Rapid Antibiotic Susceptibility Determination by Fluorescence Lifetime Tracking of Bacterial Metabolism.

To combat the rise of antibiotic-resistance in bacteria and the resulting effects on healthcare worldwide, new technologies are needed that can perform rapid antibiotic susceptibility testing (AST). Conventional clinical methods for AST rely on growth-based assays, which typically require long incubation times to obtain quantitative results, representing a major bottleneck in the determination of the optimal antibiotic regimen to treat patients. Here, we demonstrate a rapid AST method based on the metabolic activity measured by fluorescence lifetime imaging microscopy (FLIM).

A versatile bioluminescent probe with tunable color.

Bioluminescence is a powerful method for imaging , but applications at the microscale are far from routine. This is due, in part, to a lack of versatile tools for visualizing dynamic events. To address this void, we developed a new platform-Bioluminescence Resonance Energy mAKe over with a Fluorescence-Activating absorption-Shifting Tag (BREAKFAST).

Hydrogel crosslinking modulates macrophages, fibroblasts, and their communication, during wound healing.

Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels.

Multimodal Phasor Approach to study breast cancer cells invasion in 3D spheroid model.

We implemented a multimodal set of functional imaging techniques optimized for deep-tissue imaging to investigate how cancer cells invade surrounding tissues and how their physiological properties change in the process. As a model for cancer invasion of the extracellular matrix, we created 3D spheroids from triple-negative breast cancer cells (MDA-MB-231) and non-tumorigenic breast epithelial cells (MCF-10A). We analyzed multiple hallmarks of cancer within the same spheroid by combining a number of imaging techniques, such as metabolic imaging of NADH by Fluorescence Lifetime Imaging Microscopy (NADH-FLIM), hyperspectral imaging of a solvatochromic lipophilic dye (Nile Red) and extracellular matrix imaging by Second Harmonic Generation (SHG).

Building Fluorescence Lifetime Maps Photon-by-Photon by Leveraging Spatial Correlations.

Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative characterization of subcellular environments. However, quantitative FLIM analyses face several challenges. First, spatial correlations between pixels are often ignored as signal from individual pixels is analyzed independently thereby limiting spatial resolution.

Microenvironmental stiffness induces metabolic reprogramming in glioblastoma.

The mechanical properties of solid tumors influence tumor cell phenotype and the ability to invade surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state, which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peritumoral brain tissues, but tumor stiffness is heterogeneous where tumor edges are softer than the tumor core.

The microenvironment dictates glycocalyx construction and immune surveillance.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with models that do not match the microenvironmental characteristics of human tissues. Using models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.

Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells.

The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor.

Superoxide Release by Macrophages through NADPH Oxidase Activation Dominating Chemistry by Isoprene Secondary Organic Aerosols and Quinones to Cause Oxidative Damage on Membranes.

Oxidative stress mediated by reactive oxygen species (ROS) is a key process for adverse aerosol health effects. Secondary organic aerosols (SOA) account for a major fraction of fine particulate matter, and their inhalation and deposition into the respiratory tract causes the formation of ROS by chemical and cellular processes, but their relative contributions are hardly quantified and their link to oxidative stress remains uncertain. Here, we quantified cellular and chemical superoxide generation by 9,10-phenanthrenequinone (PQN) and isoprene SOA using a chemiluminescence assay combined with electron paramagnetic resonance spectroscopy as well as kinetic modeling.

Disruption of the circadian clock drives loss of heterozygosity to accelerate colorectal cancer.

An alarming rise in young onset colorectal cancer (CRC) has been reported; however, the underlying molecular mechanism remains undefined. Suspected risk factors of young onset CRC include environmental aspects, such as lifestyle and dietary factors, which are known to affect the circadian clock. We find that both genetic disruption and environmental disruption of the circadian clock accelerate driven CRC pathogenesis in vivo.

High Resolution Fluorescence Lifetime Maps from Minimal Photon Counts.

Fluorescence lifetime imaging microscopy (FLIM) may reveal subcellular spatial lifetime maps of key molecular species. Yet, such a quantitative picture of life necessarily demands high photon budgets at every pixel under the current analysis paradigm, thereby increasing acquisition time and photodamage to the sample. Motivated by recent developments in computational statistics, we provide a direct means to update our knowledge of the lifetime maps of species of different lifetimes from direct photon arrivals, while accounting for experimental features such as arbitrary forms of the instrument response function (IRF) and exploiting information from empty laser pulses not resulting in photon detection.

Multiplexed bioluminescence microscopy via phasor analysis.

Bioluminescence imaging with luciferase-luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores.

Patient-derived xenograft culture-transplant system for investigation of human breast cancer metastasis.

Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the growth and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing (RNA-seq) analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells.

UTX condensation underlies its tumour-suppressive activity.

UTX (also known as KDM6A) encodes a histone H3K27 demethylase and is an important tumour suppressor that is frequently mutated in human cancers. However, as the demethylase activity of UTX is often dispensable for mediating tumour suppression and developmental regulation, the underlying molecular activity of UTX remains unknown. Here we show that phase separation of UTX underlies its chromatin-regulatory activity in tumour suppression.

Automated segmentation and tracking of mitochondria in live-cell time-lapse images.

Mitochondria display complex morphology and movements, which complicates their segmentation and tracking in time-lapse images. Here, we introduce Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer requires only the pixel size and the time between frames to identify mitochondrial motion and morphology, including fusion and fission events.

Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry.

Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques.

Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single particle tracking.

The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT).

Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis.

It remains unknown if biophysical or material properties of biomolecular condensates regulate cancer. Here we show that AKAP95, a nuclear protein that regulates transcription and RNA splicing, plays an important role in tumorigenesis by supporting cancer cell growth and suppressing oncogene-induced senescence. AKAP95 forms phase-separated and liquid-like condensates in vitro and in nucleus.

Caffeine and Cisplatin Effectively Targets the Metabolism of a Triple-Negative Breast Cancer Cell Line Assessed via Phasor-FLIM.

Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM).

The phasor FLIM method reveals a link between a change in energy metabolism and mHtt protein spread in healthy Mammalian cells when co-cultured with Huntington diseased cells.

Huntington Disease (HD) is a late-onset autosomal neurodegenerative disease characterized by the aggregations of mutant Huntingtin proteins (mHTT). A glutamine stretch (PolyQ) at the N-terminal of the Huntingtin protein is generated by the abnormal expansion of CAG trinucleotide repeats in exon 1 of the HTT gene. While the resulting polyQ aggregates are the predominate feature of HD, the intercellular spread of the expanded protein and the effect upon this transfer inside healthy cells have not yet fully understood.