Leonel Malacrida

• PhD
• Professor (Associate) at Clinical Hospital

Fluorescence is one of the most widely used techniques in biological sciences. Its exceptional sensitivity and versatility make it a tool of first choice for quantitative studies in biophysics. The concept of phasors, originally introduced by Charles Steinmetz in the late nineteenth century for analyzing alternating current circuits, has since foun...

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...

Fluorescence is one of the most widely used techniques in biological sciences. Its exceptional sensitivity and versatility make it a tool of first choice for quantitative studies in biophysics. The concept of phasors, originally introduced by Charles Steinmetz in the late 19th century for analyzing alternating current circuits, has since found appl...

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. Th...

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...

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....

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1‐week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision‐makers from imaging core facilities across the Americas. The meeting was...

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global...

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 analyze...

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 pote...

Membrane wetting by biomolecular condensates recently emerged as a key phenomenon in cell biology, playing an important role in a diverse range of processes across different organisms. However, an understanding of the molecular mechanisms behind condensate formation and interaction with lipid membranes is still missing. To study this, we exploited...

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...

Acridine Orange (AO) has rich photophysics that has been exploited to study its interaction with RNA and DNA. Traditionally AO was used to stain cells differentiating nucleus and cytoplasm due to the differences in DNA and RNA differential composition. AO cell labeling shows complex behavior depending on several parameters, such as concentration, p...

Over the past decade, the utilization of advanced fluorescence microscopy technologies has presented numerous opportunities to study or re-investigate autofluorescent molecules and harmonic generation signals as molecular biomarkers and biosensors for in vivo cell and tissue studies. The label-free approaches benefit from the endogenous fluorescent...

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...

Membrane wetting by biomolecular condensates recently emerged as a critical phenomenon in cell biology, involved in a great diversity of processes across different organisms. However, understanding the molecular mechanism behind this process is still missing. Exploiting ACDAN and LAURDAN properties as nano-environmental sensors in combination with...

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...

Fluorescent proteins are standard tools for addressing biological questions in a cell biology laboratory. The genetic tagging of protein of interest with fluorescent proteins opens the opportunity to follow them in vivo and to understand their interactions and dynamics. In addition, the latest advances in optical microscopy image acquisition and pr...

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)naphthalen...

Protein self-assembly is a common feature in biology and is often required for a myriad of fundamental processes, such as enzyme activity, signal transduction, and transport of solutes across membranes, among others. There are several techniques to find and assess homo-oligomer formation in proteins. Naturally, all these methods have their limitati...

Hyperspectral imaging (HSI) is paramount technique in biomedical science, however, dealing with the data it produces is challenging. Traditional unmixing relies on algorithms that need spectroscopic parameters from the fluorescent species in the sample. The phasor-based multi-harmonic unmixing method is a model-free and intuitive approach, that req...

Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and water dynamics are needed to understand their roles in cellular physiology and fate determination. Macromolecular crowding in the lens is essential for normal optics, and an understanding of its regulation will help prevent cataract and presb...

The phasor approach to fluorescence lifetime imaging, and more recently hyperspectral fluorescence imaging, has increased the use of these techniques, and improved the ease and intuitiveness of the data analysis. The fit-free nature of the phasor plots increases the speed of the analysis and reduces the dimensionality, optimization of data handling...

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...

Hyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters for biomedical imaging not feasible wit...

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 p...

The phasor approach to fluorescence lifetime imaging has become a common method to analyze complicated fluorescence signals from biological samples. The appeal of the phasor representation of complex fluorescence decays in biological systems is that a visual representation of the decay of entire cells or tissues can be used to easily interpret fund...

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.

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...

Although oxygen (O 2 ) is essential for aerobic life, it can also be an important source of cellular damage. Supra-physiological levels of O 2 determine toxicity due to exacerbated reactive oxygen species (ROS) production, impairing the homeostatic balance of several cellular processes. Furthermore, injured cells activate inflammation cascades, amp...

The activity-regulated cytoskeletal-associated protein (Arc, also known as Arg3.1) is an immediate early gene product induced by activity/experience and required for multiple modes of synaptic plasticity. Both long-term potentiation (LTP) and long-term depression (LTD) are impaired upon Arc deletion, as well as the ability to form long-term spatial...

ConspectusAny chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In bi...

Hyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters to overcome the limitations of conventi...

Transient barriers are fundamental to cell supramolecular organization and assembly. Discontinuities between spaces can be generated by a physical barrier but also by thermodynamic barriers achieved by phase separation of molecules. However, because of the transient nature and the lack of a visible barrier, the existence of phase separation is diff...

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We synthesized an Anthracene derivative with solvatochromic properties to be used as a molecular probe for membrane dynamics and supramolecular organization. A ten carbon atom acyl chain and a dimethyl amino substitution were introduced into positions 2 and 6 of the anthracene ring, respectively. This derivative, (2-nonanoyl-6-(dimethylamino) anthr...

We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detectio...

The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH) is commonly used in conjunction with the phasor approach as a molecular biomarker to provide information on cellular metabolism of autofluorescence imaging of cells and tissue. However in the phasor approach, the bound and free lifetime defining the phasor metabolic trajectory is...

How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain 5 (StarD5) leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the...

Background and purpose Atherosclerosis is characterized by chronic low‐grade inflammation with concomitant lipid accumulation in the arterial wall. Anti‐inflammatory and anti‐atherogenic properties have been described for a novel class of endogenous nitroalkenes (nitrated‐unsaturated fatty acids), formed during inflammation and digestion/absorption...

The phasor approach to FLIM (Fluorescence Lifetime Imaging Microscopy) is becoming popular due to the powerful fit free analysis and the visualization of the decay at each point in images of cells and tissues. However, although several implementation of the method are offered by manufactures of FLIM accessories for microscopes, the details of the c...

Fluorescence lifetime imaging microscopy (FLIM) is used in diverse disciplines, including biology, chemistry and biophysics, but its use has been limited by the complexity of the data analysis. The phasor approach to FLIM has the potential to markedly reduce this complexity and at the same time provide a powerful visualization of the data content....

On the cover: The cover image is based on the Research Article Differences between FLIM phasor analyses for data collected with the Becker and Hickl SPC830 card and with the FLIMbox card by Suman Ranjit et al., DOI: https://doi.org/10.1002/jemt.23061.

Fluorescence spectroscopy, coupled with microscopy, opens new frontiers for the study of dynamic processes with high spatio-temporal resolution. The application of phasor plots to FLIM and hyperspectral imaging demonstrate unprecedented capabilities to study complex photophysics at the subcellular level. Using these approaches we studied the effect...

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cel...

Image correlation analysis has evolved to become a valuable method of analysis of the diffusional motion of molecules in every points of a live cell. Here we compare the iMSD and the 2D-pCF approaches that provide complementary information. The iMSD method provides the law of diffusion and it requires spatial averaging over a small region of the ce...

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the...

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...

Despite recent advances in optical super-resolution, we lack a method that can visualize the path followed by diffusing molecules in the cytoplasm or in the nucleus of cells. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level by averaging the behavior of many molecules over time at a single spot, th...

Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location. Among the dyes used in membrane studies, LAURDAN has the advantage to be sensitive to the lipid composition as well as to membrane fluidity. The LAURDAN spectrum is sensitive to the...