Effects of Photodynamic Therapy on Tumor Metabolism and Oxygenation Revealed by Fluorescence and Phosphorescence Lifetime Imaging.

This work was aimed at the complex analysis of the metabolic and oxygen statuses of tumors in vivo after photodynamic therapy (PDT). Studies were conducted on mouse tumor model using two types of photosensitizers-chlorin e6-based drug Photoditazine predominantly targeted to the vasculature and genetically encoded photosensitizer KillerRed targeted to the chromatin. Metabolism of tumor cells was assessed by the fluorescence lifetime of the metabolic redox-cofactor NAD(P)H, using fluorescence lifetime imaging.

Multiphoton microscopy assessment of the structure and variability changes of dermal connective tissue in vulvar lichen sclerosus: A pilot study.

In this article, we offer a novel classification of progressive changes in the connective tissue of dermis in vulvar lichen sclerosus (VLS) relying on quantitative assessment of the second harmonic generation (SHG) signal received from formalin fixed and deparaffinized tissue sections. We formulate criteria for distinguishing four degrees of VLS development: Initial-Mild-Moderate-Severe. Five quantitative characteristics (length and thickness type I Collagen fibers, Mean SHG signal intensity, Skewness and Coherence SHG signal) are used to describe the sequential degradation of connective tissue (changes in the structure, orientation, shape and density of collagen fibers) up to the formation of specific homogeneous masses.

Insight into redox regulation of apoptosis in cancer cells with multiparametric live-cell microscopy.

Cellular redox status and the level of reactive oxygen species (ROS) are important regulators of apoptotic potential, playing a crucial role in the growth of cancer cell and their resistance to apoptosis. However, the relationships between the redox status and ROS production during apoptosis remain poorly explored. In this study, we present an investigation on the correlations between the production of ROS, the redox ratio FAD/NAD(P)H, the proportions of the reduced nicotinamide cofactors NADH and NADPH, and caspase-3 activity in cancer cells at the level of individual cells.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences.

FUCCI-Red: a single-color cell cycle indicator for fluorescence lifetime imaging.

The phase of the cell cycle determines numerous aspects of cancer cell behaviour including invasiveness, ability to migrate and responsiveness to cytotoxic drugs. To non-invasively monitor progression of cell cycle in vivo, a family of genetically encoded fluorescent indicators, FUCCI (fluorescent ubiquitination-based cell cycle indicator), has been developed. Existing versions of FUCCI are based on fluorescent proteins of two or more different colors fused to cell-cycle-dependent degradation motifs.

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis.

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type.

Monitoring membrane viscosity in differentiating stem cells using BODIPY-based molecular rotors and FLIM.

Membrane fluidity plays an important role in many cell functions such as cell adhesion, and migration. In stem cell lines membrane fluidity may play a role in differentiation. Here we report the use of viscosity-sensitive fluorophores based on a BODIPY core, termed "molecular rotors", in combination with Fluorescence Lifetime Imaging Microscopy, for monitoring of plasma membrane viscosity changes in mesenchymal stem cells (MSCs) during osteogenic and chondrogenic differentiation.

Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose.

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module.

In vivo metabolic and SHG imaging for monitoring of tumor response to chemotherapy.

Although chemotherapy remains one of the main types of treatment for cancer, treatment failure is a frequent occurrence, emphasizing the need for new approaches to the early assessment of tumor response. The aim of this study was to search for indicators based on optical imaging of cellular metabolism and of collagen in tumors in vivo that enable evaluation of their response to chemotherapy. The study was performed on a mouse colorectal cancer model with the use of cisplatin, paclitaxel, and irinotecan.

Fluorescence time-resolved macroimaging.

While laser scanning fluorescence lifetime imaging (FLIM) is a powerful approach for cell biology, its small field of view (typically less than 1 mm) makes it impractical for the imaging of large biological samples that is often required for biomedical applications. Here we present a system that allows performing FLIM on macroscopic samples as large as 18 mm with a lateral resolution of 15 μm. The performance of the system is verified with FLIM of endogenous metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and genetically encoded fluorescent protein mKate2 in a mouse tumor in vivo.

Water-soluble cyclometalated platinum(ii) and iridium(iii) complexes: synthesis, tuning of the photophysical properties, and and phosphorescence lifetime imaging.

This paper presents synthesis and photophysical investigation of cyclometalated water-soluble Pt(ii) and Ir(iii) complexes containing auxiliary sulfonated diphosphine (bis(diphenylphosphino)benzene (dppb), P^P*) ligand. The complexes demonstrate considerable variations in excitation (extending up to 450 nm) and emission bands (with maxima ranging from 450 to 650 nm), as well as in the sensitivity of excited state lifetimes to molecular oxygen (from almost negligible to more than 4-fold increase in degassed solution). Moreover, all the complexes possess high two-photon absorption cross sections (400-500 GM for Pt complexes, and 600-700 GM for Ir complexes).

Metabolic cofactors NAD(P)H and FAD as potential indicators of cancer cell response to chemotherapy with paclitaxel.

Paclitaxel, a widely used antimicrotubular agent, predominantly eliminates rapidly proliferating cancer cells, while slowly proliferating and quiescent cells can survive the treatment, which is one of the main reasons for tumor recurrence and non-responsiveness to the drug. To improve the efficacy of chemotherapy, biomarkers need to be developed to enable monitoring of tumor responses. In this study we considered the auto-fluorescent metabolic cofactors NAD(P)H and FAD as possible indicators of cancer cell response to therapy with paclitaxel.

Multimodal label-free imaging of living dermal equivalents including dermal papilla cells.

Background: Despite the significant progress in the development of skin equivalents (SEs), the problem of noninvasively assessing the quality of the cell components and the collagen structure of living SEs both before and after transplantation remains. Undoubted preference is given to in vivo methods of noninvasive, label-free monitoring of the state of the SEs. Optical bioimaging methods, such as cross-polarization optical coherence tomography (CP OCT), multiphoton tomography (MPT), and fluorescence lifetime imaging microscopy (FLIM), present particular advantages for the visualization of such SEs.

Photodynamic therapy of mouse tumor model using chlorin e6- polyvinyl alcohol complex.

The use of polymeric carriers to deliver hydrophobic photosensitizers has been widely discussed as a way to improve both fluorescence diagnostic and photodynamic therapy (PDT) of cancers; however, the photophysical and pharmacokinetic parameters, as well as the PDT activity, of such modifications have, until now, only been poorly investigated. The purpose of the present study was to explore the efficacy of PDT with the formulation of the photosensitizer chlorin e6 (Ce6) in combination with polyvinyl alcohol (PVA) in comparison with Ce6 alone and with the clinical drug, Photodithazine in a mouse tumor model. We also investigated the photoactivity of the Ce6-PVA in a model reaction of tryptophan oxidation, analyzed the polymer-Ce6 interaction using fluorescence spectroscopy and atomic-force microscopy, and tested the phototoxicity in vitro.

Pharmacokinetics of Chlorin e₆-Cobalt Bis(Dicarbollide) Conjugate in Balb/c Mice with Engrafted Carcinoma.

The necessary precondition for efficient boron neutron capture therapy (BNCT) is control over the content of isotope B in the tumor and normal tissues. In the case of boron-containing porphyrins, the fluorescent part of molecule can be used for quantitative assessment of the boron content. We performed a study of the biodistribution of the chlorin ₆-Cobalt bis(dicarbollide) conjugate in carcinoma-bearing Balb/c mice using ex vivo fluorescence imaging, and developed a mathematical model describing boron accumulation and release based on the obtained experimental data.

Monitoring of hydrogen peroxide production under photodynamic treatment using protein sensor HyPer.

An interest to HO accumulation under photodynamic treatment can be explained by its participation in intracellular signal cascades. It is important not only to detect HO generation, but also to trace the dynamics of its intracellular content. In the present study the dynamics of cellular HO content under photodynamic treatment was analyzed using genetically encoded reversible HO-sensitive sensor HyPer.

Imaging of Intracellular pH in Tumor Spheroids Using Genetically Encoded Sensor SypHer2.

Intracellular pH (pHi) is one of the most important parameters that regulate the physiological state of cells and tissues. pHi homeostasis is crucial for normal cell functioning. Cancer cells are characterized by having a higher (neutral to slightly alkaline) pHi and lower (acidic) extracellular pH (pHe) compared to normal cells.

Chemotherapy with cisplatin: insights into intracellular pH and metabolic landscape of cancer cells in vitro and in vivo.

Although cisplatin plays a central role in cancer chemotherapy, the mechanisms of cell response to this drug have been unexplored. The present study demonstrates the relationships between the intracellular pH (pHi), cell bioenergetics and the response of cervical cancer to cisplatin. pHi was measured using genetically encoded sensor SypHer2 and metabolic state was accessed by fluorescence intensities and lifetimes of endogenous cofactors NAD(P)H and FAD.

Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation.

Background: Metabolic plasticity and the versatility of different lineages of stem cells as they satisfy their energy demands are not completely understood. In this study we investigated the metabolic changes in mesenchymal stem cells (MSCs) undergoing differentiation in two directions, osteogenic and chondrogenic, using two-photon fluorescence microscopy combined with FLIM.

Methods: Differentiation was induced by incubating the human bone marrow MSCs in osteogenic or chondrogenic mediums.

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.

Quantitative evaluation of atherosclerotic plaques using cross-polarization optical coherence tomography, nonlinear, and atomic force microscopy.

A combination of approaches to the image analysis in cross-polarization optical coherence tomography (CP OCT) and high-resolution imaging by nonlinear microscopy and atomic force microscopy (AFM) at the different stages of atherosclerotic plaque development is studied. This combination allowed us to qualitatively and quantitatively assess the disorganization of collagen in the atherosclerotic arterial tissue (reduction and increase of CP backscatter), at the fiber (change of the geometric distribution of fibers in the second-harmonic generation microscopy images) and fibrillar (violation of packing and different nature of a basket-weave network of fibrils in the AFM images) organization levels. The calculated CP channel-related parameters are shown to have a statistically significant difference between stable and unstable (also called vulnerable) plaques, and hence, CP OCT could be a potentially powerful, minimally invasive method for vulnerable plaques detection.

Fluorescence Imaging of Actin Fine Structure in Tumor Tissues Using SiR-Actin Staining.

Background: The rearrangement of actin cytoskeleton is being increasingly considered a marker of cancer cell activity, but the fine structure and remodeling of microfilaments within tumor tissue still remains unclear.

Materials And Methods: We used the recently introduced silicon-rhodamine (SiR)-actin dye to visualize endogenous actin within tissues by confocal or total internal reflection fluorescence microscopy. We established imaging conditions for robust blinking of SiR-actin, which makes this dye applicable for super-resolution localization microscopy, as well as for an efficient background elimination.

The metabolic interaction of cancer cells and fibroblasts - coupling between NAD(P)H and FAD, intracellular pH and hydrogen peroxide.

Alteration in the cellular energy metabolism is a principal feature of tumors. An important role in modifying cancer cell metabolism belongs to the cancer-associated fibroblasts. However, the regulation of their interaction has been poorly studied to date.

Probing metabolic states of differentiating stem cells using two-photon FLIM.

The ability of stem cells to differentiate into specialized cell types presents a number of opportunities for regenerative medicine, stem cell therapy and developmental biology. Because traditional assessments of stem cells are destructive, time consuming, and logistically intensive, the use of a non-invasive, label-free approach to study of cell differentiation provides a powerful tool for rapid, high-content characterization of cell and tissue cultures. Here, we elucidate the metabolic changes in MSCs during adipogenic differentiation, based on the fluorescence of the metabolic co-factors NADH, NADPH, and FAD using the methods of two-photon fluorescence microscopy combined with FLIM.