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Fluorescence Lifetime Macro Imager for Biomedical Applications

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Published on: April 7, 2023

Calibration approaches for fluorescence lifetime applications using time-domain measurements.

Anca Margineanu¹

¹Advanced Light Microscopy, Max-Delbrück-Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft, Robert-Rössle-Straße 10, 13125 Berlin, Germany.

Methods in Microscopy

|April 7, 2025

Summary

This tutorial provides experimental protocols for acquiring instrument response functions (IRF) and calibrating instruments using reference dyes for time-domain fluorescence lifetime measurements. It details sample preparation, instrument settings, and troubleshooting for accurate data analysis and comparison across biological systems.

Keywords:

FLIM instrument response function (IRF)reference dyes

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Area of Science:

Spectroscopy
Biophysics
Analytical Chemistry

Background:

Accurate fluorescence lifetime measurements are crucial for understanding biological systems.
Instrument response functions (IRF) and reference dye calibration are essential for reliable time-domain fluorescence lifetime data.
Standardized protocols are needed for consistent instrument performance and data interpretation.

Purpose of the Study:

To present detailed experimental protocols for acquiring IRF and calibrating instruments for time-domain fluorescence lifetime measurements.
To guide researchers in sample preparation and instrument setting optimization for one- and two-photon excitation.
To provide troubleshooting strategies for common issues encountered during instrument calibration and data acquisition.

Main Methods:

Detailed step-by-step protocols for preparing calibration samples (scatter solutions, second harmonic generating crystals, reference dyes).
Explanation of instrument settings for both one- and two-photon excitation modes.
Demonstration of expected IRF curves and reference decays with experimental examples.

Main Results:

Experimentally acquired examples of IRF curves and reference decays are presented.
Troubleshooting guidance is provided for distorted experimental results.
The importance of IRF and reference dye calibration for data accuracy is highlighted.

Conclusions:

Implementing these protocols ensures accurate instrument response function acquisition and calibration.
Proper calibration is vital for reliable time-resolved instrument parameter adjustment and data analysis.
This work facilitates the comparison and extrapolation of fluorescence lifetime values across diverse biological systems.