TY - JOUR
T1 - Unsupervised clustering of multiparametric fluorescent images extends the spectrum of detectable cell membrane phases with sub-micrometric resolution
AU - Bianchetti, Giada
AU - De Spirito, Marco
AU - Maulucci, Giuseppe
PY - 2020
Y1 - 2020
N2 - Solvatochromic probes undergo an emission shift when the hydration level of the membrane environment increases and are commonly used to distinguish between solid-ordered and liquid-disordered phases in artificial membrane bilayers. This emission shift is currently limited in unraveling the broad spectrum of membrane phases of natural cell membranes and their spatial organization. Spectrally resolved fluorescence lifetime imaging can provide pixel-resolved multiparametric information about the biophysical state of the membranes, like membrane hydration, microviscosity and the partition coefficient of the probe. Here, we introduce a clustering based analysis that, leveraging the multiparametric content of spectrally resolved lifetime images, allows us to classify through an unsupervised learning approach multiple membrane phases with sub-micrometric resolution. This method extends the spectrum of detectable membrane phases allowing to dissect and characterize up to six different phases, and to study real-time phase transitions in cultured cells and tissues undergoing different treatments. We applied this method to investigate membrane remodeling induced by high glucose on PC-12 neuronal cells, associated with the development of diabetic neuropathy. Due to its wide applicability, this method provides a new paradigm in the analysis of environmentally sensitive fluorescent probes.
AB - Solvatochromic probes undergo an emission shift when the hydration level of the membrane environment increases and are commonly used to distinguish between solid-ordered and liquid-disordered phases in artificial membrane bilayers. This emission shift is currently limited in unraveling the broad spectrum of membrane phases of natural cell membranes and their spatial organization. Spectrally resolved fluorescence lifetime imaging can provide pixel-resolved multiparametric information about the biophysical state of the membranes, like membrane hydration, microviscosity and the partition coefficient of the probe. Here, we introduce a clustering based analysis that, leveraging the multiparametric content of spectrally resolved lifetime images, allows us to classify through an unsupervised learning approach multiple membrane phases with sub-micrometric resolution. This method extends the spectrum of detectable membrane phases allowing to dissect and characterize up to six different phases, and to study real-time phase transitions in cultured cells and tissues undergoing different treatments. We applied this method to investigate membrane remodeling induced by high glucose on PC-12 neuronal cells, associated with the development of diabetic neuropathy. Due to its wide applicability, this method provides a new paradigm in the analysis of environmentally sensitive fluorescent probes.
KW - Fluorescence Lifetime Microscopy
KW - Solvatochromic probes
KW - Two Photon Microscopy
KW - Unsupervised Learning
KW - Fluorescence Lifetime Microscopy
KW - Solvatochromic probes
KW - Two Photon Microscopy
KW - Unsupervised Learning
UR - http://hdl.handle.net/10807/165494
U2 - 10.1364/BOE.399655
DO - 10.1364/BOE.399655
M3 - Article
SN - 2156-7085
VL - 11
SP - 5728
EP - 5744
JO - Biomedical Optics Express
JF - Biomedical Optics Express
ER -