Colleagues:

I write to bring to your attention the availability of a wide-field fluorescence lifetime imaging microscopy (FLIM) system administered by the NYULH Microscopy Laboratory as shared equipment.  The system is a Lambert Instruments FLIM Attachment (LIFA) Toggel camera and associated lasers that have been mounted on a Nikon Eclipse TE2000 in Smilow 1208A and is now available to the NYULH research community.  https://axiomoptics.com/advanced-light-microscopy/lifa-toggel-fluorescence-lifetime-imaging-camera/.

FRET is commonly done here by measuring the ratios of two fluorescent probes.  This intensity ratio changes depending on whether there is FRET.  FLIM has benefits, such as being faster being more sensitive to molecules in mixed FRET or non-FRET states.  The toggle FLIM system images by widefield fluorescence and gives a direct readout of lifetime.

As many of you know, fluorescence resonance energy transfer (FRET) has many applications in biomedical research, not the least of which is as a modality capable of determining molecular proximity in living cells.  It is ideally suited to measure macromolecular interactions such as protein-protein interactions.   As such, it constitutes an orthogonal approach to biochemical methods such as affinity capture, co-immunoprecipitation and proximity labeling and has the advantage of allowing measurements in live cells and tissues.  In addition to macromolecular proximity, there are many genetically encoded biosensors now available that read out as gain or loss of FRET.  These include biosensors that report pH, [Ca++], second messenger concentration (e.g. cAMP), DNA damage, autophagy, caspase activation, kinase activity, and activation of G proteins.
There are several ways of measuring FRET with a microscope, including sensitized emission, release of donor quenching upon acceptor photobleaching, and FLIM.  The most common method used here is ratio FRET (standard  FLIM offers distinct advantages over other methods of measuring FRET (see PMID 32406215, 22264545, 25815593).  The new LIFA system measures FLIM by frequency-domain, which makes it much faster than time-resolved systems and therefore superior for monitoring signaling events in real time.  It is also much simpler to use than most other systems.  Indeed, the new system requires only brief training by the staff of the microscope core.
Please refer to the Microscopy Laboratory web pages for more information and to book time and/or training on the instrument.
https://med.nyu.edu/research/scientific-cores-shared-resources/microscopy-laboratory

Here are some references in which FRET (measured primarily as FLIM-FRET) was used to interrogate cellular processes:

• Protein-protein interaction:  PMID 27502489.
• Chromatin compaction: PMID 28199849.
• DNA Damage PMID 33053171.
• cAMP Signaling:  PMID 34671065.
• Caspase Activation PMID 12527749.
• Monitoring Autophagy PMID 22302004.
• Kinase and GTPase activation PMID 21976697.
• ZAP-70 activation in T cells PMID 25735979.
• Integrin activation on T cells PMID 14500982.

From Dr. Mark Reid Philips