The microEye

The microEye is a Python toolkit for fluorescence microscopy that supports super-resolution single-molecule localization microscopy and single-particle tracking. It features hardware control, data analysis, and visualization.

This toolkit is compatible with the hardware used in our microscope. For further details, refer to the miEye microscope paper and OSF project.

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Table of Contents

• The microEye
  • Table of Contents
  • How to Install microEye
    • Troubleshooting Installation
  • microEye Launcher
    • Usage
  • Modules
    • The miEye Module
      • Experiment Designer (Beta)
    • The Multi Viewer Module
  • Uses Packages
  • Microscope Scheme
  • Hardware
    • Supported Cameras
    • Additional Hardware
  • Pycro-Manager Integration
    • Features
    • Integrated Hardware
    • How to Use
    • Step-by-step Instructions
  • Authors
  • People Involved
  • Acknowledgement
  • Citation

How to Install microEye

1. Install Python:

   Download and install the latest Python stable release (version ≥3.11.9).

2. Install microEye package:

   Open a terminal and execute the following command to install microEye using pip:

   pip install microEye --upgrade

   Or for a specific version:

   pip install microEye==version

3. Install specific hardware drivers: (Optional)

   • Allied Vision CMOS cameras:

     Install the Vimba X SDK (avoid installing it inside the Program Files directory). After installation, navigate to the Python API directory (e.g., C:\Allied Vision\Vimba X\api\python) where the vmbpy wheel file (.whl) is located, and install it using:

     python -m pip install vmbpy-1.1.1-py3-none-win_amd64.whl

     Important note

     It has been observed that vmbpy can hang when initializing GenICAM transport layers (TLs) if the GENICAM_GENTL64_PATH environment variable contains .cti folders from other providers (e.g., IDS Peak or pylon). To avoid this, restrict Vimba X to load only its own TLs by explicitly specifying the Vimba X .cti directory in VmbC.xml.

     Steps

     1. Auto (recommended)

        Set the VIMBA_X_HOME environment variable to your Vimba X installation directory. microEye will read VIMBA_X_HOME at startup and use it to point the Vimba X transport-layer (.cti) configuration (e.g., VIMBA_X_HOME/cti).

        Note: On Windows the Vimba X installer typically creates this variable automatically — verify it before manually setting it.

     2. Manual

        If the automatic method is not prefered, create the environment variable VIMBA_X_CTI and point it to the directory (or directories) that contain the Vimba X .cti transport-layer files. Use directory paths (not individual .cti files). Multiple directories are allowed — separate them with the platform-specific path separator.

   • Basler CMOS cameras: Install pylon (avoid installing it inside the Program Files directory).

   • IDS CMOS cameras:

     • Install IDS Software Suite 4.96.1 for Windows 32/64-bit.

     • Alternatively, IDS Peak Extended Setup is prefered as it support old uEye models and the new uEye+ ones.

       • The installation package also includes the uEye camera drivers for UI models in addition to the standard setup. Therefore, no installation of the IDS Software Suite is required for the operation of UI models.
       • If the process cannot read the system-wide GENICAM_GENTL_PATH/GENICAM_GENTL64_PATH, add the same variable to your User environment variables (not just System) and restart any open terminals or applications for the change to take effect.

   • Integrated Optics Lasers: Install Laser control software.

   • PCO cameras: Install pco.sdk, pco.runtime.

     Note: Support is under development.

   • Thorlabs CMOS cameras: Install Thorcam in its default directory. Note: Some Thorlabs cameras may be identified as IDS uEye cameras by Windows and may run without Thorcam.

   • Thorlabs hardware, install Kinesis® Software and Elliptec™ Software.

4. Open a terminal and execute microEye: 🥳

   usage: microEye.exe [-h] [--module {mieye,viewer}] [--QT_API {PySide6,PyQt6,PyQt5}] [--theme {None,qdarktheme,qdarkstyle,...}]
                    [--log-level {CRITICAL,ERROR,WARNING,INFO,DEBUG}] [--no-log-file] [--no-log-console]
   
   options:
     -h, --help            show this help message and exit
     --module {mieye,viewer}
                           The module to launch [mieye|viewer], If not specified, launcher is executed.
     --QT_API {PySide6,PyQt6,PyQt5}
                           Select QT API [PySide6|PyQT6|PyQt5], If not specified, the environment variable QT_API is used.
     --theme {None,qdarktheme,qdarkstyle,...}
                           The theme of the app, if not specified, the environment variable MITHEME is used.
     --log-level {CRITICAL,ERROR,WARNING,INFO,DEBUG}
                           Root logging level (default WARNING).
     --no-log-file         Disable logging to file.
     --no-log-console      Disable logging to console.

Note: Ensure all necessary drivers are installed for microEye to function properly.

Troubleshooting Installation

If you encounter any issues during the installation process, please check the following:

• Ensure that you have the latest version of Python installed (3.9 ≤ version ≤3.11).
• Try installing the required packages repeating step (2) or individually using pip install <package_name>.
• Check the system requirements for specific hardware drivers and follow the installation instructions provided by the manufacturers.
• Refer to the project's issue tracker on GitHub for any known installation issues and solutions.

If the issue persists, feel free to open a new issue on the project's GitHub repository, providing detailed information about the problem and any error messages you encountered.

microEye Launcher

The microEye Launcher allows you to launch either the miEye Module or the Viewer Module, and provides options to select the Qt API and theme for the application.

Usage

Upon running the launcher, you will be presented with the following interface:

• miEye Module: Launches the miEye module for microscope control and acquisition.
• Viewer Module: Launches the viewer module for image/data anlysis and visualization.
• QT API (dropdown): Select the Qt API to use. Options are PySide6, PyQt6, or PyQt5.
• Theme (dropdown): Select the theme for the application. Options are None (default), qdarktheme, or qdarkstyle.

To launch a module, simply click on the respective button (miEye Module or Viewer Module). If you wish to change the Qt API or theme, select the desired option from the dropdown menus before launching.

Modules

The miEye Module

The miEye_module provides the primary graphical user interface (GUI) for microscope control and data acquisition, combining the functionalities of the deprecated Acquisition and Control modules.

miEye module (NOT UP TO DATE)	Acquisition Camera

How to use:

To launch the miEye module, run the following command:

microEye --module mieye

Experiment Designer (Beta)

The Experiment Designer is a new feature within the miEye Module that allows users to create and manage complex acquisition protocols through a graphical interface.

Key features include:

• GUI-based design of acquisition protocols;
• Support for loops and device parameter adjustments/actions;
• Export and import capabilities for protocols;
• Threaded execution with pause functionality;
• Configurable delays between protocol steps
• Context menu for easy adjustments
• Nested protocol structure
• Acquisition action wait option
• Cell reordering and deletion via keyboard shortcuts
• Real-time execution progress view

To access the Experiment Designer:

1. Launch the miEye module.
2. Look for the Protocols view in the interface.

Note: This feature is currently in beta. We value your feedback to enhance its functionality and improve the user experience.

The Multi Viewer Module

The multi_viewer Module is an improved GUI that replaces the deprecated tiff_viewer module. It allows users to process multiple files and provides data analysis and visualization tools for super-resolution single-molecule localization microscopy and single-particle tracking.

Raw Data	Localizations

How to use:

To launch the Multi Viewer module, run the following command:

microEye --module viewer

Uses Packages

The microEye uses the following Python packages:

Code Quality and Formatting	Data Analysis and Visualization	File and Data Storage	GUI and UI Development	Image and Video Processing	Microscopy	Other Utilities
autopep8	dask	h5py	PyQt5 (optional)	opencv_python	pco	hidapi
pyflakes	matplotlib	ome_types	PyQt6 (optional)	PyOpenGL	pycromanager	mmpycorex
	numba	tables	PyQt6-QScintilla (optional)		pyueye	pyfiglet
	numpy	tifffile	PySide6		pypylon	pyjokes
	pandas	zarr	pyqtgraph			pyserial
	pystackreg		pyqtdarktheme			setuptools
	scikit-image		QDarkStyle			tabulate
	scikit_learn		QScintilla			tqdm
	scipy

Note: The VimbaPython package is bundled with the Vimba SDK and must be installed manually.

IMPORTANT: Starting from microEye v2.4.0, the legacy Vimba SDK is no longer supported. Please use the Vimba X SDK and its accompanying vmbpy Python package instead.

Microscope Scheme

Schematic overview of the miEye instrument:

• A) Single-Mode Fiber (SMF): Excitation path for TIRF-, HILO-, and Epi-mode.
• B) Multi-Mode Fiber (MMF): Excitation path for Epi-mode when imaging MMF output on the sample plane.
• C) Fluorescence Emission: Path for fluorescence emission.
• D) Automatic Focus Stabilization: the automatic focus stabilization path using an IR laser in TIRF setting.

Scheme 1	Scheme 2

Key Components: AC: Achromat lens, AS: Aspheric lens, BFP: Back-focal plane, TL: Tube lens, B: B-coated N-BK7 optics, BS: Beamsplitter.

Hardware

Supported Cameras

Camera	Description	Driver	Link
IDS uEye UI-3060CP Rev. 2	IDS industrial-grade CMOS cameras	IDS uEye SDK 4.96.1 pyueye	Product Link
Thorlabs DCC1545M	DCx camera using UC480 driver	ThorCam 3.7.0	Product Link
Allied Vision Alvium 1800	Allied Vision industrial-grade CMOS cameras (U-158m, U-511m)	Vimba X SDK vmbpy	Product Link
Excelitas PCO sCMOS	Excelitas pco.edge 4.2 LT USB sCMOS Camera	pco.sdk pco.python	Product Link
Pycro-camera	Access to cameras via Micro-Manager drivers/adapters using Pycro-Manager	uManager Pycro-Manager	-

Additional Hardware

Hardware	Description	Driver	Link
Integrated Optics MatchBox	Multi-wavelength Laser Combiner, Single Laser MatchBox	Native	Products Link
Piezo Concept FOC	Nanopositioner for microscope objectives	Native	Piezo Concept FOC
Thorlabs Elliptec ELL6/ELL9/ELL12	Dual/Four/Six-Position Slider	Native	ELL6, ELL9, ELL12
Thorlabs Elliptec ELL14	Rotation Mount: SM1 Threaded	Native	ELL14
Thorlabs Elliptec ELL20	Linear Stage: 60 mm Travel	Native	ELL20
Thorlabs KDC101	Kinesis Controller for Z825B/Z925B actuators (Activate USB VCP to access the COM port in device manager)	Native	KDC101
Parallax TSL1401-DB (#28317)	Linescan Camera Module	Native	Parallax TSL1401-DB
RelayBox Arduino	For laser control using camera GPIO signals	Native	RelayBox
miEye OSF Project Parts List	Parts list of miEye OSF Project	-	Repo Link

Pycro-Manager Integration

Integration with Pycro-Manager is now implemented, providing support for managing headless instances and Core instances. This allows for seamless control and configuration of hardware devices through the Pycro-Manager framework.

Features

• Headless Manager: Manage multiple headless Micro-Manager instances (JavaBackend), including starting, stopping, and saving configurations.
• Core Instances Manager: Handle multiple PycroCore instances for device communication and control.

Integrated Hardware

The following table lists the hardware devices that are integrated and supported by the miEye module:

Device	Description
Camera	Access Micro-Manager cameras (mainly done for focus stabilization).
ZStage	Access Micro-Manager Z-stage devices for focus stabilization and axial positioning control.

How to Use

1. Headless Manager:

   • Open the miEye_module and navigate to Tools -> Micro-Manager Headless Manager in the main menu.
   • Start a new headless instance using a specific configuration file.
   • Stop individual running instances or terminate all instances simultaneously.
   • Save configurations for future use or load previously saved configurations for headless instances.

2. Core Instances:

   • Open the miEye_module and navigate to Tools -> Micro-Manager Core Bridges in the main menu.

   • Click on Add Core Bridge and provide the port address in the dialog box.

   • A list of connected devices will be displayed, allowing you to view each device's properties.

   • Close the window and refresh the camera list to access Pycro-Manager cameras.

     Accessing other types of devices is under development!

     Note: Currently, only acquisition and focus stabilization features are implemented. Support for additional hardware and stage-related controls is under active development.

Step-by-step Instructions

For comprehensive step-by-step instructions, please refer to the Pycro-Manager Integration Guide.

Authors

Mohammad Nour Alsamsam

People Involved

PhD supervision:

Dr. Marijonas Tutkus

Sample preparation, experiments and testing:

Aurimas Kopūstas Tutkus Lab Team

Acknowledgement

Research and access to intruments and samples is credited to:

• Vilnius University, Lithuania.
• Center For Physical Sciences and Technology, Vilnius, Lithuania.
• Research Council of Lithuania.

Special thanks to the following projects and libraries that make this work possible:

• SMAP/fit3Dcspline: The original code, provided as a part of the Ries group SMAP software. The pyfit3Dcspline module in our project is a Python adaptation of this functionality, offering both CPU and GPU accelerated fitting of Single-Molecule Localization Microscopy (SMLM) data. For more details, refer to the pyfit3Dcspline README.

• ACCéNT: a partial implementation of the photon free calibration within the acquisition pipeline which generates pixel-wise mean and variance images.

  Robin Diekmann, Joran Deschamps, Yiming Li, Aline Tschanz, Maurice Kahnwald, Ulf Matti, Jonas Ries, "Photon-free (s)CMOS camera characterization for artifact reduction in high- and super-resolution microscopy", bioRxiv 2021.04.16.440125. doi: 2021.04.16.440125

• Phasor Fit: We have implemented the 2D phasor fitting algorithm in Python for fast pre-fitting visualization of localizations.

  K.J.A. Martens, A.N. Bader, S. Baas, B. Rieger, J. Hohlbein. "Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): an algorithm for MHz localization rates using standard CPUs," bioRxiv, 2017. DOI: 10.1101/191957.

• Endesfelder Lab/SMLMComputational: A numba accelerated adaptation of Drift Correction, Fourier Ring Correlation (FRC) structural resolution and Nearest Neighbour Analysis (NeNA) for localization precision from Endesfelder Lab.

  Raw data to results: a hands-on introduction and overview of computational analysis for single-molecule localization microscopy", Martens et al., (2022), Frontiers in Bioinformatics. Paper

• TARDIS (Temporal Analysis of Relative Distances): We have developed a partial Python implementation of TARDIS without fitting for now. For more information, refer to the TARDIS software releases. The underlying algorithms and scientific details of TARDIS are detailed in the manuscript:

  Martens et al., “Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particle tracking”, Nature Methods (2024). Article

Note: I'm committed to maintaining an accurate acknowledgment list for our project. However, if I inadvertently miss acknowledging your work, please don't hesitate to reach out to us. I appreciate your understanding, and I'm doing my best to manage all acknowledgments amidst my other responsibilities.

I make it a standard practice to cite and provide credit within a function's docstring whenever I draw inspiration from any external reference.

Citation

If you find our work or software helpful in your research or project, we kindly request that you cite it appropriately. Here is the suggested citation format:

M.N. Alsamsam, A. Kopūstas, M. Jurevičiūtė, and M. Tutkus, “The miEye: Bench-top super-resolution microscope with cost-effective equipment,” HardwareX 12, e00368 (2022). Paper

Additionally, we would appreciate it if you could provide a link to our GitHub repository or any relevant publication associated with the software.

Alsamsam, M. N. microEye, https://github.com/samhitech/microEye [Computer software]

Thank you for your support!