Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

• MeSH
• Flavin-Adenine Dinucleotide * MeSH
• Microscopy, Fluorescence, Multiphoton MeSH
• Sperm Motility MeSH
• NADP * MeSH
• Spermatozoa drug effects   MeSH
• Bedbugs MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Cancer cells facilitate tumor growth by creating favorable tumor micro-environments (TME), altering homeostasis and immune response in the extracellular matrix (ECM) of surrounding tissue. A potential factor that contributes to TME generation and ECM remodeling is the cytoskeleton-associated human death-associated protein kinase 1 (DAPK1). Increased tumor cell motility and de-adhesion (thus, promoting metastasis), as well as upregulated plasminogen-signaling, are shown when functionally analyzing the DAPK1 ko-related proteome. However, the systematic investigation of how tumor cells actively modulate the ECM at the tissue level is experimentally challenging since animal models do not allow direct experimental access while artificial in vitro scaffolds cannot simulate the entire complexity of tissue systems. Here, we used the chorioallantoic membrane (CAM) assay as a natural, collagen-rich tissue model in combination with all-optical experimental access by multiphoton microscopy (MPM) to study the ECM remodeling potential of colorectal tumor cells with and without DAPK1 in situ and even in vivo. This approach demonstrates the suitability of the CAM assay in combination with multiphoton microscopy for studying collagen remodeling during tumor growth. Our results indicate the high ECM remodeling potential of DAPK1 ko tumor cells at the tissue level and support our findings from proteomics.

• Publication type
• Journal Article MeSH

Decellularized human pericardium is under study as an allogenic material for cardiovascular applications. The effects of crosslinking on the mechanical properties of decellularized pericardium were determined with a uniaxial tensile test, and the effects of crosslinking on the collagen structure of decellularized pericardium were determined by multiphoton microscopy. The viability of human umbilical vein endothelial cells seeded on decellularized human pericardium and on pericardium strongly and weakly crosslinked with glutaraldehyde and with genipin was evaluated by means of an MTS assay. The viability of the cells, measured by their metabolic activity, decreased considerably when the pericardium was crosslinked with glutaraldehyde. Conversely, the cell viability increased when the pericardium was crosslinked with genipin. Coating both non-modified pericardium and crosslinked pericardium with a fibrin mesh or with a mesh containing attached heparin and/or fibronectin led to a significant increase in cell viability. The highest degree of viability was attained for samples that were weakly crosslinked with genipin and modified by means of a fibrin and fibronectin coating. The results indicate a method by which in vivo endothelialization of human cardiac allografts or xenografts could potentially be encouraged.

• MeSH
• Allografts MeSH
• Biocompatible Materials * chemistry   MeSH
• Biomechanical Phenomena MeSH
• Human Umbilical Vein Endothelial Cells cytology   metabolism   MeSH
• Fibrin MeSH
• Fibronectins MeSH
• Glutaral MeSH
• Heterografts MeSH
• Iridoids MeSH
• Collagen chemistry   ultrastructure   MeSH
• Humans MeSH
• Microscopy, Fluorescence, Multiphoton MeSH
• Pericardium chemistry   transplantation   ultrastructure   MeSH
• Tensile Strength MeSH
• Surface Plasmon Resonance MeSH
• Cross-Linking Reagents MeSH
• Materials Testing MeSH
• Cell Survival MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Allergy and Immunology MeSH
• Immune System MeSH
• Publication type
• Textbook MeSH

• Conspectus
• Patologie. Klinická medicína
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• alergologie a imunologie

Východiska: Viscerálne aferentné nervy monitorujú vnútorné orgány a sprostredkujú informácie centrálnemu nervovému systému. Úloha aferentných nervov v patogenéze a symptómoch gastrointestinálnych ochorení je často podceňovaná. Preto treba zdôrazniť, že bolesť, pálenie záhy, plnosť, nevoľnosť a vracanie sú všetko symptómy sprostredkované viscerálnymi aferentnými nervami. Dôležité je tiež vyzdvihnúť, že stimulácia viscerálnych aferentných nervov zápalom pri chorobách narúša normálnu reguláciu sekrécie a motility, čím prispieva k symptómom od dysfágie po hnačku a zápchu. Z tohto výpočtu symptómov je zrejmé, že terapeutické ovplyvnenie viscerálnych aferentných nervov by mohlo pomôcť pri liečbe mnohých gastrointestinálnych ochorení. Vývoj nových liekov zameraných na viscerálne aferentné nervy však brzdí nedostatok informácií o týchto nervoch. Napríklad ani receptory pre kyselinu v pažerákových nervoch sprostredkujúcich pyrózu, ako ani iónové kanály zodpovedné za mechanickú aktiváciu v nervoch sprostredkujúcich koliku neboli celkom objasnené. Nedostatok informácií o viscerálnych aferentných nervoch možno vo veľkej miere pripísať ťažkostiam spojeným s ich štúdiom, typicky po jednom nervovom vlákne metódou klasickej elektrofyziológie. Cieľ: V tomto článku prezentujeme adaptáciu špičkovej vysoko výkonnej metódy na štúdium gastrointestinálnych aferentných nervov, ktorá umožňuje súčasné zobrazovanie aktivity v stovkách neurónov. Táto metóda je založená na genetickej expresii fluorescenčného vápnikového indikátora GCaMP6 a multifotónovej mikroskopie v transgénnej myši. Očakáva sa, že okrem poskytovania základných informácií o viscerálnych aferentných nervoch budúce iterácie tejto metódy umožnia štúdium nervovej aktivity v biopsiách od pacientov s gastrointestinálnymi ochoreniami.

Background: Visceral afferent nerves project from internal organs and provide information to the central nervous system. The role of afferent nerves in the pathogenesis and symptoms of gastrointestinal diseases is often underappreciated. Nonetheless, pain, heartburn, fullness, nausea, and vomiting are all mediated by visceral afferent nerves. Furthermore, stimulation of visceral afferent nerves by inflammation in diseases disrupts the normal regulation of gastrointestinal secretion and motility, thereby contributing to symptoms ranging from dysphagia to diarrhea and constipation. Thus, therapeutic targeting of visceral afferent nerves may aid the treatment of many gastrointestinal diseases. However, the development of novel drugs targeting visceral afferent nerves is hindered by the limited knowledge of these nerves. For example, the identities of the acid receptors in esophageal nerves that cause heartburn and the ion channels responsible for mechanical activation in nerves that mediate colic pain have not been fully elucidated. The limited knowledge of visceral afferent nerves is largely attributable to difficulties associated with their study; only one nerve fiber can typically be studied at a time by classical electrophysiology. Purpose: Here, we report the adaptation of a cutting-edge high-throughput method to study gastrointestinal afferent nerves that allows the activity in hundreds of neurons to be imaged simultaneously. This method is based on genetic expression of the fluorescent calcium indicator GCaMP6 and multiphoton microscopy in transgenic mice. In addition to providing fundamental information about visceral afferent nerves, future iterations of this method are expected to enable studies of nerve activity in biopsies from patients with gastrointestinal diseases.

• MeSH
• Microscopy, Fluorescence methods   MeSH
• Gastrointestinal Tract * innervation   pathology   MeSH
• Models, Animal MeSH
• Mice MeSH
• Calcium physiology   metabolism   MeSH
• Visceral Afferents diagnostic imaging   drug effects   MeSH
• Visceral Pain * diagnostic imaging   MeSH
• Check Tag
• Mice MeSH
• Publication type
• Review MeSH

Investigating cerebral metabolism in vivo at a microscopic level is essential for understanding brain function and its pathological alterations. The intricate signaling and metabolic dynamics between neurons, glia, and microvasculature requires much more detailed understanding to better comprehend the mechanisms governing brain function and its disease-related changes. We recently demonstrated that pharmacologically-induced alterations to different steps of cerebral metabolism can be distinguished utilizing 2-photon fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence in vivo. Here, we evaluate the ability of the phasor analysis method to identify these pharmacological metabolic alterations and compare the method's performance with more conventional nonlinear curve-fitting analysis. Visualization of phasor data, both at the fundamental laser repetition frequency and its second harmonic, enables resolution of pharmacologically-induced alterations to mitochondrial metabolic processes from baseline cerebral metabolism. Compared to our previous classification models based on nonlinear curve-fitting, phasor-based models required fewer parameters and yielded comparable or improved classification accuracy. Fluorescence lifetime imaging of NADH and phasor analysis shows utility for detecting metabolic alterations and will lead to a deeper understanding of cerebral energetics and its pathological changes.

• MeSH
• Bicuculline analogs & derivatives   pharmacology   MeSH
• Biomarkers metabolism   MeSH
• Models, Biological MeSH
• Rodentia physiology   MeSH
• Intravital Microscopy methods   MeSH
• Humans MeSH
• Microscopy, Fluorescence, Multiphoton methods   MeSH
• Mitochondria drug effects   metabolism   MeSH
• Disease Models, Animal MeSH
• Cerebral Cortex drug effects   metabolism   MeSH
• NAD metabolism   MeSH
• Nonlinear Dynamics MeSH
• Rats, Sprague-Dawley MeSH
• Seizures chemically induced   diagnostic imaging   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Comparative Study MeSH

Cyclodextrin (CD) polymers are interesting nanoparticulate systems for pharmaceutical delivery; however, knowledge regarding their applications towards delivery into complex microbial biofilm structures is so far limited. The challenge is to demonstrate penetration and transport through the biofilm and its exopolysaccharide matrix. The ideal functionalization for penetration into mature biofilms is unexplored. In this paper, we present a novel set of rhodamine labelled βCD-polymers, with different charge moieties, i.e., neutral, anionic, and cationic, and explore their potential delivery into mature Staphylococcus epidermidis biofilms using multiphoton laser scanning microscopy (MPM). The S. epidermidis biofilms, being a medically relevant model organism, were stained with SYTO9. By using MPM, three-dimensional imaging and spectral investigation of the distribution of the βCD-polymers could be obtained. It was found that the cationic βCD-polymers showed significantly higher integration into the biofilms, compared to neutral and anionic functionalized βCDs. None of the carriers presented any inherent toxicity to the biofilms, meaning that the addition of rhodamine moiety does not affect the inertness of the delivery system. Taken together, this study demonstrates a novel approach by which delivery of fluorescently labelled CD nanoparticles to bacterial biofilms can be explored using MPM. Future studies should be undertaken investigating the potential in using cationic functionalization of CD based delivery systems for targeting anti-microbial effects in biofilms.

• MeSH
• Biofilms * MeSH
• Cellulose chemistry   MeSH
• Cyclodextrins chemistry   MeSH
• Fluorescent Dyes MeSH
• Nanoparticles chemistry   MeSH
• Rhodamines MeSH
• Staphylococcus epidermidis MeSH
• Publication type
• Journal Article MeSH

INTRODUCTION: Remodeling of human placental membranes (amniochorionic or fetalmembrane) throughout gestation, a necessity to accommodate increasing uterine volume, involves continuous alterations (replacement of cells and remodeling of extracellular matrix). Methodologic limitations have obscured microscopic determination of cellular and layer-level alterations. This study used a combination of advanced imaging by multiphoton autofluorescence microscopy (MPAM) and second harmonic generation (SHG) microscopy along with tissue optical clearing to characterize the 3Dimensional multilayer organization of placental membranes. METHODS: Placental membranes biopsies (6 mm) collected from term, not-in-labor cesarean deliveries (n = 7) were fixed in 10% formalin (native) or treated with 2,2'-thiodiethanol to render them transparent for deeper imaging. Native and cleared tissues were imaged using MPAM (cellular autofluorescence) and SHG (fibrillar collagen). Depth z-stacks captured the amnion epithelium, underlying matrix layers, and in the cleared biopsies, the decidua layer. RESULTS: MPAM and SHG revealed fetal membrane epithelial topography and collagen organization in multiple matrix layers. Term amnion layers showed epithelial shedding and gaps. Optical clearing provided full-depth imaging with improved visualization of collagen structure, mesenchymal cells in extracellular matrix layers, and decidua morphology. Layer thicknesses measured by imaging corroborated with histology. Mosaic tiling of MPAM/SHG image stacks allowed large area visualization of entire biopsies. CONCLUSION: MPAM-SHG microscopy allowed for study of this multi-layered tissue and revealed shedding, gap formation, and other structural changes. This approach could be used to study structural changes associated with membranes as well as other uterine tissues to better understand events in normal and abnormal parturition.

• MeSH
• Extracellular Matrix MeSH
• Extraembryonic Membranes anatomy & histology   diagnostic imaging   MeSH
• Histocytological Preparation Techniques MeSH
• Humans MeSH
• Microscopy, Fluorescence, Multiphoton * MeSH
• Second Harmonic Generation Microscopy * MeSH
• Placenta anatomy & histology   diagnostic imaging   MeSH
• Pregnancy MeSH
• Imaging, Three-Dimensional MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Two-Photon Processor (TPP) is a versatile, ready-to-use, and freely available software package in MATLAB to process data from in vivo two-photon calcium imaging. TPP includes routines to search for cell bodies in full-frame (Search for Neural Cells Accelerated; SeNeCA) and line-scan acquisition, routines for calcium signal calculations, filtering, spike-mining, and routines to construct parametric fields. Searching for somata in artificial in vivo data, our algorithm achieved better performance than human annotators. SeNeCA copes well with uneven background brightness and in-plane motion artifacts, the major problems in simple segmentation methods. In the fast mode, artificial in vivo images with a resolution of 256 × 256 pixels containing ≈ 100 neurons can be processed at a rate up to 175 frames per second (tested on Intel i7, 8 threads, magnetic hard disk drive). This speed of a segmentation algorithm could bring new possibilities into the field of in vivo optophysiology. With such a short latency (down to 5-6 ms on an ordinary personal computer) and using some contemporary optogenetic tools, it will allow experiments in which a control program can continuously evaluate the occurrence of a particular spatial pattern of activity (a possible correlate of memory or cognition) and subsequently inhibit/stimulate the entire area of the circuit or inhibit/stimulate a different part of the neuronal system. TPP will be freely available on our public web site. Similar all-in-one and freely available software has not yet been published.

• MeSH
• Algorithms * MeSH
• Aniline Compounds analysis   MeSH
• Fluoresceins analysis   MeSH
• Fluorescent Dyes MeSH
• Microscopy, Fluorescence, Multiphoton methods   MeSH
• Cerebral Cortex chemistry   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Software * MeSH
• Calcium analysis   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Multifunctional two-photon laser scanning microscopy provides attractive advantages over conventional two-photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. RESULTS: These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two-photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. CONCLUSIONS: With the multifunctional two-photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.

• MeSH
• Chloroplasts chemistry   ultrastructure   MeSH
• Microscopy, Confocal methods   MeSH
• Plant Leaves chemistry   ultrastructure   MeSH
• Microscopy, Fluorescence, Multiphoton methods   MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Selaginellaceae chemistry   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH