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

Fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique offering profound insights into a broad spectrum of biological processes such as metabolic imaging, protein–protein interactions and live-cell intracellular dynamics. The future of FLIM appears promising, with continuous technological advancements for time-resolved measurements pushing the boundaries for spatiotemporal information. However, the growth of the FLIM community has been slower, owing to the requirement for specialized training and technology. This Primer aims to address this gap by providing a comprehensive overview of FLIM principles, methods and analysis. We discuss various methods for measuring fluorescence lifetimes, including time-tagging and phase-modulation shift methods, along with their implementations and setup variations. Additionally, we explore different avenues for data analysis, with a specific focus on the phasor approach and its crucial considerations. Furthermore, we present a range of applications demonstrating versatility and usability of FLIM. Limitations and optimization strategies are also discussed, covering methodological constraints, equipment limitations and potential errors, along with their solutions. By sharing our expertise, we aim to expand FLIM to broader audiences while reinforcing concepts within the FLIM community. This Primer seeks to inspire bioimaging researchers to fully embrace FLIM, thereby advancing our understanding of complex biological systems

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

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

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

Desarrollo de una estrategia proteómica basada en el marcado por proximidad in vivo para la identificación de vecinos de la proteína de división celular FtsZ de Corynebacterium glutamicum

La división celular bacteriana es un proceso dirigido por el divisoma, un complejo macromolecular cuyo ensamblaje comienza con la polimerización de la proteína FtsZ en el sitio de división. FtsZ participa en el posterior reclutamiento de otras proteínas componentes del divisoma, que en el caso de Escherichia coli y Bacillus subtilis fueron identificadas y caracterizadas. Sin embargo, el suborden Corynebacterineae (que incluye importantes patógenos humanos) carece de homólogos reconocibles para muchas de estas proteínas de división celular, sugiriendo una composición y arquitectura diferente del divisoma. Este trabajo se centra en el desarrollo de estrategias proteómicas basadas en la biotinilación por proximidad para estudiar el divisoma de Corynebacterium glutamicum. Para ello generamos una cepa que expresa FtsZ unida a una ascorbato peroxidasa ingenierizada (APEX2). APEX2 cataliza la oxidación de fenol biotina en presencia de H2O2 dando lugar a un radical que reacciona con aminoácidos de proteínas cercanas y permite su purificación por afinidad e identificación por espectrometría de masa (MS). La utilidad de esta estrategia depende en forma crítica de diseño experimental y la inclusión de controles adecuados. Evaluamos los niveles de expresión FtsZ-APEX2 y sus efectos sobre la división celular y la composición del proteoma. Además, optimizamos las condiciones del marcado in vivo y de la purificación e identificación de péptidos biotinilados por MS. Esto nos permitió obtener una lista de proteínas en la vecindad de FtsZ, incluyendo su principal interactor reportado, lo que valida la estrategia experimental. Futuros estudios permitirán seleccionar candidatos a validar como nuevos integrantes del divisomaAgencia Nacinal de Investigación e Innovació

FhaA plays a key role in mycobacterial polar elongation and asymmetric growth

Mycobacteria, including pathogens like Mycobacterium tuberculosis, exhibit unique growth patterns and cell envelope structures that challenge our understanding of bacterial physiology. This study sheds light on FhaA, a conserved protein in Mycobacteriales, revealing its pivotal role in coordinating cell envelope biogenesis and asymmetric growth. The elucidation of the FhaA interactome in living mycobacterial cells reveals its participation in the protein network orchestrating cell envelope biogenesis and cell elongation/division. By manipulating FhaA levels, we uncovered its influence on cell morphology, cell envelope organization, and the localization of peptidoglycan biosynthesis machinery. Notably, fhaA deletion disrupted the characteristic asymmetric growth of mycobacteria, highlighting its importance in maintaining this distinctive feature. Our findings position FhaA as a key regulator in a complex protein network, orchestrating the asymmetric distribution and activity of cell envelope biosynthetic machinery. This work not only advances our understanding of mycobacterial growth mechanisms but also identifies FhaA as a potential target for future studies on cell envelope biogenesis and bacterial growth regulation. These insights into the fundamental biology of mycobacteria may pave the way for novel approaches to combat mycobacterial infections addressing the ongoing challenge of diseases like tuberculosis in global health.Agencia Nacional de Investigación e InnovaciónFondo para la Convergencia Estructural del MERCOSURECOS-Sud France-Uruguay (contract U20B02)Agence Nationale de la Recherche (ANR,France

A global view of standards for open image data formats and repositories

Imaging technologies are used throughout the life and biomedical sciences to understand mechanisms in biology and diagnosis and therapy in animal and human medicine. We present criteria for globally applicable guidelines for open image data tools and resources for the rapidly developing fields of biological and biomedical imaging.Chan Zuckerberg Initiative 2019-210874 Horizon 2020 Framework Programme of the European Union 653493Versión publicada - versión final del edito