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Fluorescence and Phosphorescence: Instrumentation

Fluorescence and Phosphorescence: Instrumentation

Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.

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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...

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Related Experiment Video

Updated: Jun 16, 2025

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Published on: August 9, 2024

FLIMPA: A Versatile Software for Fluorescence Lifetime Imaging Microscopy Phasor Analysis.

Sofia Kapsiani¹, Nino F Läubli¹, Edward N Ward¹

¹Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB3 0AS, U.K.

Analytical Chemistry

|May 23, 2025

Summary

This summary is machine-generated.

We developed FLIMPA, an open-source software for analyzing fluorescence lifetime imaging microscopy (FLIM) data using phasor plots. FLIMPA offers advanced visualization and analysis, enabling new insights into molecular interactions and drug effects.

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Area of Science:

Biophysics
Microscopy
Computational Biology

Background:

Fluorescence lifetime imaging microscopy (FLIM) provides insights into fluorophore environments.
Phasor plot analysis is a powerful, model-independent method for FLIM data interpretation.
Existing commercial phasor plot software is often closed-source and proprietary.

Purpose of the Study:

To introduce FLIMPA, an accessible, open-source software for FLIM phasor plot analysis.
To provide advanced data analysis and visualization tools for FLIM data.
To enable comparative analysis across multiple experimental conditions and interactive exploration of samples.

Main Methods:

Development of FLIMPA in Python.
Implementation of phasor plot analysis for FLIM data.
Application of FLIMPA to a novel cell-based assay for microtubule depolymerization.

Main Results:

FLIMPA offers features comparable to commercial software, with enhanced data integration and visualization capabilities.
The software successfully visualizes fluorescence lifetime changes associated with microtubule depolymerization.
A novel cell-based assay for quantifying microtubule depolymerization using SiR-tubulin and FLIMPA was established.

Conclusions:

FLIMPA provides a powerful, open-source alternative for FLIM phasor plot analysis.
The developed software facilitates deeper understanding of molecular environments and cellular processes.
FLIMPA enables novel applications, such as the quantification of drug-induced microtubule depolymerization.