flim-imager-releases

FLIM Imager v1.0

Table of Contents

1. Introduction
2. Application Usage
   • Preview Mode
   • Scouting Mode
   • FLIM Mode
   • FLIM Phasors Mode
   • ROI
   • Colormaps
   • Intensity Histogram
   • Zoom
   • Magnification
   • CPS
   • SBR
   • Settings
3. Diagnostic Messages
4. Data export
5. License
6. Contact

Introduction

Welcome to FLIM Imager v1.0 usage guide. In this documentation section, you will find all the necessary information for the proper use of the application. For a general introduction to the aims, technical requirements and installation of the project, read the FLIM Imager Homepage. You can also follow the Data export dedicated guide link.

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Application Usage

FLIM Imager provides users with four distinct analysis modes to suit various experimental and analytical needs:

• Preview Mode;
• Scouting Mode;
• FLIM Mode;
• FLIM Phasors Mode

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Preview Mode

Preview Mode is designed to help users achieve the perfect calibration for their acquisition setup. This mode offers a convenient toolbox that allows users to easily select and adjust the scan region (image) they wish to acquire.

Key features of Preview Mode include:

• Scan Region Adjustment: Users can configure the dimensions of:

  • Scan (width/height): The entire scannable area.
  • Image (width/height): The specific section of the scan to be acquired.
• Channel-by-Channel Exploration: Preview Mode enables users to explore and adjust parameters one channel at a time for precise tuning.
• Real-Time Feedback: The software includes a practical warning system to alert users if the set scan dimensions differ from the dimensions detected by the processor. These warnings provide the actual scan dimensions, helping users adjust their parameters accordingly for optimal accuracy.

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Scouting Mode

Scouting Mode provides users with an additional step to explore the sample frame by frame after calibrating the dimensions and offsets of the scan area.

Key features of Scouting Mode include:

• Frame-by-Frame Exploration: Unlike cumulative acquisition, in this mode, the pixel intensities of each frame are displayed individually without summation. This ensures a clear and distinct view of each acquired frame, allowing for precise inspection of the sample.

• Simultaneous Dual-Channel Visualization: Scouting Mode enables users to visualize two acquisition channels simultaneously, making it easier to compare and analyze different aspects of the sample in real-time.

This mode is designed to refine the exploration process and help users identify key features or regions of interest before moving to more in-depth analysis in FLIM Mode.

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FLIM Mode

FLIM Mode is one of the software core features, enabling in-depth analysis of the acquired sample. This mode uses a cumulative image reconstruction approach, where the intensities of individual frames are summed during acquisition to provide a comprehensive view of the sample.

Key features of FLIM Mode include:

• Cumulative Frame Acquisition: The pixel intensities from all frames are added together during acquisition, offering a complete representation of the scanned area.

• Global TCSPC Histogram: Users can view the global Time-Correlated Single Photon Counting (TCSPC) histogram in real-time, enabling decay analysis of the acquired sample during the acquisition process.

• Pixel-by-Pixel Decay Analysis: After the acquisition is complete, users can hover over individual pixels to view the TCSPC histogram for each pixel, providing a detailed spatial breakdown of decay characteristics.

• Calibration: Possibility to acquire in Calibration Mode by specifying the number of harmonics and the tau value (ns) to obtain reference data for conducting phasor analysis on the FLIM Phasors tab.

• Channel Visualization: Users can analyze up to two acquisition channels. To ensure clarity and ease of visualization, the channels are displayed one at a time in a carousel-like interface, allowing users to toggle between them.

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FLIM Phasors Mode

FLIM Phasors Mode is the other core feature of the software, designed to extract detailed insights from the acquired images for conducting phasor analysis. This mode enables a graphical representation of the lifetime distribution in the form of a phasor plot, providing a powerful tool for lifetime characterization.

Key features of FLIM Phasors Mode include:

• Phasor Plot Visualization: View the lifetime distribution associated with the acquired image in an intuitive and interactive phasor plot.

• Harmonics & Quantization: Users can customize the phasor plot by selecting the specific harmonic to visualize and adjusting the quantization level (bins) applied to the represented points.

• Circlets & Clustering: Users can apply segmentation and clustering directly on the phasor plot using customizable circlets. Circlets can be adjusted for position, radius, color, and alpha channel. Moreover, the corresponding clusters are dynamically visualized on the original image, with the pixels matching each cluster displayed in the circlet’s selected color. This real-time feedback facilitates detailed spatial analysis of the clusters.

• Median Filter: To enhance the robustness of phasor-based analysis, a recursive median filter can be applied to the 𝑔 and 𝑠 coordinates using a 3×3 kernel. This operation effectively reduces noise while preserving spatial structures, ensuring a cleaner representation of fluorescence lifetime distributions. By iteratively refining the phasor data, the filter minimizes outlier influence and enhances segmentation accuracy.

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ROI

In both Scouting, FLIM Mode and FLIM Phasors Mode, users can select a specific Region of Interest (ROI) from the acquired image after the acquisition is complete. This feature allows for detailed analysis of a particular area of the sample.

Key ROI capabilities include:

• Colormaps for enhanced visualization.
• Intensity Histogram for a graphical and intuitive representation of the image intensity map.
• Contrast adjustments for fine-tuning image clarity.
• Magnification tools to closely inspect the selected region.

In FLIM Mode, users can also view the global TCSPC histogram of the ROI and inspect the pixel-by-pixel TCSPC histograms, similar to how this functionality works for the entire image. Similarly, in FLIM Phasors Mode, users can view the corresponding phasor plot for the selected ROI, enabling a graphical representation of the lifetime distribution within the region of interest.

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Colormaps

FLIM Imager provides users with a versatile set of 83 colormaps that can be applied to the acquired images across all analysis modes. This functionality is available both in real-time during acquisition and in post-processing.

Key features include:

• Extensive Colormap Options: Users can choose from a library of 83 colormaps to enhance the visualization of their data, catering to different analytical needs and preferences.

• Contrast Adjustment: The colormap feature is paired with a contrast adjustment tool, enabling users to fine-tune image clarity and highlight specific details of interest.

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Intensity Histogram

Available across all modes, the intensity histogram provides a quick and intuitive overview of image intensity distribution. Displayed alongside the colorbar, this logarithmic-scale histogram enables users to assess intensity variations at a glance, facilitating contrast adjustments and data interpretation. By highlighting the overall brightness trends, it aids in identifying low-signal regions and optimizing visualization parameters for more accurate fluorescence analysis.

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Zoom

In both Scouting, FLIM Mode and FLIM Phasors Mode, users have the ability to perform a full-screen zoom on the acquired image, enhancing their ability to closely examine details. This functionality is available both in real-time and post-processing modes.

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Magnification

In both Scouting, FLIM Mode and FLIM Phasors Mode, users can apply magnification to the acquired sample, allowing for detailed inspection of specific areas. This feature is available both in real-time and post-processing modes. Users can choose from 4 magnification levels:

• x2
• x4
• x5
• x10

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CPS

In all acquisition modes, users can monitor the CPS (Photon Counts Per Second) for each active channel, providing real-time feedback on signal intensity. This metric is essential for assessing acquisition quality and optimizing imaging parameters.

To enable CPS visualization, the Pixel Dwell Time must be set, as it determines the temporal window over which photon counts are measured.

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SBR

The SBR (Signal-to-Background Ratio) quantifies signal quality by comparing the peak fluorescence intensity to the background noise level. It is measured in decibels (dB) and helps assess data reliability and contrast.

In FLIM and FLIM Phasors tabs, users can monitor the SBR in real-time during acquisition, enabling immediate evaluation of image quality. A higher SBR indicates a stronger signal relative to background noise, leading to more accurate lifetime measurements and phasor analysis.

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Settings

FLIM Imager includes a comprehensive settings package that allows users to configure their acquisition parameters and hardware connections with ease.

Key features of the settings system include:

• Customization: Users can tailor various parameters to match their specific acquisition needs, ensuring seamless integration with their hardware setup.

• Automatic Local Storage: Every configured setting is automatically saved in the local storage, enabling the software to retain user preferences between sessions. This feature saves time by eliminating the need to reconfigure settings with each use.

Below is a list of configurable settings.

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Setup

The setup setting allows users to run the software in Default mode or configure it for a specific microscope setup. Currently, the software supports a tailored configuration for Abberior Stedycon.

When selecting Abberior Stedycon, the acquisition and software settings are optimized for this microscope type. This includes default configurations such as:

• A maximum of 3 acquisition channels;
• Laser frequency set to 40 MHz;
• Channel type set to SMA;
• Reconstruction mode set to PLF.

Additionally, when using the Abberior Stedycon setup, users can select the desired Multi-channel mode:

• Frame: Acquired frames are alternately assigned to the active channels.
• Line: Acquired lines are alternately assigned to the active channels.
• Pixel: Individual pixels are alternately assigned to the active channels.

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Channels

FLIM Imager supports the simultaneous acquisition of up to 8 channels. While the software acquires data for all selected channels, the interface is optimized to display a maximum of 2 channels at a time. This design choice enhances UI clarity and usability during analysis.

Even though non-displayed channels are not visualized in the interface, their data is fully retained. These channels remain accessible for data export, allowing users to reconstruct and analyze them in post-processing.

To configure channels, users must specify the connection type being used (USB or SMA).

FLIM Imager also offers an automated channel detection feature. By clicking DETECT CHANNELS, the software scans for active connections and presents them to the user for confirmation. Upon approval, the detected channels are automatically configured, simplifying the setup process.

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Laser Sync In / Sync Out

Users are required to specify the laser frequency used in their experiment. This selection, along with the chosen connection type (USB or SMA), ensures that the correct firmware is triggered to support data acquisition with the configured settings.

In Sync Out mode, users can manually select a frequency from the following options: 80 MHz, 40 MHz, 20 MHz, or 10 MHz. In Sync In mode, the software automatically detects the laser frequency being used, streamlining the configuration process.

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Max Frames

Users can configure a predefined number of frames to acquire during an experiment. If this parameter is set to 0 or left unconfigured, the software will continue acquiring frames until the user manually clicks the STOP button.

This feature provides flexibility for both time-limited acquisitions and continuous, user-controlled data collection.

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Skip Frames

Users have the option to configure a number of initial frames to skip before starting the actual data acquisition. This feature allows greater control over the data by enabling the user to discard any initial noisy or unstable frames, ensuring that only clean, relevant data is acquired for analysis.

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Pixel Dwell Time (μs)

The Pixel Dwell Time defines the amount of time, measured in microseconds, that the laser beam illuminates a specific point on the specimen while the signal from that point is being detected.

This optional setting is particularly useful for performing advanced analysis and photon counting estimates over time, providing more precise control over the data acquisition process and enabling in-depth temporal analysis. Additionally, Pixel Dwell Time must be set to calculate CPS (photon counts per second) and is also required for acquisition in Line-Frame (LF) mode.

In Pixel-Line-Frame mode, the minimum dwell time is 0.1 μs, whereas in Line-Frame mode, the minimum dwell time is 1 μs.

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Scan Width (px) / Scan Height (px)

Configurable in Preview Mode, the Scan Width and Scan Height settings allow users to define the overall dimensions of the available scanned area.

If the specified scan area does not match the actual dimensions detected by the processor, the user will receive a warning notification. This warning will indicate the actual dimensions detected, allowing the user to adjust the settings accordingly for accurate data acquisition.

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Image Width (px) / Image Height (px)

Configurable in Preview Mode, the Image Width and Image Height settings allow users to configure the specific region of the scannable area that they wish to actually acquire.

These settings work in conjunction with the offset calculations, enabling the processor to determine the exact portion of the image to reconstruct.

Calibration

Configurable in FLIM Mode, the Calibration setting allow users to decide whether to generate a reference file for Phasors analysis or not.

Harmonics

Configurable in FLIM Mode, the Harmonics setting allow users to decide the number of harmonics for the reference file and so, for the Phasors analysis. This parameter is activated only when Calibration is enabled.

Tau (ns)

Configurable in FLIM Mode, the Harmonics setting allow users to decide the tau (ns) value for the reference file and so, for the Phasors analysis. This parameter is activated only when Calibration is enabled.

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Here a table summary of the configurable parameters:

param	description	data type	required
setup	the type of acquisition setup	Default/Abberior	true
abberiorMultichannelMode	the type of Abberior multi-channel image assigment	Frame/Pixel	true if in Abberior setup mode
enabledChannels	number of active channels	number[]	true
channelType	channels connection type	USB/SMA	true
frequencyMhz	Laser frequency in Mhz	number	true
selectedFirmware	The selected firmware based on laser frequency and channels connection	string	true
frameWidth	Width of the scannable area (px)	number	true
frameHeight	Height of the scannable area (px)	number	true
imageWidth	Width of the scan portion to acquire (px)	number	true
imageHeight	Height of the scan portion to acquire (px)	number	true
offsetTop	Distance of the image region from the top of the scanned area	number	true
offsetRight	Distance of the image region from the right of the scanned area	number	true
offsetBottom	Distance of the image region from the bottom of the scanned area	number	true
offsetLeft	Distance of the image region from the left of the scanned area	number	true
maxFrames	Max number of frames to acquire	number/null	false
skipFrames	Number of initial frames to skip	number/null	false
dwellTime	Pixel dwell time in microseconds	number	required only to calculate CPS and in LF mode
exportData	Whether to export acquired data or not	boolean	false
exportPath	The folder destination for exported data	string	only if exportData is set to true
exportFilename	the filename for exported data	string	only if exportData is set to true
channelsToShow	channels to visualize in the GUI (up to 2)	number[]	true
colormaps	colormaps settings for each channel	object[]	false
roiColormaps	colormaps settings for each channel (ROI mode)	object[]	false
phasorColormaps	colormaps settings for each channel (phasors plots)	object[]	false
phasorRoiColormaps	colormaps settings for each channel (phasors plots ROI mode)	object[]	false
intensityThresholds	intensity thresholds for each channel	object[]	false
roiIntensityThresholds	intensity thresholds for each channel (ROI mode)	object[]	false
curveLogMode	scale mode for TCSPC plots for each channel (linear/log)	boolean[]	false
calibration	whether to generate a reference file for phasors analysis or not	boolean	false
harmonics	number of harmonics (1 to 4) for phasors analysis. Activated only when “calibration” is set to True	number/null	required for calibration
tauNs	tau (ns) value for phasors analysis. Activated only when “calibration” is set to True	number/null	required for calibration
selectedHarmonic	the harmonic to visualize in the phasor plot	number	true
selectedHarmonic	the harmonic to visualize in the phasor plot (ROI mode)	number	true
bins	The number of bins for phasor plot points quantization	number	true
roiBins	The number of bins for phasor plot points quantization (ROI mode)	number	true

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Diagnostic Messages

FLIM Imager provides two modes for displaying diagnostic messages:

• Toast Messages: These are brief, non-intrusive notifications that appear temporarily on the screen to inform users of important events and errors.

• Log Console: For more persistent tracking, the software includes an extendable log console at the bottom of the application. This console retains a history of up to 500 log messages, including Info, Warning, Success, and Error messages. Each message is timestamped for precise tracking of events.

The diagnostic messages help users stay informed about the system’s status and troubleshoot potential issues during data acquisition and analysis.

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Data Export

In all modes, users have the option to export acquired data for further analysis in post-processing, independent of the software. When the Export data option is selected, the user will be prompted before each acquisition to specify a file path and filename for the exported data. In FLIM and FLIM Phasors Mode user will be also prompted to decide whether to export frame by frame data and/or global image data.

The exported data varies depending on the analysis mode:

• Preview Mode: Only the JPEG image of the acquired sample is exported.
• Scouting Mode: The software exports .json files for each acquired frame and active channel, along with a Python script for analyzing the frames.
• FLIM Mode: The exported data includes, depending on the option selected (frame and/or global):
  • .json files for each acquired frame and active channel;
  • global cumulative file of all acquired frames
  • Python scripts: one for analyzing individual frame data and one for analyzing global data (image + TCSPC).
  • Matlab scripts: one for analyzing individual frame data and one for analyzing global data (image + TCSPC).
• FLIM Phasors Mode: The exported data includes, depending on the option selected (frame and/or global):
  • .json imaging files for each acquired frame and active channel;
  • global imaging cumulative file of all acquired frames
  • .json files with phasors data (g/s points) for each acquired frame, active channel and harmonic;
  • global phasors data (g/s points) file for each active channel;
  • Python scripts: one for analyzing individual frame data and one for analyzing global data (image + TCSPC + Phasors).
  • Matlab scripts: one for analyzing individual frame data and one for analyzing global data (image + TCSPC + Phasors).

For detailed information on the structure of the .bin files and instructions for using the provided Python scripts, refer to:

• FLIM Imager Data Export guide

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License

Distributed under the MIT License.

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Contact

FLIM LABS: info@flimlabs.com

Project Link: FLIM Imager

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