PIEScope GUI controls

piescope_gui is a python package for controlling the Photon-Ion-Electron microscope (PIE-scope) Gorelick et al., eLife 2019 via a PyQt graphical user interface.

Features

• Integrated control of fluorescence and FIB/SEM microscopes.
• Semi-automated registration & alignment of fluorescenc and FIB/SEM images, and overlaid images display.
• Create ion beam milling patterns on the registered and overlaid images.

Citation

If you find this useful, please cite our work. See CITATION for details.

Software license

This software is released under the terms of the GPLv3 license. There is NO WARRAYNTY either express or implied. See LICENSE for details.

Dependencies

There are a number of essential hardware and software dependencies.

Hardware requirements

• FIB/SEM microscope (a commercial product by ThermoFisher FEI)
• Basler detector (https://www.baslerweb.com)
• Toptica lasers (for the fluorescence microscope)
• SMARACT stage (controlling the fluorescence objective lens position)

Software requirements

• Python 3.6
• Autoscript software (a commercial product by ThermoFisher FEI)
• The Basler Pylon Software Suite
• The Basler pypylon python package
• The piescope python package containing the back-end functionalities
• Other python packages, as specified in the requirements.txt and requirements-dev.txt

Installation

See INSTALLATION for a detailed guide.

Software developers may also like to refer to the development installation guide.

Running the program

First activate your python environment (the same one you installed piescope into). Eg: open the Anaconda Prompt from the start menu and type:;;

conda activate piescope

Then you can launch the GUI using the command:

piescope

You will then see the main GUI screen appear:

Initially both the FIB/SEM and Fluorescence image display areas in the GUI are empty. The next screenshot shows the GUI with example C. Elegans data displayed:

Blue outline (left): Controls for flourescence image acquisition.

Red outline (center left): Fluorescence image display area and controls.

Green outline (right): FIB/SEM image display area and controls.

Semi-automated image alignment

An image registration feature is provided, using control point matching to align images of different modalities (i.e. fluorescenc and FIB/SEM images). A screenshot of that interface is shown here:

Note: the image correlation utility is also available separately as a standalone package.

Aligned images are then displayed as a transparent overlay.

Creating ion beam milling patterns

Finally, users may create ion beam milling patterns directly from the registered fluorescence and FIB/SEM images. In the screenshot below, a rectangle pattern is drawn onto the image display and can be run immediately to ablate the sample.

This is a key advantage of the PIE-scope, as not only can we use the surface structural information from the FIB/SEM but also the internal functional information provided by fluorescence imaging.

Hardware control diagram

This diagram shows the relationship between the hardware components of the PIE-scope.

The FIB/SEM system, controlled via the AutoScript API by FEI ThermoFisher Scientific, encompasses:

• Sample stage movement
• Electron imaging
• Ion beam imaging
• Ion beam milling (ablation of selected regions in the sample)

The fluorescence imaging system includes:

• Toptica lasers, controlled by serial communication (USB connection)
• Basler detector, controlled with the Pylon Software Suite and the pypylon python API
• SMARACT objective lens stage, controlled via a socket connection

Suggested end-to-end workflow

This is the suggested end-to-end workflow for experiments, consisting of six steps: