Quantifying the Qualitative: Differential partition of DiI-C18 in Natural Membranes.

TURINA, ANAHI DEL VALLE; SANCHEZ-BORZONE, MARIELA; COLMANO, G. NICOLAS

Buenos Aires

Jornada; Primeras Jornadas virtuales SAB 2020; 2020

Sociedad Argentina de Biofísica

Epifluorescence microscopy (EFM) applied to Langmuir Films (LF) provide images that allow to evince macroscopic domains and phase coexistence. Given their quantum yield, photostability and more disordered phase preference, dialkylcarbocianine probes are used to examine liquid phases in model membranes. Due to compositional complexity of natural membranes it is expected to show Lo-Ld phase coexistence making DiI-C18 probe adequate for their study. However, the partition of the probe in a particular liquid phase difficult EFM images interpretation, often reducing the analysis to a qualitative description.We analyzed a considerable amount of EFM images of Langmuir-Schaefer films constructed from LF of biomimetic (PC:PE:PS:Cho) and synaptosomal membranes (Bt= Bovine; Ti= Triatomine) labeled with DiI-C18 for quantifying the macroscopic phases at different lateral pressures. Moreover, images of insecticide Fipronil treated membranes were also analyzed.The processing and analysis were performed with the image processing package Fiji. Briefly, images were grey scaled and three grey ranges (Darker, Lighter and Brighter) were defined as different membrane environments that affect the fluorescence and/or the partition of the probe. We observed a significative Dark Gray Area increase with the Bt membrane packing, suggesting that: a) the more ordered phase is growing along with compression and b) DiI-C18 partitioned preferentially in the disordered phase of these membranes. The probe behaves similar in biomimetic membranes. On the other hand, DiI-C18 seems to prefer ordered phase in Ti membranes. At 35 mN/m, the more ordered liquid phase represents the 65±2% (Bt) and 59±3% (Ti) and decreased to 51±3% (Bt) and 40±1% (Ti) by the presence of Fipronil. Biomimetic membranes reproduced that behavior. This analysis allowed us to gather numerical data obtained from EFM images to compare with data obtained from other techniques. Also, we determined that DiI-C18 behaves differentially in each natural membrane. Acknowledgements to Centro de Referencia de Vectores( CeReVe), CONICET, SECyT and FONCyT.