Biology subjects

Malacrida, L. S.

Publications and source records attributed to Malacrida, L. S..

8 recordsLinked to original sources

bioRxiv2026

High-Throughput Single-Cell Spectroscopy Using Phasor Analysis of Spectral Flow Cytometry

Phasor analysis is a well-established tool in hyperspectral and lifetime microscopy, providing a powerful, fit-free approach for interpreting complex fluorescence. However, its application has remained largely restricted to imaging-based modalities. Spectral flow cytometry (SFC) enables acquisition of full emission spectra from large numbers of independent single-cell events, offering superior statistical power compared to microscopy, albeit at the expense of spatial and temporal information. Here, we present the first implementation of spectral phasor analysis for SFC (phSFC), establishing a unified analytical framework that preserves interpretative continuity with hyperspectral microscopy while extending phasor-based analysis to high-throughput, single-cell measurements. Using the membrane-sensitive probe LAURDAN as a benchmark, we demonstrate that SFC reproduces phasor signatures of membrane order previously reported by hyperspectral confocal microscopy (HSI). We performed comparative analyses using multilamellar lipid vesicles (MLVs) prepared from known physical order compositions. Both modalities, SFC and HSI, accurately resolved MLVs with fluid, gel and liquid-ordered and liquid-disorder membrane phases, capturing cholesterol-dependent spectral shifts, including trajectories associated with mixtures of the different lipid phase behavior. Although absolute phasor coordinates differed between modalities due to distinct spectral sampling and detector configurations, the relative organization of membrane physical states was preserved. Notably, SFC produced more compact phasor distributions, consistent with larger sample size and enhanced statistical robustness. To further extend phSFC, we first evaluated its capacity to resolve membrane changes in live cultured cells following cholesterol depletion, establishing consistency between HSI and SFC measurements. We then applied phSFC to detect membrane dynamics in primary leukocytes isolated from bronchoalveolar lavage of mice with inflammation-associated lung pathology. LAURDAN fluorescence in the presence of autofluorescence and antibody-derived signals is quantified and discussed with simple solution by n-harmonic phasor analysis unmixing. Together, these results establish SFC as a robust and complementary extension of LAURDAN phasor analysis, bridging HSI and high-throughput flow cytometry measurements.

biophysics↗

bioRxiv2026

Phasor analysis of RGB camera data enables fluorescence microscopy unmixing and brightfield segmentation in a commercial microscope

Spectral information plays a crucial role in biological imaging, yet conventional epifluorescence and histological techniques often rely on RGB image acquisition, limiting the resolution of spectrally overlapping components. Here, we present a phasor-based spectral analysis framework adapted for RGB images, enabling unsupervised segmentation and unmixing without the need for hyperspectral systems or sequential acquisition. By applying a discrete Fourier transform to the red, green, and blue intensities at each pixel, we generate a two-dimensional phasor plot where spectral relationships are encoded in modulation and phase. We demonstrate the utility of this approach across three distinct applications: segmentation of lung histology images stained with hematoxylin and eosin to quantify alveolar collapse, analysis of autofluorescence in skin lesions (nevi and melanoma) to highlight pathological spectral signatures, and spectral unmixing in multicolor-labeled U2OS cells to resolve overlapping fluorophores. Our method improves signal separation, reduces noise, and enhances biological interpretability using standard RGB acquisition. These findings establish RGB phasor analysis as a practical and powerful tool for spectral decomposition and segmentation in microscopy, bridging the gap between conventional imaging and advanced spectral analysis.

biophysics↗

bioRxiv2026

Exploring the effects of Golgi Reassembly and Stacking Proteins in lipid membranes

Golgi Reassembly and Stacking Proteins (GRASP) have been associated with Golgi-ribbon structure and unconventional vesicular protein secretion. In performing these functions, GRASPs consistently interact with membranes. The presence of lipid modifications, such as myristoylation, is a crucial consideration for obtaining detailed information about the interactions between GRASPs and membranes. Nonetheless, it has been overlooked in the literature so far. Here, we describe a reconstitution protocol for myristoylated human GRASP65 and GRASP55 in lipid model membranes, enabling investigation of their interactions using techniques ranging from structural characterization to spectroscopy and microscopy. Our results showed that myristoyl-anchored GRASPs can influence membrane dynamics, suggesting a possible role for their disordered SPR domain in this interaction.

biophysics↗

bioRxiv2025

Quantitative and unbiased lung alveolar septum assessment in a LPS experimental mouse model using 2D-spatial correlation image analysis from hematoxylin and eosin slides.

Quantitative assessment of lung tissue architecture is essential for evaluating disease progression in experimental models of acute lung injury (ALI). However, conventional methods for measuring alveolar septum (tabique) thickness rely on manual annotation, which is time-consuming and observer-dependent, compromising comparability and reproducibility. In this study, we introduce a novel, fully automated approach based on two-dimensional spatial autocorrelation function (2D-ACF) analysis to quantify septum thickness from hematoxylin and eosin (H&E)-stained lung tissue sections. After validating the method with a simulated data set, the new approach was applied to a murine model of Acute Lung Injury (ALI) induced by intratracheal instillation of lipopolysaccharide (LPS), under two dietary conditions: normal (ND) and high-fat diet (HFD), thereby testing our approach with a double hit protocol for ALI development. The 2D-ACF analysis provided a robust metric of structural organization, allowing for an unbiased estimation of mean septum thickness across entire tissue images. Compared to controls, LPS instillation increased septal thickness more than six-fold, with further thickening observed in the HFD+Instilled group. These findings were consistent across >400 images and captured subtle additive effects of metabolic stress on lung injury. Traditional manual measurements of septum thickness exhibited substantial inter-observer variability, particularly in the LPS-instilled group, where structural heterogeneity made consistent interpretation challenging. This subjectivity limits the reproducibility of histopathological evaluations, especially under pathological conditions with subtle anatomopathological differences. In contrast, the 2D-ACF method offered a standardized and observer-independent approach. A direct comparison between methods revealed a strong and statistically significant correlation in the instilled group (r = 0.89, p < 0.001), indicating that the 2D-ACF captures key structural features aligned with expert assessments while reducing user bias. In summary, the 2D-ACF framework offers a powerful alternative to conventional image analysis for studying lung pathology. Its adaptability to different experimental settings and potential to be extended to other tissues with spatial heterogeneity makes it a valuable tool for translational biomedical research and digital pathology.

biophysics↗

bioRxiv2024

Modulation of Host Cell Membrane Biophysics Dynamics by Neospora caninum: A Study Using LAURDAN Fluorescence with Hyperspectral Imaging and Phasor Analysis

Neospora caninum is known to manipulate host cell organelles and recruit lipids for its survival. However, the impact of this lipid redistribution on host cell membranes remains poorly understood. This study used LAURDAN fluorescence, hyperspectral imaging, and phasor plot analysis to investigate how N. caninum modifies membrane order in Vero cells. The results revealed a significant decrease in host cell plasma and internal membrane order upon infection, suggesting that cholesterol is redistributed from the host plasma membrane to the parasitophorous vacuoles. To mimic cholesterol depletion, uninfected cells were treated with methyl-{beta}-cyclodextrin (MBCD), which increased membrane fluidity. Conversely, replenishing infected cells with cholesterol-loaded MBCD restored membrane fluidity to levels lower than control cells, indicating cholesterol enrichment. These findings provide novel insights into how N. caninum modulates host cell membrane dynamics through lipid manipulation, potentially aiding its intracellular survival.

biophysics↗

bioRxiv2023

Spectral phasor plot for hyperspectral imaging of acridine orange photophysics within the cell

Acridine Orange (AO) exhibits complex photophysical properties that have been utilized to study its interactions with RNA and DNA. Traditionally, AO has been used to stain cells, distinguishing nucleus and cytoplasm based on differences in DNA and RNA composition. Its labeling behavior varies depending on factors such as concentration and pH. AO fluorescence is rich and complex; therefore, acquiring the full spectrum or the fluorescence lifetime is beneficial for understanding its intricate photophysics. Nevertheless, handling 4D datasets can be challenging and often relies on sophisticated or highly parameterized models for their analysis. Model-free approaches, such as spectral phasor (SP) analysis for hyperspectral imaging (HSI), are well suited to navigating these analytical challenges. Using SPs principles of linear combination and reciprocity, we identified spectral fingerprints of RNA and DNA in in vitro experiments that remain consistent in both live and fixed cells. Interestingly, SP analysis of AO fluorescence in live cells revealed a third spectral component at 640 nm. The linear properties of SP analysis enabled the quantification of AO bound to RNA, DNA, and the third component. Co-labeling with AO and LysoTracker suggested that some of these subcellular structures are associated with acidic compartments. Moreover, SP analysis distinguished AO self-interactions from its RNA binding in cytoplasmic compartments. This study establishes SP analysis as a robust tool for hyperspectral imaging of AO, thereby enhancing our understanding of its intricate photophysics and unlocking new opportunities for advanced in vivo cell imaging applications.

biophysics↗

bioRxiv2022

Phasor-Based Multi-Harmonic Unmixing for in-vivo Hyperspectral Imaging

Hyperspectral imaging (HSI) is a paramount technique in biomedical science, however, unmixing and quantification of each spectral component is a challenging task. Traditional unmixing relies on algorithms that need spectroscopic parameters from the fluorescent species in the sample. The phasor-based multi-harmonic unmixing method requires only the empirical measurement of the pure species to compute the pixel-wise photon fraction of every spectral component. Using simulations, we demonstrate the feasibility of the approach for up to 5 components and explore the use of adding a 6th unknown component representing autofluorescence. The simulations show that the method can be successfully used in typical confocal imaging experiments (with pixel photon counts between 101 and 103). As a proof of concept, we tested the method in living cells, using 5 common commercial dyes for organelle labeling and we easily and accurately separate them. Finally, we challenged the method by introducing a solvatochromic probe, 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (LAURDAN), intended to measure membrane dynamics on specific subcellular membrane-bound organelles by taking advantage of the linear combination between the organelle probes and LAURDAN. We succeeded in monitoring the membrane order in the Golgi apparatus, Mitochondria, and plasma membrane in the same in-vivo cell and quantitatively comparing them. The phasor-based multi-harmonic unmixing method can help expand the outreach of HSI and democratize its use by the community for it does not require specialized knowledge.

biophysics↗

bioRxiv2021

In vivo macromolecular crowding is differentially modulated by Aquaporin 0 in zebrafish lens: insights from a nano-environment sensor and spectral imaging.

Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and ultimately water-dynamics are needed to understand their role in cellular fates. The macromolecular crowding in the lens is essential for understanding normal optics of the lens, and moreover for understanding and prevention of cataract and presbyopia. Here we combine the use of the water nano-environmentally sensitive sensor (6-acetyl-2-dimethylaminonaphthalene, ACDAN) with in vivo studies of Aquaporin zero zebrafish mutants to understand the lens macromolecular crowding. Spectral phasor analysis of ACDAN fluorescence reveal the extent of water dipolar relaxation and demonstrate that the mutations in the duplicated zebrafish Aquaporin 0s, Aqp0a and Aqp0b, alter the water state and macromolecular crowding in the living zebrafish lens. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This work opens new perspectives for in vivo studies on macromolecular crowding. TeaserIn this study we uncover the roles of Aquaporin 0 in macromolecular crowding required for lens development and vision.

biophysics↗