Confocal Fluorescence Microscopy Imaging Research Articles

Mono- or di- naphthalimides as fluorophore to detect hypochlorous acid (HOCl) by ratiometric fluorescent signal and their biological application

Hypochlorous acid (HClO), a biologically significant part of the reactive oxygen species (ROS), has antibacterial effects on the biological immune-defense system and participates in a variety of physiological process. Thus the detection of HOCl has become an important issue. In this work, we prepared two novel fluorescent probes based on mono- or di- naphthalimides as fluorophore with ratiometric fluorescent signal and both of them could response to the amount of hypochlorous acid for the HOCl-promoted oxidative removal of the CN bond accompanied by good sensitivity and selectivity. The turn-on Probe I and ratiometric Probe II showed low detection limit and rapid response time respectively. Moreover, confocal fluorescence microscopy image using HepG2 cells showed that both probes could be used in living cells.

Differential response of human dermal fibroblast subpopulations to visible and near-infrared light: Potential of photobiomodulation for addressing cutaneous conditions.

The past decade has witnessed a rapid expansion of photobiomodulation (PBM), demonstrating encouraging results for the treatment of cutaneous disorders. Confidence in this approach, however, is impaired not only by a lack of understanding of the light-triggered molecular cascades but also by the significant inconsistency in published experimental outcomes, design of the studies and applied optical parameters. This study aimed at characterizing the response of human dermal fibroblast subpopulations to visible and near-infrared (NIR) light in an attempt to identify the optical treatment parameters with high potential to address deficits in aging skin and non-healing chronic wounds. Primary human reticular and papillary dermal fibroblasts (DF) were isolated from the surplus of post-surgery human facial skin. An in-house developed LED-based device was used to irradiate cell cultures using six discrete wavelengths (450, 490, 550, 590, 650, and 850 nm). Light dose-response at a standard oxygen concentration (20%) at all six wavelengths was evaluated in terms of cell metabolic activity. This was followed by an analysis of the transcriptome and procollagen I production at a protein level, where cells were cultured in conditions closer to in vivo at 2% environmental oxygen and 2% serum. Furthermore, the production of reactive oxygen species (ROS) was accessed using real-time fluorescence confocal microscopy imaging. Here, production of ROS in the presence or absence of antioxidants, as well as the cellular localization of ROS, was evaluated. In terms of metabolic activity, consecutive irradiation with short-wavelength light (⇐530 nm) exerted an inhibitory effect on DF, while longer wavelengths (>=590 nm) had essentially a neutral effect. Cell behavior following treatment with 450 nm was biphasic with two distinct states: inhibitory at low- to mid- dose levels (<=30 J/cm2 ), and cytotoxic at higher dose levels (>30 J/cm2 ). Cell response to blue light was accompanied by a dose-dependent release of ROS that was localized in the perinuclear area close to mitochondria, which was attenuated by an antioxidant. Overall, reticular DFs exhibited a greater sensitivity to light treatment at the level of gene expression than did papillary DFs, with more genes significantly up- or down- regulated. At the intra-cellular signaling pathway level, the up- or down- regulation of vital pathways was observed only for reticular DF, after treatment with 30 J/cm2 of blue light. At the cellular level, short visible wavelengths exerted a greater inhibitory effect on reticular DF. Several genes involved in the TGF-β signaling pathway were also affected. In addition, procollagen I production was inhibited. By contrast, 850 nm near-infrared (NIR) light (20 J/cm2 ) exerted a stimulatory metabolic effect in these cells, with no detectable intracellular ROS formation. Here too, reticular DF were more responsive than papillary DF. This stimulatory effect was only observed under in vivo-like low oxygen conditions, corresponding to normal dermal tissue oxygen levels (approximately 2%). This study highlights a differential impact of light on human skin cells with upregulation of metabolic activity with NIR light, and inhibition of pro-collagen production and proliferation in response to blue light. These findings open-up new avenues for developing therapies for different cutaneous conditions (e.g., treatment of keloids and fibrosis) or differential therapy at distinct stages of wound healing. Lasers Surg. Med. 50:859-882, 2018. © 2018 Wiley Periodicals, Inc.

In-situ Observation of Hierarchical Self-Assembly Driven by Bicontinuous Gelation in Mixed Nanodisc Dispersions

The search for new functional soft materials with precise and reconfigurable structures at the nano and meso-scale is a major challenge as well as objective of the current science. Patchy colloids of different shapes and functionalities are considered important new building blocks of a bottom-up approach towards rational design of new soft materials largely governed by anisotropic interactions. Herein, we investigate the self-assembly, growth of hierarchical microstructures and aging dynamics of 2D nano-platelets of two different aspect ratios (Laponite ~25 and Montmorillonite ~250) which form gels with different porosity that is achieved by tuning their mixing ratios. Qualitative in situ real-space studies are carried out, including fluorescent confocal microscopy imaging of the bicontinuous gelation process or final states, which provides dynamic visualization of the self-organization. The bicontinuous gels exhibit a foam-like morphology having pores of a few micrometers in size that can be tuned by varying the mixing ratio of nanoplatelets. It is shown that this new class of clay gels has unique and tunable physical properties that will find potential applications in the development of low cost lithium ion batteries, nanocomposites and nuclear waste management.

VOTING-BASED SEGMENTATION OF OVERLAPPING NUCLEI IN CLARITY IMAGES.

New tissue-clearing techniques and improvements in optical microscopy have rapidly advanced capabilities to acquire volumetric imagery of neural tissue at resolutions of one micron or better. As sizes for data collections increase, accurate automatic segmentation of cell nuclei becomes increasingly important for quantitative analysis of imaged tissue. We present a cell nucleus segmentation method that is formulated as a parameter estimation problem with the goal of determining the count, shapes, and locations of nuclei that most accurately describe an image. We applied our new voting-based approach to fluorescence confocal microscopy images of neural tissue stained with DAPI, which highlights nuclei. Compared to manual counting of cells in three DAPI images, our method outperformed three existing approaches. On a manually labeled high-resolution DAPI image, our method also outperformed those methods and achieved a cell count accuracy of 98.99% and mean Dice coefficient of 0.6498.

PURE-LET Image Deconvolution.

We propose a non-iterative image deconvolution algorithm for data corrupted by Poisson or mixed Poisson-Gaussian noise. Many applications involve such a problem, ranging from astronomical to biological imaging. We parameterize the deconvolution process as a linear combination of elementary functions, termed as linear expansion of thresholds. This parameterization is then optimized by minimizing a robust estimate of the true mean squared error, the Poisson unbiased risk estimate. Each elementary function consists of a Wiener filtering followed by a pointwise thresholding of undecimated Haar wavelet coefficients. In contrast to existing approaches, the proposed algorithm merely amounts to solving a linear system of equations, which has a fast and exact solution. Simulation experiments over different types of convolution kernels and various noise levels indicate that the proposed method outperforms the state-of-the-art techniques, in terms of both restoration quality and computational complexity. Finally, we present some results on real confocal fluorescence microscopy images and demonstrate the potential applicability of the proposed method for improving the quality of these images.We propose a non-iterative image deconvolution algorithm for data corrupted by Poisson or mixed Poisson-Gaussian noise. Many applications involve such a problem, ranging from astronomical to biological imaging. We parameterize the deconvolution process as a linear combination of elementary functions, termed as linear expansion of thresholds. This parameterization is then optimized by minimizing a robust estimate of the true mean squared error, the Poisson unbiased risk estimate. Each elementary function consists of a Wiener filtering followed by a pointwise thresholding of undecimated Haar wavelet coefficients. In contrast to existing approaches, the proposed algorithm merely amounts to solving a linear system of equations, which has a fast and exact solution. Simulation experiments over different types of convolution kernels and various noise levels indicate that the proposed method outperforms the state-of-the-art techniques, in terms of both restoration quality and computational complexity. Finally, we present some results on real confocal fluorescence microscopy images and demonstrate the potential applicability of the proposed method for improving the quality of these images.

Ex Vivo Confocal Fluorescence Microscopy for Rapid Evaluation of Tissues in Surgical Pathology Practice.

- Optical imaging techniques are currently available for imaging tissues without the need for any type of extensive tissue preparation. There are several applications for their potential use in surgical pathology practice. - To evaluate the feasibility of using a confocal fluorescence microscopy (CFM) platform for ex vivo examination of tissues obtained from surgical resections of breast, lung, kidney, and liver. - Tissue fragments (0.5-1.0 cm) were immersed in 0.6 mM acridine orange for 6 seconds and imaged using a CFM platform at a 488-nm wavelength. The imaged tissues were subsequently fixed in formalin and processed routinely to generate hematoxylin-eosin-stained tissue sections. Mosaics of the grayscale CFM images were studied at different magnifications for recognition of the tissue and were compared with conventional histopathologic examination of hematoxylin-eosin tissue sections. - We imaged 55 tissue fragments obtained from 16 breast (29%), 18 lung (33%), 14 kidney (25%), and 7 liver (13%) surgical excision specimens. Acridine orange labeled the nuclei, creating the contrast between nucleus and cytoplasm and thereby recapitulating the tissue architecture. We could obtain CFM images of good quality within 5 to 10 minutes that allowed recognition of the cytomorphologic details for categorization of the imaged tissue and were similar to histologic examination of hematoxylin-eosin tissue sections. - The ease and speed of acquisition of CFM images together with the resolution and resemblance of the CFM images to hematoxylin-eosin sections suggest that the CFM platform has excellent potential for use in surgical pathology practice.

Modeling, validation and verification of three-dimensional cell-scaffold contacts from terabyte-sized images

BackgroundCell-scaffold contact measurements are derived from pairs of co-registered volumetric fluorescent confocal laser scanning microscopy (CLSM) images (z-stacks) of stained cells and three types of scaffolds (i.e., spun coat, large microfiber, and medium microfiber). Our analysis of the acquired terabyte-sized collection is motivated by the need to understand the nature of the shape dimensionality (1D vs 2D vs 3D) of cell-scaffold interactions relevant to tissue engineers that grow cells on biomaterial scaffolds.ResultsWe designed five statistical and three geometrical contact models, and then down-selected them to one from each category using a validation approach based on physically orthogonal measurements to CLSM. The two selected models were applied to 414 z-stacks with three scaffold types and all contact results were visually verified. A planar geometrical model for the spun coat scaffold type was validated from atomic force microscopy images by computing surface roughness of 52.35 nm ±31.76 nm which was 2 to 8 times smaller than the CLSM resolution. A cylindrical model for fiber scaffolds was validated from multi-view 2D scanning electron microscopy (SEM) images. The fiber scaffold segmentation error was assessed by comparing fiber diameters from SEM and CLSM to be between 0.46% to 3.8% of the SEM reference values. For contact verification, we constructed a web-based visual verification system with 414 pairs of images with cells and their segmentation results, and with 4968 movies with animated cell, scaffold, and contact overlays. Based on visual verification by three experts, we report the accuracy of cell segmentation to be 96.4% with 94.3% precision, and the accuracy of cell-scaffold contact for a statistical model to be 62.6% with 76.7% precision and for a geometrical model to be 93.5% with 87.6% precision.ConclusionsThe novelty of our approach lies in (1) representing cell-scaffold contact sites with statistical intensity and geometrical shape models, (2) designing a methodology for validating 3D geometrical contact models and (3) devising a mechanism for visual verification of hundreds of 3D measurements. The raw and processed data are publicly available from https://isg.nist.gov/deepzoomweb/data/ together with the web -based verification system.

Fluorescent Phase-Pure Zero-Dimensional Perovskite-Related Cs4PbBr6 Microdisks: Synthesis and Single-Particle Imaging Study.

Quantum-confined perovskites are a new class of promising materials in optoelectronic applications. In this context, a zero-dimensional perovskite-related substance, Cs4PbBr6, having high exciton binding energy can be an important candidate, but its photoluminescence (PL) is a topic of recent debate. Herein, we report an ambient condition controlled synthesis of Cs4PbBr6 microdisks of different shapes and dimensions which exhibit fairly strong green PL (quantum yield up to 38%, band gap ∼2.43 eV) in the solid state. Using confocal fluorescence microscopy imaging of the single particles, we show that the fluorescence of Cs4PbBr6 microdisks is inherent to these particles. Fluorescence intensity and lifetime imaging of the microdisks reveals significant spatial heterogeneity with a bright central area and somewhat dimmer edges. This intensity and lifetime distribution is attributed to enhanced trap-mediated nonradiative deactivation at the edges compared to the central region of the microdisks. Our results, which unambiguously establish the PL of these Cs4PbBr6 and suggest its possible origin, brighten the potential of these materials in photon-emitting applications.

Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response to nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. We observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.

Identification and functional characterization of the pheromone biosynthesis activating neuropeptide receptor isoforms from Mamestra brassicae

In most moth species, including Mamestra brassicae, pheromone biosynthesis activating neuropeptide (PBAN) regulates pheromone production. Generally, PBAN acts directly on the pheromone gland (PG) cells via its specific G protein-coupled receptor (i.e. PBANR) with Ca2+ as a second messenger. In this study, we identified cDNAs encoding three variants (A, B and C) of the M. brassicae PBANR (Mambr-PBANR). The full-length coding sequences were transiently expressed in cultured Trichoplusia ni cells and Sf9 cells for functional characterization. All three isoforms dose-dependently mobilized extracellular Ca2+ in response to PBAN analogs with Mambr-PBANR-C exhibiting the greatest sensitivity. Fluorescent confocal microscopy imaging studies demonstrated binding of a rhodamine red-labeled ligand (RR10CPBAN) to all three Mambr-PBANR isoforms. RR10CPBAN binding did not trigger ligand-induced internalization in cells expressing PBANR-A, but did in cells expressing the PBANR-B and -C isoforms. Furthermore, activation of the PBANR-B and -C isoforms with the 18 amino acid Mambr-pheromonotropin resulted in co-localization with a Drosophila melanogaster arrestin homolog (Kurtz), whereas stimulation with an unrelated peptide had no effect. PCR-based profiling of the three transcripts revealed a basal level of expression throughout development with a dramatic increase in PG transcripts from the day of adult emergence with PBANR-C being the most abundant.

Accurate Detection of Dysmorphic Nuclei Using Dynamic Programming and Supervised Classification.

A vast array of pathologies is typified by the presence of nuclei with an abnormal morphology. Dysmorphic nuclear phenotypes feature dramatic size changes or foldings, but also entail much subtler deviations such as nuclear protrusions called blebs. Due to their unpredictable size, shape and intensity, dysmorphic nuclei are often not accurately detected in standard image analysis routines. To enable accurate detection of dysmorphic nuclei in confocal and widefield fluorescence microscopy images, we have developed an automated segmentation algorithm, called Blebbed Nuclei Detector (BleND), which relies on two-pass thresholding for initial nuclear contour detection, and an optimal path finding algorithm, based on dynamic programming, for refining these contours. Using a robust error metric, we show that our method matches manual segmentation in terms of precision and outperforms state-of-the-art nuclear segmentation methods. Its high performance allowed for building and integrating a robust classifier that recognizes dysmorphic nuclei with an accuracy above 95%. The combined segmentation-classification routine is bound to facilitate nucleus-based diagnostics and enable real-time recognition of dysmorphic nuclei in intelligent microscopy workflows.

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis.

Correlated mass spectrometry and confocal microscopy imaging verifies the dual-targeting action of an organoruthenium anticancer complex.

An addressable single cell imaging strategy combining ToF-SIMS and confocal fluorescence microscopy imaging has been developed, and sucessfully applied to visualize the subcellular distribution of an organoruthenium anticancer complex, [(η6-benzene)Ru(N,N-L)Cl]+ (1; L: 4-anilinoquinazoline ligand), showing its accumulation in both cell membrane and nuclei, and verifying its dual-targeting feature.

Monitoring of seawater immersion degradation in glass fibre reinforced polymer composites using quantum dots

Abstract In this paper, a simple and innovative method is proposed to monitor the effects of seawater ageing in glass fibre reinforced polymer (GFRP) composites using quantum dots (QDs). The cadmium sulfide (CdS) QDs modified with 3-(trimethoxysilyl)-1-propanethiol (MPS) were firstly synthesized, and then the modified CdS QDs were labeled on the glass fibre by chemical bond. GFRP composites with QDs at the interface between the fibre and matrix were obtained. The sigle-fibre-fragment tests of specimens were done to verify the modified CdS QDs did not affect the interfacial bonding of the matrix and fiber. The effects of seawater ageing on the adhesion behaviour between the fibre and the matrix of QD/GFRP hybrids were investigated based on confocal fluorescence microscopy images. After 6 months of immersion in seawater, the glass transition temperature ( T g ), tensile strength, and flexural strength of specimens decreased 2.5%, 13.8%, and 9.8%, respectively. Furthermore, the thermal and mechanical properties of specimens can be calculated by the fluorescence intensity quantificationally.

Encapsulation of Pancreatic Lipase in Hydrogel Beads with Self-Regulating Internal pH Microenvironments: Retention of Lipase Activity after Exposure to Gastric Conditions.

Oral delivery of lipase is important for individuals with exocrine pancreatic insufficiency; however, lipase loses activity when exposed to the highly acidic gastric environment. In this study, pancreatic lipase was encapsulated in hydrogel beads fabricated from alginate (gel former), calcium chloride (cross-linker), and magnesium hydroxide (buffer). Fluorescence confocal microscopy imaging was used to map the pH microclimate within the hydrogel beads under simulated gastrointestinal tract (GIT) conditions. The pH within buffer-free beads rapidly decreased when they moved from mouth (pH 6.3) to stomach (pH <4), leading to a loss of lipase activity in the small intestine. Conversely, the pH inside buffer-loaded beads remained close to neutral in the mouth (pH 7.33) and stomach (pH 7.39), leading to retention of lipase activity in the small intestine, as shown by pH-stat analysis of lipid digestion. The presence of the encapsulated buffer also reduced bead shrinkage under gastric conditions.

PH-switchable bacteria detection using zwitterionic fluorescent polymer

A zwitterionic fluorescent polymer with high sensitivity to pH changes was constructed for the detection and imaging of both gram-positive and gram-negative pathogenic bacteria. A detection probe using the zwitterionic fluorescent polymer was synthesized with single boron dipyrromethane (BODIPY) as a hydrophobic dye and bromoethane as a cationic group for bacteria binding with conjugated poly(sulfobetaine methacrylate) (BOD/BE-PSM). The zwitterionic fluorescent polymer bound to bacteria through ionic complexes between anionic groups on the bacterial surface and cationic BOD/BE-PSM groups after 1h incubation. This finding demonstrated that the fluorescence on/off system operated via changes in the hydrophilic and hydrophobic nature of the zwitterionic fluorescent polymer, depending on the pH (6.0, 7.4, or 9.0), at a fixed 1mg/mL polymer concentration. The system showed good stability with a limit of detection of 1mg/mL. Quenching caused by interactions with the hydrophobic BODIPY dye was also observed, enabling bacteria detection, as shown by fluorescence spectroscopy and confocal microscopy images. Our results indicated that the zwitterionic fluorescent polymer could be used to detect bacteria over a wide range of pH values.

Red-Emitting Tetracoordinate Organoboron Chelates: Synthesis, Photophysical Properties, and Fluorescence Microscopy.

Seven tetracoordinate organoboron fluorophores with heterobiaryl N,O- or N,N-chelate ligands were prepared and photophysically characterized (in toluene). The electronic variation of the heteroaromatic moiety provided a means for the fine-tuning of the UV/vis absorption and emission spectra. In the most interesting cases, the spectra were red-shifted to maximum absorbance at wavelengths longer than 500 nm and emission maxima between 620 and 660 nm. The pronounced intramolecular charge-transfer character of the dyes yielded large Stokes shifts (3500-5100 cm-1), while maintaining appreciable fluorescence quantum yields of up to 0.2 for emission maxima longer than 600 nm. The lipophilic character of the dyes enabled their application as stains of vesicle substructures in confocal fluorescence microscopy imaging.

Gold Nanoantenna-Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer.

In this work, we demonstrate controlled drug delivery from low-temperature-sensitive liposomes (LTSLs) mediated by photothermal heating from multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. The unique geometry of MGNs enables the generation of mild hyperthermia (∼42 °C) by converting near-infrared light to heat and effectively delivering doxorubicin (DOX) from the LTSLs in breast cancer cells. We confirmed the cellular uptake of MGNs by using both fluorescence confocal Z-stack imaging and transmission electron microscopy (TEM) imaging. We performed a cellular viability assay and live/dead cell fluorescence imaging of the combined therapeutic effects of MGNs with DOX-loaded LTSLs (DOX-LTSLs) and compared them with free DOX and DOX-loaded non-temperature-sensitive liposomes (DOX-NTSLs). Imaging of fluorescent live/dead cell indicators and MTT assay outcomes both demonstrated significant decreases in cellular viability when cells were treated with the combination therapy. Because of the high phase-transition temperature of NTSLs, no drug delivery was observed from the DOX-NTSLs. Notably, even at a low DOX concentration of 0.5 μg/mL, the combination treatment resulted in a higher (33%) cell death relative to free DOX (17% cell death). The results of our work demonstrate that the synergistic therapeutic effect of photothermal hyperthermia of MGNs with drug delivery from the LTSLs can successfully eradicate aggressive breast cancer cells with higher efficacy than free DOX by providing a controlled light-activated approach and minimizing off-target toxicity.

Abstract B01: The metabolic adaptations during cancer-stroma co-evolution: The intracellular pH, NAD(P)H changes and hydrogen peroxide production in cancer cells

Abstract Intoduction: It is generally accepted that cancer cells and cancer-associated fibroblasts (CAFs) can change their metabolism to maintain tumor progression and adapt cancer cells to unfavorable conditions. Recently it was found that in some tumors cancer cells have oxidative metabolism and CAFs switch their metabolism to aerobic glycolysis when interplay with cancer cells (“reverse Warburg effect”) (1). It is supposed that cancer cells produce hydrogen peroxide to initiate oxidative stress in CAFs and drive aerobic glycolysis (2). Also it is known that intracellular pH (pHi) level is an important regulator of many cells functions. And the pHi in cancer cells differ from pHi in normal cells. The pHi alterations in cancer cell may be a result of metabolic changes during neoplastic processes (3). The goal of our study was to investigate metabolic interaction between cancer cells and fibroblasts in the co-culture model. The following parameters were studied In cancer cells: pHi hydrogen peroxide level and NAD(P)H. Materials and Methods: The experiments were performed on Hela Kyoto cancer cells and normal skin fibroblasts. HeLa Kyoto, stably expressing genetically encoded fluorescent pHi-sensor SypHer2 (HeLa-SypHer2) or H2O2-sensor HyPer2 (HeLa-HyPer2) were used for detection of intracellular and hydrogen peroxide levels, correspondently (4). Fluorescence of NAD(P)H was detected using two-photon excitation, and fluorescence lifetime and relative contributions of free and protein-bound forms were measured. Cancer cells and fibroblasts were co-plated with a 1:5 fibroblast-to-cancer cell ratio or plated in parallel as a monotypic culture (5). Cells were analyzed daily for 3 days. Fluorescence confocal microscopy and fluorescence lifetime imaging (FLIM) were performed using the fluorescence laser-scanning microscope LSM 710. For FLIM investigation FLIM module based on time-correlated single photon counting Simple Tau 152 TCSPC was used. The obtained images were processed using ImageJ 1.39p and SPCImage software. Results: In our work we demonstrate that during tumor-stroma co-evolution in model system based on Hela Kyoto cells and fibroblasts cancer cells switch their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis while fibroblast switch their metabolism from glycolysis to OXPHOS. We found that these metabolic changes happened after hydrogen peroxide production in cancer cells and spreading of H2O2 to adjusted fibroblasts. The peak of hydrogen peroxide production took place at the second day of co-cultivation. In the corresponded mono-culture the flash of H2O2 was not observed. It was shown that the pHi level in cancer cells became slightly more acidic in co-culture conditions in comparison with corresponded mono-culture. Conclusions: To our knowledge, these three parameters reflecting cancer cells metabolism were analyzed for the first time in a model of tumor-stroma co-evolution. The understanding of metabolic features of cancer and normal cells during neoplastic transformation have fundamental and practical importance as it can be used for tumor diagnostics, development of new anticancer drugs targeting metabolic pathways.

Resolution and signal-to-noise ratio improvement in confocal fluorescence microscopy using array detection and maximum-likelihood processing

Confocal fluorescence microscopy (CFM) is widely used in biological sciences because of its enhanced 3D resolution that allows image sectioning and removal of out-of-focus blur. This is achieved by rejection of the light outside a detection pinhole in a plane confocal with the illuminated object. In this paper, an alternative detection arrangement is examined in which the entire detection/image plane is recorded using an array detector rather than a pinhole detector. Using this recorded data an attempt is then made to recover the object from the whole set of recorded photon array data; in this paper maximum-likelihood estimation has been applied. The recovered object estimates are shown (through computer simulation) to have good resolution, image sectioning and signal-to-noise ratio compared with conventional pinhole CFM images.