Measurements of absolute concentrations of NADH in cells using the phasor FLIM method.

We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution

Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis.

Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening

Novel opportunities from bioimaging to understand the trafficking and maturation of intracellular pulmonary surfactant and its role in lung diseases

Pulmonary surfactant (PS), a complex mixture of lipids and proteins, is essential for maintaining proper lung function. It reduces surface tension in the alveoli, preventing collapse during expiration and facilitating re-expansion during inspiration. Additionally, PS has crucial roles in the respiratory system’s innate defense and immune regulation. Dysfunction of PS contributes to various respiratory diseases, including neonatal respiratory distress syndrome (NRDS), adult respiratory distress syndrome (ARDS), COVID-19-associated ARDS, and ventilator-induced lung injury (VILI), among others. Furthermore, PS alterations play a significant role in chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The intracellular stage involves storing and releasing a specialized subcellular organelle known as lamellar bodies (LB). The maturation of these organelles requires coordinated signaling to organize their intracellular organization in time and space. LB’s intracellular maturation involves the lipid composition and critical processing of surfactant proteins to achieve proper functionality. Over a decade ago, the supramolecular organization of lamellar bodies was studied using electron microscopy. In recent years, novel bioimaging tools combining spectroscopy and microscopy have been utilized to investigate the in cellulo intracellular organization of lamellar bodies temporally and spatially. This short review provides an up-to-date understanding of intracellular LBs. Hyperspectral imaging and phasor analysis have allowed identifying specific transitions in LB’s hydration, providing insights into their membrane dynamics and structure. A discussion and overview of the latest approaches that have contributed to a new comprehension of the trafficking and structure of lamellar bodies is presented

Insights into in vivo adipocyte differentiation through cell-specific labeling in zebrafish.

White adipose tissue hyperplasia has been shown to be crucial for handling excess energy in healthy ways. Though adipogenesis mechanisms have been underscored in vitro, we lack information on how tissue and systemic factors influence the differentiation of new adipocytes. While this could be studied in zebrafish, adipocyte identification currently relies on neutral lipid labeling, thus precluding access to cells in early stages of differentiation. Here we report the generation and analysis of a zebrafish line with the transgene fabp4a(- 2.7):EGFPcaax. In vivo confocal microscopy of the pancreatic and abdominal visceral depots of transgenic larvae, revealed the presence of labeled mature adipocytes as well as immature cells in earlier stages of differentiation. Through co-labeling for blood vessels, we observed a close interaction of differentiating adipocytes with endothelial cells through cell protrusions. Finally, we implemented hyperspectral imaging and spectral phasor analysis in Nile Red-labeled transgenic larvae and revealed the lipid metabolic transition towards neutral lipid accumulation of differentiating adipocytes. Altogether our work presents the characterization of a novel adipocyte-specific label in zebrafish and uncovers previously unknown aspects of in vivo adipogenesis.Agencia Nacional de Investigación e InnovaciónPrograma de Desarrollo de las Ciencias BásicasFondo de Convergencia Estructural del Mercosu

Quantitative melanoma diagnosis using Spectral Phasor Analysis of Hyperspectral Imaging from label-free slices

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 differentiatemelanoma 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.Los autores agradecen el apoyo del PEDECIBA y de la Agencia Nacional de Investigación e Innovación (ANII) como miembros del Sistema Nacional de Investigadores (SNI

A FRET-based cGMP biosensor in Drosophila

CUTie2 is a FRET-based cGMP biosensor tested so far only in cells. To expand its use to multicellular organisms we generated two transgenic Drosophila melanogaster strains that express the biosensor in a tissue-dependent manner. CUTie2 expression and subcellular localization was verified by confocal microscopy. The performance of CUTie2 was analyzed on dissected larval brains by hyperspectral microscopy and flow cytometry. Both approaches confirmed its responsivity, and the latter showed a rapid and reversible change in the fluorescence of the FRET acceptor upon cGMP treatment. This validated reporter system may prove valuable for studying cGMP signaling at organismal level.Agencia Nacional de Investigación e InnovaciónDirección Nacional de Innovación, Ciencia y Tecnologí