ABSTRACT
In this study, fluorescence recovery after photobleaching (FRAP) experiments were performed on RBC labeled by lipophilic fluorescent dye CM-DiI to evaluate the role of adenylyl cyclase cascade activation in changes of lateral diffusion of erythrocytes membrane lipids. Stimulation of adrenergic receptors with epinephrine (adrenaline) or metaproterenol led to the significant acceleration of the FRAP recovery, thus indicating an elevated membrane fluidity. The effect of the stimulation of protein kinase A with membrane-permeable analog of cAMP followed the same trend but was less significant. The observed effects are assumed to be driven by increased mobility of phospholipids resulting from the weakened interaction between the intermembrane proteins and RBC cytoskeleton due to activation of adenylyl cyclase signaling cascade.
Subject(s)
Adenylyl Cyclases , Erythrocyte Membrane , Fluorescence Recovery After Photobleaching , Membrane Fluidity , Adenylyl Cyclases/metabolism , Membrane Fluidity/drug effects , Humans , Erythrocyte Membrane/metabolism , Enzyme Activation , Signal Transduction/drug effects , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Epinephrine/pharmacology , Epinephrine/metabolismABSTRACT
Multiphoton microscopy (MPM) is a method of molecular imaging and specifically of intravital imaging that is characterized by high spatial resolution in combination with a greater depth of penetration into the tissue. MPM is a multimodal method based on detection of nonlinear optical signals - multiphoton fluorescence and optical harmonics - and also allows imaging with the use of the parameters of fluorescence decay kinetics. This review describes and discusses photophysical processes within major reporter molecules used in MPM with endogenous contrasts and summarizes several modern experiments that illustrate the capabilities of label-free MPM for molecular imaging of biochemical processes in connective tissue and cells.
Subject(s)
Biochemical Phenomena , Cells/metabolism , Connective Tissue/metabolism , Fluorescent Dyes/chemistry , Microscopy, Fluorescence, Multiphoton/methods , Optical Imaging/methods , HumansABSTRACT
We performed comparative analysis of the morphology of chondrocytes in normal cartilage, after their isolation from the tissue, and at different stages of culturing; structural dynamics of cells during culturing was also studied. Significant morphological differences in chondrocytes at the specified stages of their preparation to in vivo use were revealed. Pronounced structural changes (blebbing and cytoplasm swelling) were found in chondrocytes before their implantation, which can affect the formation of cartilage regenerate. The study was performed using light microscopy methods including time-lapse recording of the cell cultures with differential interference Nomarski contrasting combined with transmission electron microscopy.