Microscopic bioluminescence imaging has been historically challenging due to a lack of detection methods and easily resolved probes. Here we combine bioluminescence with phasor analysis, an optical method commonly used to distinguish spectrally similar fluorophores. Bioluminescent phasor enabled rapid differentiation of multiple luciferase reporters and resonance energy transfer processes. The merger of bioluminescence and phasor analysis provides a platform for routine, time-lapse imaging of collections of cellular features.
Current assays for Clostridioides difficile in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a more rapid point-of-care assay featuring a “turn-on” bioluminescent readout of a C. difficile-specific protease, PPEP-1. NanoLuc, a bright and stable luciferase, was “caged” with a PPEP-1-responsive peptide tail that inhibited luminescence. Upon proteolytic cleavage, the peptide was released and NanoLuc activity was restored, providing a visible readout. The bioluminescent sensor detected PPEP-1 concentrations as low as 10 nM. Sensor uncaging was achieved within minutes, and signal was captured using a digital camera. Importantly, the sensor was also functional at ambient temperature and compatible with fecal material, suggesting that it can be readily deployed in a variety of settings.
Cell-to-cell communications are critical to biological
processes
ranging from embryonic development to cancer progression. Several
imaging strategies have been developed to capture such interactions,
but many are challenging to deploy in thick tissues and other complex
environments. Here, we report a platform termed Luminescence to Observe
and Track Intercellular Interactions (LOTIIS). The approach features
split fragments of a luciferase enzyme that reassemble when target
cells come into proximity. One fragment is secreted by “sender”
cells, and the complementary piece is secreted by “receiver”
cells. Split reporter assembly is facilitated by a single chain variable
fragment (scFv)–peptide interaction on the receiver cell, resulting
in localized light production. We demonstrate that LOTIIS can rapidly
label cells in close proximity in a time- and distance-dependent fashion.
The platform is also compatible with bioluminescence resonance energy
transfer probes for multiplexed imaging. Collectively, these data
suggest that LOTIIS will enable a variety of cellular interactions
to be tracked in biological settings.
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