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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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Related Experiment Video

Updated: May 4, 2026

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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Practical guide to fluorescence lifetime imaging microscopy.

Nadia Sarfraz1, Esther Braselmann1

  • 1Department of Chemistry, Georgetown University, Washington, DC 20057.

Molecular Biology of the Cell
|May 27, 2025
PubMed
Summary
This summary is machine-generated.

Fluorescence lifetime imaging microscopy (FLIM) offers advanced cellular analysis by sensing molecular environments and enabling RNA quantification. This guide empowers researchers to utilize FLIM for diverse biosensors and microscopy applications.

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Area of Science:

  • Biophotonics and advanced microscopy techniques.

Background:

  • Fluorescence lifetime imaging microscopy (FLIM) provides unique advantages over traditional intensity-based methods, including sensitivity to molecular environments and concentration independence.
  • FLIM has been recently adapted for quantitative RNA sensing in live cells, creating the Riboglow-FLIM platform.
  • The technology holds significant potential to complement and enhance existing microscopy platforms.

Purpose of the Study:

  • To provide a practical guide for utilizing FLIM with diverse fluorescence sensors.
  • To introduce researchers to the fundamental concepts and best practices for designing and executing FLIM experiments.
  • To demonstrate the broad applicability of FLIM workflows through real-world case studies.

Main Methods:

  • Discussion of FLIM basics, including principles of operation and data acquisition.
  • Explanation of experimental setup considerations for FLIM.
  • Overview of data-fitting principles and analysis techniques relevant to FLIM data.

Main Results:

  • Demonstration of FLIM's capability for quantitative RNA sensing in live cells using the Riboglow-FLIM platform.
  • Case studies showcasing the application of FLIM with well-characterized fluorescent proteins.
  • Validation of FLIM workflows for diverse fluorescence sensing applications.

Conclusions:

  • FLIM is a powerful modality for sensing molecular environments, binding events, and metabolism.
  • The Riboglow-FLIM platform represents a significant advancement for quantitative RNA analysis in live cells.
  • This guide equips researchers with the knowledge to effectively implement FLIM for various biological sensing applications.