Lorenzo Scipioni
High-content determination of the cell state by Phasor S-FLIM
Scipioni Lorenzo1, Giulia Tedeschi1, Alessandro Rossetta2, Michelle Digman1, Enrico Gratton1
1- UCI-BME. 2- FLIM Labs
Many high throughput “-omics” techniques (genomics, transcriptomics, proteomics, metabolomics) have been developed to characterize the state of a cell. However, such methods either require highly specific expertise and instrumentation or they’re not applicable to single cells, let alone over multiple time points. We aim to propose a novel, fluorescence-based, high throughput method that allows for the acquisition of multiple physiological parameters over extended periods of time for the characterization of the physiological state of a cell.
Environment-sensitive probes can report on important physiological parameters including mitochondrial activity (membrane potential), lipid metabolism (lipid droplet concentration), transcriptional activity (chromatin compaction) and cellular homeostasis (intracellular pH), to name a few. Despite being well characterized, many technical challenges (spectral overlap, sequential acquisition, need for lifetime information) prevent their combination and therefore the acquisition of multiple physiological parameters at the same time.
One way to solve this issue is the simultaneous acquisition of spectral and lifetime information relative to the probes (S-FLIM), which would also provide a much higher level of detail about their photophysical response to the environment. Unfortunately, only a handful of commercial microscopes allow for S-FLIM acquisition, which is obtained either sequentially or with poor spectral resolution, therefore limiting the sensitivity as well as the acquisition speed. Moreover, it results in unnecessarily long exposure times, which inevitably leads to photobleaching and photodamage.
We constructed a microscope which features spectral acquisition over 32 detectors, each of which capable of acquiring lifetime information in parallel with very high precision and photon efficiency. We will show how the Phasor S-FLIM characterization can be an invaluable tool for the characterization, design and multiplexing of environment-sensitive probes and how this approach will open the doors to a completely new way of looking at biological samples.
