of
radius R and large energy of adhesion (Maugis 1992).
P=
4Ea 3
Ea 3
2
2
3R 1- 2
1- 2
a2
2 a
E
(1 2 )
17. Sphere, Maugis solution. Maugis (1995) derived the load indentation relationship in the
presence of adhesion, which takes into account the true shape of a spherical tip (as opposed to
the JKR model, which uses parabolic approximation).
2 2 + 2
+
2
Log [
]
2
1
4
2
1 2
(
)
=
a
+
2
Log [
]
1 2
18. Hyperboloid, Sun-Akhremitchev-Walker (SAW). Sun et al (2004) derived the load contact
radius and indentation contact radius relations for indentation with a hyperboloidal tip in the
presence of adhesion.
2
2
( ) 1
23 (1 2 )
+
(
+
ArcSin
[
])
2
)
2
1 2
2
2
( ) + 1
(
)
where = Cot[]
2
(
) 1
2(1 2 )
=
( + ArcSin [ 2
])
2 2
() + 1
121
References:
1. Akhremitchev BB and Walker GC (1999) Finite Sample Thickness Effects on Elasticity
Determination Using Atomic Force Microscopy. Langmuir 15: 5630 5634.
2. Bilodeau G (1992) Regular Pyramid Punch Problem. ASME J Appl Mech. 59: 519 523.
3. Briscoe DJ, Sebastian KS and Adams MJ (1994) The effect of indenter geometry on the elastic
response to indentation. J Phys D: Appl Phys 27: 1156 1162.
4. Derjaquin BV, Muller VM, Toporov YP (1975) Effect of contact deformations on the adhesion of
particles. J Colloid Interface Sci 53: 314-326.
5. Dimitriadis EK, Horkay F, Maresca J, Kachar B, Chadwick RS (2002) Determination of elastic
moduli of thin layers of soft material using the atomic force microscope. Biophys J 82:2798
2810.
6. Fung YC, Fronek K, Patitucci P (1979) Pseudoelasticity of arteries and the choice of its
mathematical expression. Am J Physiol-Heart C 237:H620H631
7. Galin LA (1946) Spatial contact problems of the theory of elasticity for punches of circular shape
in planar projection. J. Appl. Math. Mech. (PMM), 10: 425448.
8. Gavara N and Chadwick RS (2012) Determination of the elastic moduli of thin samples and
adherent cells using conical AFM tips. Nat Nanotechnol. 7: 733736.
9. Harding JW, Sneddon IN (1945) The elastic stresses produced by the indentation of the plane
surface of a semi-infinite elastic solid by a rigid punch. Proc Camb Philol Soc. 41:16.
10. Johnson KL, Kendall K, Roberts AD (1971) Surface energy and the contact of elastic solids. Proc.
R. Soc. Lond. A 324: 301-312.
11. Lin DC, Schreiber DI, Dimitriadis EK, Horkay F (2009) Spherical indentation of soft matter beyond
the Hertzian regime: numerical and experimental validation of hyperelastic models. Biomech
Model Mechanobiol 8:345358.
12. Maugis D (1992) Adhesion of spheres: The JKR-DMT transition using a Dugdale model. J Colloid
Interface Sci 150: 243 269.
13. Maugis D (1995) Extension of the Johnson-Kendall-Roberts theory of the elastic contact of
spheres to large contact radii. Langmuir 11: 679 682.
14. Maugis D (1995) Extension of the Johnson-Kendall-Roberts theory of the elastic contact of
spheres to large contact radii. Langmuir 11: 679 682.
15. Ogden RW (1972) Large deformation isotropic elasticityon the correlation of theory and
experiment for incompressible rubberlike solids. Proc R Soc Lond A Math Phys Sci 326: 565584.
16. Rico F, Roca-Cusachs P, Gavara N, Farre R, Rotger M and Navajas D (2005) Probing Mechanical
Properties of Living Cells by Atomic Force Microscopy With Blunted Pyramidal Cantilever Tips.
Phys Rev E 72, 021914.
17. Sneddon IN (1965) The relation between load and penetration in the axisymmetric Boussinesq
problem for a punch of arbitrary profile. Int J Eng Sci 3: 47 - 57.
18. Sun Y, Akhremitchev B, Walker G (2004) Using the adhesive interaction between atomic force
microscopy tips ad polymer surfaces to measure the elastic modulus of compliant samples.
Langmuir 20: 5837 5845.
122
AtomicJ supports plain text files, as well as files generated by microscopes from Agilent
Technologies, Bruker, JPK, Asylum Research, Park Systems and NT-MDT. It also supports some
file formats generated by the Nanotec Electronica WSxM software.
If you use another microscope, please send me a few sample files generated by your
microscope, ideally both in binary and text format (pawel.hermanowicz@uj.edu.pl). If possible,
please attach print-screens showing how the files are displayed by the software that operates
your AFM. I will then add support for this type of files.
You can also convert your files to plain text (ASCII) files. AtomicJ can open comma-separated
(.csv) and text separated (.tsv) files. Their structure is very simple:
Plain text files for force curves consist of two columns of numbers, separated by commas
(.csv) or tabs (.tsv) (Fig. 90). The left column contains values of scanner position (or
alternatively cantilever position), while the right on the corresponding deflections or
forces. Each column begins with the name of the quantity and a SI unit in brackets. You
will find curves saved in this format among the files available at
https://sourceforge.net/projects/jrobust/files/TestFiles/AFM Recordings.zip.
Fig. 90. A force curve saved in plain text format (.csv), here viewed in MS Excel. The file consists of two
columns - the left contains values of cantilver/scanner position, the right one the corresponding
deflections or forces.
Plain text files for images (Fig. 91) should consist of a header and data for one or more
image channels. Header should contain eight fields:
1. Type obligatory field, it should be always Image
123
2. XQuantity the quantity on X-axis. Usually it is length in microns. The unit should
be written in brackets and only SI units will be recognized.
3. YQuantity the quantity on Y-axis. Usually it is length in microns. The unit should
be written in brackets and only SI units will be recognized.
4. XCount- the number of pixels along the X-axis (i.e. the number of column in the
image).
5. YCount- the number of pixels along the Y-axis (i.e. the number of rows in the
image).
6. XLength the length of the image along the X-axis.
7. YLength the length of the image along the Y-axis.
8. Data obligatory field, marking the header and beginning of the channel data
After the header you should specify channels, one by one. For each channel, you have
to include :
1. Channel - obligatory field, marking the beginning of the channel data.
2. ZQuantity - the quantity visualized in the channel. The unit should be specified in
brackets. For example, if the channel shows topography in microns, ZQuantity
can be 'Topography (um)', if the channel shows phase shift, ZQuantity can be
'Phase (deg)'
3. The array of pixel values. Each row of pixels in the channel should be written in a
separate row in the file.
An image saved in plain text (CSV) format is included the among the files available at
https://sourceforge.net/projects/jrobust/files/TestFiles/AFM Recordings.zip.
Fig. 91. An image saved in plain text format (CSV), here viewed in MS Excel. The file consists of a header,
where the image dimensions and quantities on axes are specified, and data for channels.
124