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Cells are constantly adapting to maintain their identity in response to the surrounding media's temporal and spatial heterogeneity. The plasma membrane, which participates in the transduction of external signals, plays a crucial role in this adaptation. Studies suggest that nano and micrometer areas with different fluidities at the plasma membrane change their distribution in response to external mechanical signals. However, investigations linking fluidity domains with mechanical stimuli, specifically matrix stiffness, are still in progress. This report tests the hypothesis that the stiffness of the extracellular matrix can modify the equilibrium of areas with different order in the plasma membrane, resulting in changes in overall membrane fluidity distribution. We studied the effect of matrix stiffness on the distribution of membrane lipid domains in NIH-3 T3 cells immersed in matrices of varying concentrations of collagen type I, for 24 or 72 h. The stiffness and viscoelastic properties of the collagen matrices were characterized by rheometry, fiber sizes were measured by Scanning Electron Microscopy (SEM) and the volume occupied by the fibers by second harmonic generation imaging (SHG). Membrane fluidity was measured using the fluorescent dye LAURDAN and spectral phasor analysis. The results demonstrate that an increase in collagen stiffness alters the distribution of membrane fluidity, leading to an increasing amount of the LAURDAN fraction with a high degree of packing. These findings suggest that changes in the equilibrium of fluidity domains could represent a versatile and refined component of the signal transduction mechanism for cells to respond to the highly heterogeneous matrix structural composition. Overall, this study sheds light on the importance of the plasma membrane's role in adapting to the extracellular matrix's mechanical cues.

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Introduction: Melanoma diagnosis traditionally relies on microscopic examination of hematoxylin and eosin (H&E) slides by dermatopathologists to search for specific architectural and cytological features. Unfortunately, no single molecular marker exists to reliably differentiate melanoma from benign lesions such as nevi. This study explored the potential of autofluorescent molecules within tissues to provide molecular fingerprints indicative of degenerated melanocytes in melanoma. Methods: Using hyperspectral imaging (HSI) and spectral phasor analysis, we investigated autofluorescence patterns in melanoma compared to intradermal nevi. Using UV excitation and a commercial spectral confocal microscope, we acquired label-free HSI data from the whole-slice samples. Results: Our findings revealed distinct spectral phasor distributions between melanoma and intradermal nevi, with melanoma displaying a broader phasor phase distribution, signifying a more heterogeneous autofluorescence pattern. Notably, longer wavelengths associated with larger phases correlated with regions identified as melanoma by expert dermatopathologists using H&E staining. Quantitative analysis of phase and modulation histograms within the phasor clusters of five melanomas (with Breslow thicknesses ranging from 0.5 mm to 6 mm) and five intradermal nevi consistently highlighted differences between the two groups. We further demonstrated the potential for the discrimination of several melanocytic lesions using center-of-mass comparisons of phase and modulation variables. Remarkably, modulation versus phase center of mass comparisons revealed strong statistical significance among the groups. Additionally, we identified the molecular endogenous markers responsible for tissue autofluorescence, including collagen, elastin, NADH, FAD, and melanin. In melanoma, autofluorescence is characterized by a higher phase contribution, indicating an increase in FAD and melanin in melanocyte nests. In contrast, NADH, elastin, and collagen dominate the autofluorescence of the nevus. Discussion: This work underscores the potential of autofluorescence and HSI-phasor analysis as valuable tools for quantifying tissue molecular fingerprints, thereby supporting more effective and quantitative melanoma diagnosis.

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This article reviews the use of the 6-acetyl-2-(dimethylamino)naphthalene (ACDAN) fluorophore to study dipolar relaxation in cells, tissues, and biomimetic systems. As the most hydrophilic member of the 6-acyl-2-(dimethylamino)naphthalene series, ACDAN markedly partitions to aqueous environments. In contrast to 6-lauroyl-2-(dimethylamino)naphthalene (LAURDAN), the hydrophobic and best-known member of the series used to explore relaxation phenomena in biological (or biomimetic) membranes, ACDAN allows mapping of spatial and temporal water dipolar relaxation in cytosolic and intra-organelle environments of the cell. This is also true for the 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) derivative which, unlike LAURDAN, partitions to both hydrophobic and aqueous environments. We will (i) summarize the mechanism which underlies the solvatochromic properties of the DAN probes, (ii) expound on the importance of water relaxation to understand the intracellular environment, (iii) discuss technical aspects of the use of ACDAN in eukaryotic cells and some specialized structures, including liquid condensates arising from processes leading to liquid immiscibility and, (iv) present some novel studies in plant cells and tissues which demonstrate the kinds of information that can be uncovered using this approach to study dipolar relaxation in living systems.

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The use of phasors to analyze fluorescence data was first introduced for time-resolved studies for a simpler mathematical analysis of the fluorescence-decay curves. Recently, this approach was extended to steady-state experiments with the introduction of the spectral phasors (SP), derived from the Fourier transform of the fluorescence emission spectrum. In this work, we revise key mathematical aspects that lead to an interpretation of SP as the characteristic function of a probability distribution. This formalism allows us to introduce a new tool, called multi-dimensional spectral phasor (MdSP) that seize, not only the information from the emission spectrum, but from the full excitation-emission matrix (EEM). In addition, we developed a homemade open-source Java software to facilitate the MdSP data processing. Due to this mathematical conceptualization, we settled a mechanism for the use of MdSP as a tool to tackle spectral signal unmixing problems in a more accurate way than SP. As a proof of principle, with the use of MdSP we approach two important biophysical questions: protein conformational changes and protein-ligand interactions. Specifically, we experimentally measure the EEM changes upon denaturation of human serum albumin (HSA) or during its association with the fluorescence dye 1,8-anilinonaphtalene sulphate (ANS) detected via tryptophan-ANS Förster Resonance Energy Transfer (FRET). In this sense, MdSP allows us to obtain information of the system in a simpler and finer way than the traditional SP. Specifically, understanding a protein's EEM as a molecular fingerprint opens new doors for the use of MdSP as a tool to analyze and comprehend protein conformational changes and interactions.

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The group-specific antigen (GAG) polyprotein of HIV-1 is the main coordinator of the virus assembly process at the plasma membrane (PM) and is directed by its N-terminal matrix domain (MA). MA is myristoylated and possess a highly basic region (HBR) responsible for the interaction with the negative lipids of the PM, especially with PIP2. In addition, MA binds RNA molecules proposed as a regulatory step of the assembly process. Here we study the interaction of a synthetic peptide (N-terminal 21 amino acids of MA) and liposomes of different compositions using a variety of biophysical techniques. Particularly, we use the fluorescence properties of the single tryptophan of the peptide to analyze its partition to membranes, where we harness for first time the analytical ability of spectral phasors method to study this interaction. We found that electrostatic interactions play an important role for peptide partition to membranes and myristoylation reduces the free energy of the process. Interestingly, we observe that while the presence of PIP2 does not cause measurable changes on the peptide-membrane interaction, the interaction is favored by cholesterol. Additionally, we found that the partition process goes through a transition state involving peptide disaggregation and changes in the peptide secondary structure. On the other hand, we found that the presence of oligonucleotides competes with the interaction with lipids by increasing peptide solubility. In summary, we think that our results, in context of the current knowledge of the role of HIV-1 MA, contribute to a better molecular understanding of the membrane association process.

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The study of surfactant and bio membranes interaction is particularly complex due to the diversity in lipid composition and the presence of proteins in natural membranes. Even more difficult is the study of this interaction in vivo since cellular damage may complicate the interpretation of the results, therefore for most of the studies in this field either artificial or model systems are used. One of the model system most used to study biomembranes are erythrocytes due to their relatively simple structure (they lack nuclei and organelles having only the plasma membrane), their convenient experimental manipulation and availability. In this context, we used rabbit erythrocytes as a model membrane and Laurdan (6-lauroyl-2-dimethylaminonaphthalene) as the fluorescent probe to study changes promoted in the membrane by the interaction with the sucrose monoester of myristic acid, ß-d-fructofuranosyl-6-O-myristoyl-α-d-glucopyranoside (MMS). Surfactant and erythrocytes interaction was studied by measuring hemoglobin release and the changes in water content in the membrane sensed by Laurdan. Using two-photon excitation, three types of measurements were performed: Generalized Polarization (analyzed as average GP values), Fluorescence Lifetime Imaging, FLIM (analyzed using phasor plots) and Spectral imaging (analyzed using spectral phasor). Our data indicate that at sublytical concentration of surfactant (20µM MMS), there is a decrease of about 35% in erythrocytes size, without changes in Laurdan lifetime or emission spectra. We also demonstrate that as hemolysis progress, Laurdan lifetime increased due to the decrease in hemoglobin (strong quencher of Laurdan emission) content inside the erythrocytes. Under these conditions, Laurdan spectral phasor analyses can extract the information on the water content in the membrane in the presence of hemoglobin. Our results indicate an increase in membrane fluidity in presence of MMS.

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Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment. In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral phasor analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.

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In this note, we present a discussion of the advantages and scope of model-free analysis methods applied to the popular solvatochromic probe LAURDAN, which is widely used as an environmental probe to study dynamics and structure in membranes. In particular, we compare and contrast the generalized polarization approach with the spectral phasor approach. To illustrate our points we utilize several model membrane systems containing pure lipid phases and, in some cases, cholesterol or surfactants. We demonstrate that the spectral phasor method offers definitive advantages in the case of complex systems.