Simple drift compensation for point measurements with SymPhoTime64

Overview

This script, written in a PicoQuant SPT64 scripting language, is designed to perform fluorescence lifetime imaging (FLIM) and time-correlated single-photon counting (TCSPC) measurements using a PI-Scanner (Piezo Scanner) and a TCSPC module. It systematically scans points and images, analyzes intensity, and determines new scanning positions dynamically.

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Key Components

1. Device Initialization and Setup

• The script starts by obtaining instances of required hardware:
  • MyTCSPC → TCSPC device (for photon counting)
  • Scan → PI-Scanner (for positioning and imaging)
  • WS → Workspace for storing measurement data
• It verifies if both devices are properly connected.

2. Measurement Methods

The script defines three core measurement functions:

2.1 WaitForMeasEnd(WithScanner: Bool)

• Waits for the measurement to complete, ensuring both TCSPC and Scanner finish their processes.

2.2 MeasurePoint(x: Float; y: Float; Time_in_ms: Int)

• Moves the scanner to a specific (x, y) position and records TCSPC data for Time_in_ms.
• Data is saved in a .ptu file.

2.3 MeasureImage(x: Float; y: Float; Size: Float; Pix: Int; Frames: Int)

• Scans an area around (x, y), capturing a fluorescence intensity image.
• Uses the fastest scanner speed, saving results as .ptu files.

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3. Image Analysis and Adaptive Measurement

MagicHappens()

• Analyzes the acquired image to identify the maximum intensity point.
• Uses image metadata (pixel resolution, offsets) to determine new coordinates (Nx, Ny).
• This dynamically updates the next measurement location.

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4. Main Measurement Loop (DoMeasurementProgram())

• Repeats the following 4 times:
  1. Measure a single point.
  2. Measure a small image region around that point.
  3. Analyze the image and adjust Nx, Ny dynamically for the next iteration.
• Saves the results in .ptu files.

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Execution Flow

1. Initialize Devices & Check Connectivity

   • Ensures both TCSPC and PI-Scanner are ready.
   • Retrieves the current workspace directory for storing data.

2. Run the Measurement Cycle

   • Starts with (Nx, Ny) = (0,0).
   • Alternates between point measurement and image scanning.
   • Dynamically determines the next measurement point based on the brightest detected area.

3. End of Execution

   • Closes all files and outputs "Measurement Done".

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Main Purpose

This script automates a fluorescence lifetime measurement process by:

• Scanning an initial point and image.
• Analyzing the intensity distribution.
• Moving to a new position based on the brightest detected area.
• Iterating this process 4 times.

This approach is useful for experiments requiring adaptive scanning—for example, detecting fluorescent molecules or biological samples in microscopy.

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Disclaimer

This script is experimental code provided as-is with no warranties or guarantees of correctness.
It is intended for demonstration purposes only. Use at your own risk.
No official support is provided for this script.