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Microscopy & Imaging JoVE (Open Access) Citable · DOI

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

DOI: 10.3791/55119-v
What you'll learn
  • ✓ Set up and configure open-source FLIM-HCA instrument using µManager software
  • ✓ Acquire fluorescence lifetime imaging data from multiwell plates for FRET analysis
  • ✓ Analyze protein interaction data using FIMfit software and interpret FLIM results
Protocol

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in µManager and data analysis is undertaken in FLIMfit.

Difficulty
advanced
Total time
~4–6 hours per multiwell plate (including setup, IRF calibration, acquisition, and analysis)
Model organism
HeLa, COS cells
Biosafety
BSL-1

Steps

1
Install and configure openFLIM-HCA plugin

Set up the FLIM-HCA instrument control software plugin in µManager. This establishes the graphical interface and automation parameters needed for high-content imaging.

▶ 01:03
2
Acquire instrument response function and reference dye data

Collect IRF calibration images and reference dye fluorescence lifetime measurements to establish baseline parameters for FLIM analysis. These data are essential for downstream lifetime calculations.

▶ 01:51
3
Perform automated FLIM imaging on multiwell plates

Execute automated fluorescence lifetime image acquisition across multiple wells containing FRET-based protein interaction samples. The software controls stage movement, laser timing, and detector settings.

▶ 04:32
4
Analyze FLIM data and visualize FRET results

Process acquired FLIM images using FIMfit to extract lifetime values, calculate FRET efficiency, and generate representative pseudo-colored lifetime maps in COS and HeLa cell populations.

▶ 06:29
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