Projects and experiments

After starting the application, you are presented with the project dialog. You can create a new project, create a new experiment or continue an experiment. They are useful to group results.

Project

A project is a collection of experiments and helps you grouping similar experiments.

Experiment

An experiment groups the results of your research. There can be multiple experiments assigned to a project.

It is possible to resume experiments. However, certain environmental parameters may have changed and need to be taken into account when doing so: If plates are re-inserted, points recorded in a previous session might show a positioning offset when being accessed again or a different spring constant applies if another probe is mounted.

To avoid plate mixups the software supports assignment of plate identifiers on an experiment level. If set, the identifiers will be displayed in the experiment list.

Tools

Toolbar

The toolbar is located on the left side of the main window and gives quick access to a set of functions described below:

Tool	Name	Description
	Scalebar	Toggles the scale bar at the bottom of the sample display
	Navigation tool	Tool to navigate the worktable
	Take screenshot	The screenshot of the current microscope image can be exported later in different formats
	Record a video
	Save Video History	Save last few seconds
	Pressure tool	Manual pressure control
	Oscilloscope	Shows signal streams
	Probe tool	Manual z positioning
	Spectroscopy tool	User-programmable z positioning
	Wash tool	Save and run wash cycles
	Record a note
	Preset tool	Save and restore settings
	Show Result Data
	Volume Calculator	Perform volume calculations on injected cells

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Navigation tool

The navigation tool marks the head’s current position with a green dot on the active plate configuration. By clicking on a well the bot will move its head to the selected well.

Positioning

Relative

A well wider and higher than 10mm allows relative positioning. Click on any point within the well and the bot will move there.

Absolute

A well containing a point pattern can always be approached with absolute positions. Simply click on the well and select the position in the dialog which just appeared. These wells are marked by a point pattern.

The dialog allows you to move to:

• the well’s center
• the point pattern’s center
• any point in the point pattern
• a point relative to the well’s center

If a well does not contain a point pattern and is less wide or less high than 10mm, the bot will always move to the center of the well. These kind of wells show a cross at the center when you hover over them with your mouse.

Activity Map

The activity map can be toggled using the button on the right. It displays previously processed positions which have a result attached in the plate view. Usually the positions are too close to each other to be displayed as single points in the overview. Instead, a black rectangular area indicates the approximate position of possibly multiple processed positions in a well.

Pressing any of the Ctrl keys on the keyboard zooms in on the area of the plate under the mouse:

Activity map shown in navigation tool with zoom enabled through the ‘Ctrl’-key

The points displayed in the activity map can be restricted using the point overlay control in the status bar.

Navigation History

A click on the bottom right button reveals the navigation history. It lists previously used positions. Entries are added automatically when using the navigation tool to move to new positions. Entries can can be added manually by clicking the button with the plus sign that adds the current position to the list. Select a position to show its location on the plate. Click on the blue point to move to the previous position.

Make a video

Pressing this icon triggers the recording of the current microscope image. Accordingly, the record icon is displayed until you click again to cap the recording.

Save past 30s

This tool allows you to conveniently save an unexpected event on video even after it occurred. By clicking the icon the past 30 seconds of your video feed are recovered and saved at a reduced frame rate.

Pressure tool

This tool allows you to directly control the pressure applied to the cantilever. The currently active pressure is highlighted in blue.

You can add, edit or delete custom pressure values by using the corresponding buttons on the right.

Add

Adds the current target pressure to the list of custom pressure values. You can directly change the pressure value using the number pad. Custom pressures are stored between sessions.

Delete

Enters “deletion mode”: When in this mode, clicking on a custom pressure will delete it.

Edit

Enters “edit mode”: When in this mode, clicking on a custom pressure allows you to change its value. If you change an active pressure value, the new value is applied immediately.

Show slider

Switches to the slider view where you can change the target pressure using a slider.

Additionally, the status of the pressure reservoir is indicated at the bottom, where a full bar (green) signals that the operational pressure levels are reached in the reservoirs.

Oscilloscope

The oscilloscope icon toggles an animated graph which displays the deflection and the pressure of the last few seconds.

Right next to the graph, you can see the current values of the deflection and the pressure.

The scaling of the rulers on either side can be adjusted. Per default, it will automatically scale to show all recent values. If you would like to see a fixed total range, you can adjust the zoom by expanding the zoom combo box and choosing a value. Additionally, you can just use your mouse scroll wheel to scroll through all available zoom levels.

Note that the deflection signal always shows uncorrected values. Therefore, one will observe a corresponding shift of the signal when applying a relative setpoint but the signal itself will not necessarily correspond to that setpoint.

Probe tool

Control the movement of the probe using the following options (left to right):

• Approach: Pressing triggers an approach operation to the configured setpoint; pressing the icon again aborts the operation
• Approach: Pressing triggers a movement to the corresponding plate’s content height. Pressing the icon again aborts the movement.
• Home position: Retracts the FluidFM probe to the uppermost position. Pressing the icon again aborts the movement.
• Retract: Retracts the probe by the configured distance. Pressing the icon again aborts the movement.
• Advance: Moves the cantilever quickly towards the sample; a second click aborts the movement. Due to the high crash risk, the operation must first be unlocked by clicking on the lock.

Spectroscopy tool

This tool allows you to quickly define and carry out a force spectroscopy.

It can be used to optimize the spectroscopy parameters used in the workflows or to run ad-hoc motion profiles.

How to use:

• First, click on the surface next to the triangle/”play” symbol to load the configuration dialog
• Second, click on the triangle/”play” symbol to start executing your program
• Finally, you can have a look at the resulting curve displayed below the tool

A spectroscopy curve taken with the tool is not automatically saved; pressing the save icon next to the curve moves it to the result history.

Wash tool

The wash tool allows you to set up a wash cycle using wells on the probe plate (except the probe holder locations) or any other plate.

The idea is to first fill empty, unused wells with washing solutions of your choice and then define a dipping time and a pressure value you wish to apply during the dipping step. Unless Return to current position after washing is checked, the probe remains in the last container to avoid drying out.

As an example, you could have:

1. Bleach in well A2, 5s at 100mbar
2. Water in well B2, 30s at 10mbar
3. Water in well C2, 2s at 0mbar (the probe will stay here).

Creating a wash sequence

Ensure the plate view is visible on the left side of the wash sequence. Press the Edit button if it is not shown.

• To add a new wash step to the sequence: click on a well in the plate view and drag it to the sequence.
• To change the pressure or duration of a wash step: first select the step you want to modify, then click on the property you want to edit.
• To change the position of a step in the sequence: click on the dotted area of a step and drag it to the new position.

Click on the button to start the wash cycle. You won’t be able to change the sequence while washing is in progress.

Return to current position after washing remembers the position of the probe before starting the wash cycle and moves the probe back to this position once the wash cycle finishes.

Persistency

If your wash sequence only contains wells from a single probe plate, the sequence is stored when the control software is closed and loaded again when you next open the wash tool if you don’t remove the probe plate.

Record Note (Text tool)

You can take a short text note during your experiment by using the Text tool. It will be saved in the result history and be available later when you are reviewing the data. In that sense, you can use the Text tool as a built-in lab journal.

Preset tool

This tool allows you to store current settings to a preset and restore the saved settings at a later time. This empowers you to switch between different settings quickly without having to change all settings manually.

Click on the button to save the current settings to a new preset. You will be presented with a window asking you for a name for the preset and showing the list of settings that will be stored. You must choose a different name for each saved preset. Saved presets will available again after restarting the software.

Clicking on a preset will immediately apply the stored settings.

enters “edit mode”: when in this mode, click on a preset to view the settings without applying them. In the opened dialog, the preset’s name can be changed and individual settings can be removed. If all settings are removed, the whole preset is deleted.

Result history

All your results (such as force curves and screenshots, except recorded videos) are stored in the database. You can view the stored results of the current experiment in the result history.

If you click on a preview icon or thumbnail an enlarged view with the full details of the result will open. This will also enable you to export the result to a file.

The result history offers different view modes, which can be chosen at the bottom of the result history:

Single column

Displays the result previews in a compact single column next to the tool bar.

Extended column

Displays the result previews in a single column as above, but with a few informative details:

1. An Id of the result in the database
2. The date and time (in minutes) when the result was acquired
3. The time since the first result of the experiment was acquired

Multiple columns

Displays the result previews in multiple columns, such that more results are visible compared to single column mode. This mode also enables you to export all results at the same time.

Point filter

Allows you to filter results by position. This view is only useful in conjunction with points that were saved during the review step of the Injection workflow.

Select a point in the upper part to see a preview of all results associated with this point. This will also enable you to directly export all these results.

If a point group is selected (by clicking on the group’s name to the left of the points), all results associated with any of the points in this group can be exported at the same time.

System Information

Located at the bottom of the video feed, this tool displays system status information

• Currently applied pressure.
• Current axes position as a (x,y,z) tuple. Note that erroneous values might be reported while an initialization procedure takes place.
• Currently measured ADC values and the resulting cantilever deflection. The deflection value might differ from the one shown in the oscilloscope because here the underlying ADC values are not normalized.
• A warning if the intensity of the cantilever’s deflection value drops below a certain threshold. This might be caused by disturbances within the laser path which implies a disabled force control mechanism. The warning is only supported after the laser was aligned and its signal maximized.
• Whether a probe appears to be mounted
• Currently used sensitivity and stiffness values
• The digital magnification level of the video feed
• Controls to display an overlay of previously processed positions

Result export

Results of the current experiment can be exported from the result history in various ways:

• a single result can be exported from the detailed result view (after clicking on a result preview in any of the views)
• all results can be exported from the multi-column view
• results associated with certain points or point groups can be exported from the point-filter view
• multiple results can be exported in the multi-column view (hold the Shift or Control keyboard-key while clicking on the results to select multiple results)

Once you click on the export button, you will be presented the following dialog:

Here you can choose which details you would like to export and how to name the exported files.

Click on the export directory to change the directory where to export the result data. If this path is too long to display, it will be truncated - hover the mouse over the path to see the full one.

You can also control whether to create sub-directories. By default, the exported data will be placed in the sub-directory using the name of the current experiment. For result groups a subfolder for the grouped results will always be created.

Next, you can choose how to name the exported files. The default is to include the result type (e.g. picture, spectroscopy, …) and the unique database Id of the result in the name.

Finally the dialog allows you to select which result types you would like to export and offers a few export options depending on the result type.

Exporting results associated with points

If exporting from the point-filter view, you can also choose to create sub-directories for each point group and point. Directories created for points will always include an Id of the point (this Id is different from the results’ Ids).

When exporting a point group an XML file containing details about the points will be created in the group’s sub-directory.

Export Formats

How data is written to the export destination is mainly controlled by built-in software parameters. However, following general rules apply:

File type	Description
Spectroscopy data	Axis positions are stored in [mm]; time values in [s]; deflection values in [V]
Spectroscopy plot	An svg file that is subsampled according to the resolution specified in the export window
Images	Uncompressed image data in native resolution and bit depth
Thermal tuning data	Collection of data blocks recorded at several kHz. Each line contains a time offset [s] and an ADC value within [0,220]
Volume calculation report	A csv file listing the calculated volume per image

Dragging

Some tools are overlaid over the video feed. These can be moved by dragging the black handle bar on their right side.

Cancellation

Buttons that start an operation usually also terminate it on a subsequent click. This is indicated by a red cross which overlays the button when cancellation is possible.

An example with the approach button:

State	Description
	An operation is ready to be started
	The operation is running - press again to abort
	The operation is currently not available; either it is being stopped or another operation is running

Arrow navigation

In various situations (e.g. during Laser Alignment) fine grained control over a movement is necessary. In this case an arrow navigation tool as depicted will be available.

Clicking on the arrow keys will perform a movement in the corresponding direction by the distance indicated on the right by step size. The step size can be changed with the + and - buttons.

Clicking on the button will reset all axes to their initial position (this button may not be available in all situations).

Number editor

Clicking on an editable number in the software brings up the number editor. You can change both the value and the unit of the current number. Changes become only effective once the number is applied.

The number editor validates your input and gives feedback if, for example, your unit is not supported or your value is out of range.

If the slider icon is present, you can click on it to show a slider instead of buttons to set your desired value.

Quick Adjust

Tool visible with activated parameter ‘Digital Zoom’ whose value is also listed in the statusbar.

This feature can be used to quickly adjust view-related parameters instead of setting them in the settings menu.

It opens with Ctrl+Space and then stays open as long as Ctrl is being pressed.

The tool displays the available parameters as tabs and one can cycle through them by pressing Ctrl+Space multiple times. While the tool is being shown one can adjust the slider with the mouse wheel so there is no need to click on it and the adjustments works independent of the mouse position.

The last used parameter can be adjusted without opening the tool by pressing Ctrl and using the mouse wheel. It is then updated as if the tool would be open. The current value and name of active parameter which will be changed is displayed in the status bar.

Stage movement

Standard dragging

While looking at a sample, clicking and maintaining pressure on the mouse’s left button will trigger a dragging process. At that point, the cursor will change to a grabbing hand and any mouse movement will be followed by the stage.

To quickly move an object below the probe one can press the control key and double click on the target object.

Infinity movement

While the standard drag is the ideal way to carry out small movements, the need to travel longer distances within a well might arise. To this end, use the so called “infinity” drag. Click and maintain the mouse’s left button while hovering around the edge of the screen and you will trigger a constant movement in the opposite direction. Why the opposite direction? To enable you to switch between standard dragging and “infinity” movement seamlessly.

A screenshot of an “infinity” movement indicated by the white arrow over a transparent border.

Boundaries

To prevent you from hitting a plate, a boundary checker is implemented for each plate type. As a result, you will see a red, transparent border appear whenever a boundary is reached while moving the stage, be it when dragging or in “infinity movement” mode.

A screenshot of a boundary warning: the red arrows show the forbidden directions while the red, transparent border indicates a boundary was reached.

Point Overlay

Previously previously processed positions which have a result attached can be displayed as overlay on the video feed.

The overlay can be shown or hidden using the button at the bottom of the video feed.

Positions displayed in the overlay can be limited to positions processed

• since the software was started
• in the scope of the active exeperiment
• since a configurable time (click on the displayed time to change the range)

This also limits the positions displayed in the activity map of the navigation tool.

If positions are too close to be displayed on the video feed, a rectangle is displayed instead, indicating the number of positions within the represented area.

Data Plotting

The plotting module of the software gives you the possibility to visualize spectroscopy curves and spring constant measurements.

The plots

To begin with, there are several ways to look at a spectroscopy curve. These are accessible through the controls on the bottom-right of the module.

Controls to modify the plotting logic of the displayed information. [t] for time, [z] for vertical distance.

Time plot

The simplest display is a (t, z(t), V(t)) plot where z(t) stands for z axis position, while V(t) represents the position of the signal on the detection. The deflection value V(t) can be interpreted as follow: +/-1 Volt represents a cantilever fully bent away from/towards the sample.

Force plot

The most readable display is a (z, V(z)) plot of the signal position on the detector against axis height. The closer the cantilever is to the surface, the more likely it is for the signal to change due to various interactions.

The time plot is available either in absolute units or in their Fourier-transformed version. Selecting the [t] icon opens two sub-icons: [s] for seconds and [Hz] for Hertz. This enables you to take a peek at possible noise sources, for instance.

Absolute units: noisy interval

Fourier spectrum: first noise frequency around 20Hz, second frequency peak at ca. 250Hz and following harmonics.

Data menu

A right-click over the plot will open a menu to choose the data you wish to display.

Data menu: Choose to see processed/unprocessed data, fits or averages depending on what is available.

The toolbar

The toolbar displays some of the options at your dispositions:

• Basic/Details
• Unzoomed view
• Position reader
• Plot dragging
• Two-dimensional measurer/ruler
• Region zoom

Not included in the toolbar is the essential “scroll-zoom” that lets you zoom in and out at any moment using the mouse wheel.

Basic/detailed view

The basic/details button lets you toggle between a view with additional information such as curve legend, points, and a more sober view that displays only the curve.

Basic view: the curves, the ranges, the controls and the toolbar.

Detailed view: special points, curve legend, point legend added to the basic view.

Unzoomed view

The button marked with 1:1 lets you go back to the unzoomed view directly in order to save you some scrolling.

Position reader

The target icon stands for a position reader that displays the position of your cursor translated in plot unit. To activate this feature, you can either click on the target icon or press on the space bar.

Position reader: when the position reader is selected, a box appears next to your cursor to provide exact values on its position.

Drag

The grabbing hand stands for the dragging feature of the tool. You can either click on the hand icon and drag, or simply click, hold and move the mouse to drag the plot.

Dragging: When dragging is selected, the cursor is a “grab-hand”; a click turns it into a “grabbing-hand”.

Measurer

Click on the ruler icon, then on a point on the plot to trigger the measuring tool. You can use it to get precise estimations of distances/intervals in the plot. This feature is also accessible by pressing “Ctrl” and clicking on a point in the plot.

Measurer: Measuring an interval becomes easy using this option.

Region zoom

If you need to take a peak at a certain region of the plot, press on the magnifier icon, then select the region by clicking on a point and moving the mouse. This feature is also accessible by double-clicking and holding the left button of the mouse.

Region zoom : Draw a rectangle to describe the region you want to further investigate.

Settings

Settings Name	Description	Affected Components	Persistent
Approach Setpoint	The probe deflection value at which the approach will be stopped. Typically around 10 mV or 30 nN. Higher values are sometimes necessary if you have noise due to floating particles in the sample, as for example in cell medium.	Probe tool, Default value for workflows	No
Approach Speed	How fast the FluidFM probe approaches the sample. Higher values can drastically reduce the time to approach, but also increase the risk of probe damage. We recommend values around 5 µm/s.		No
Retract Speed	The speed at which the probe is lifted away from the surface.		No
Safety Retraction	The dafault distance by which the probe is automatically retracted after a successful approach.	Probe tool, Sensitivity measurements	No
Long Distance Retraction	The distance by which the probe is automatically retracted before the stage moves by at least 500µm with the probe close to the surface.	Injection, Cell Isolation and Spotting (including calibration) workflows	No
Active Sensitivity	[m/V] Check and/or adjust the sensitivity anytime. Beware that it is in [m/V], i.e. a value of 1.5 E-6 or 1.5 micron per Volt is not uncommon. You can type “E” to indicate the exponent of 10 when modifying the sensitivity.		No
Active Spring Constant	[N/m] Check and/or adjust the active spring constant anytime.		No
PI Parameters	These parameters are used to keep the setpoint with a proportional-integral control loop once the probe is in contact with the surface. Too high parameters will cause the probe to oscillate, too low parameters will slow the system down. To optimize the PI parameters, monitor the cantilever deflection when in contact with the surface during a spectroscopy.
Illumination	The FluidFM BOT has an integrated LED illumination. Set its value here.		Yes
Objective	You can choose which objective you want to use here. When using a non-motorized microscope it is important to adjust this value yourself whenever changing the objective. The magnification is an important information for ARYA to know the right scale.		No
Optovar	A magnifying optovar can enlarge details on some microscopes, it will however not increase the resolution.		No
Show Help Lines	Choose whether to show help lines (a crosshair and a square indicating the size of a 100 by 100 µm area) in the center of the video view.		Yes
Idle Pressure	The pressure that will be constantly applied by the pressure system in idle situations to prevent liquid from flowing through the cantilever in either direction. This value is automatically updated after probe filling.	Pressure tool, Default value for workflows	Yes
Record Injection Data	Whether to save spectroscopy curves of injection runs.	Injection workflow	Yes
Auto Retract Lens	Whether to automatically retract the lens when passing plate borders and restore the original focus distance on move completion. Users of non-motorized microscopes are adviced with a message to retract the lens.	Any move between plates	Yes