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Application of Atomic Force Microscopy Measurements on Cardiovascular Cells

Sun, 05 Apr 2026 22:00:50 +0000

The atomic force microscope (AFM) is a state-of-the-art tool that can analyze and characterize samples on a scale from angstroms to 100 μm by physical interaction between AFM cantilever tip and sample surface. AFM imaging has been used incrementally over last decade in living cells in cardiovascular research. Beyond its high resolution 3D imaging, AFM allows the quantitative assessments on the structure and function of the underlying cytoskeleton and cell organelles, binding probability, adhesion forces, and micromechanical properties of the cell by "sensing" the cell surface with mechanical sharp cantilever tip. AFM measurements have enhanced our understanding of cell mechanics in normal physiological and pathological states.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-61779-523-7_22

Live-Cell Assessment of Mitochondrial Reactive Oxygen Species Using Dihydroethidine

Sun, 05 Apr 2026 22:00:50 +0000

Reactive oxygen species (ROS) play an important role in both physiology and pathology. Mitochondria are an important source of the primary ROS superoxide. However, accurate detection of mitochondrial superoxide especially in living cells remains a difficult task. Here, we describe a method and the pitfalls to detect superoxide in both mitochondria and the entire cell using dihydroethidium (HEt) and live-cell microscopy.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-4939-2257-4_15

Intravital Two-Photon Imaging to Understand Bacterial Infections of the Mammalian Host

Sun, 05 Apr 2026 22:00:50 +0000

Intravital two-photon microscopy (2PM) is an advanced fluorescence based imaging technique that allows for a cinematic study of physiological events occurring within tissues of the live animal. Based on this real-time imaging platform, the pathophysiology of bacterial infections can be studied in the most relevant of model systems—the live host. Whereas traditional animal models of host–pathogen interaction studies rely on end stage analysis of dissected tissues, noninvasive intravital imaging allows for real-time monitoring of infection during shorter or extended time frames. Here we describe the use of advanced surgical techniques for initiation of spatially and temporally well-controlled kidney infections in rats, and how the bacterial whereabouts can be studied while at the same time monitoring the host's altered tissue homeostasis based on real-time deep tissue imaging on the 2PM platform. Whereas this chapter focuses on pyelonephritis induced by uropathogenic Escherichia coli (UPEC) in rats, the major concepts can easily be translated to numerous infections in a variety of organs.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-4939-1261-2_5

Live Imaging of Leukocyte–Endothelium Interactions

Sun, 05 Apr 2026 22:00:50 +0000

Leukocyte extravasation is a highly dynamic, interactive, and coordinated process that plays a central role during the inflammatory response of innate immunity. The interaction of leukocytes with the activated endothelium under shear forces is comprised of many sequential events, each involving specific leukocyte and endothelial receptors, as well as chemokines and adaptor and signaling molecules. Because of its complexity, researchers studying leukocyte extravasation at the subcellular level have been forced to search for appropriate in vitro models that mimic pathophysiological conditions at sites of inflammation. We report methods for direct visualization of cellular and molecular processes of critical importance to spatiotemporally dissect the different steps in the adhesion cascade. These methodologies include techniques for the study of the dynamics of individual molecules involved in a discrete part of the process, as well as simple procedures to label molecules and cells in order to observe the extravasation process.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-461-6_2

Quantitative Analysis of Membrane Potentials

Sun, 05 Apr 2026 22:00:50 +0000

The changes that occur in electrochemical gradients across biological membranes provide us with invaluable information on physiological responses, pathophysiological processes and drug actions/toxicity. This chapter aims to provide researchers with sufficient information to carry out a quantitative assessment of mitochondrial energetics at a single-cell level thereby providing output on changes in the mitochondrial membrane potential (Δψ_m) through the utilization of potentiometric fluorescent probes (TMRM, TMRE, Rhodamine 123). As these cationic probes behave in a Nernstian fashion, changes at the plasma membrane potential (Δψ_p) need also to be accounted for in order to validate the responses obtained with Δψ_m-sensitive fluorescent probes. To this end techniques that utilize Δψ_p-sensitive anionic fluorescent probes to monitor changes in the plasma membrane potential will also be discussed. In many biological systems multiple changes occur at both a Δψ_m and Δψ_p level that often makes the interpretation of the cationic fluorescent responses much more difficult. This problem has driven the development of computational modelling techniques that utilize the redistribution properties of the cationic and anionic fluorescent probes within the cell to provide output on changes in Δψ_m and Δψ_p.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_20

Measuring the Elastic Properties of Living Cells with Atomic Force Microscopy Indentation

Sun, 05 Apr 2026 22:00:50 +0000

Atomic force microscopy (AFM) is a powerful and versatile tool for probing the mechanical properties of biological samples. This chapter describes the procedures for using AFM indentation to measure the elastic moduli of living cells. We include step-by-step instructions for cantilever calibration and data acquisition using a combined AFM/optical microscope system, as well as a detailed protocol for data analysis. Our protocol is written specifically for the BioScope™ Catalyst™ AFM system (Bruker AXS Inc.); however, most of the general concepts can be readily translated to other commercial systems.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-62703-056-4_15

Correlative Optical and Scanning Probe Microscopies for Mapping Interactions at Membranes

Sun, 05 Apr 2026 22:00:49 +0000

Innovative approaches for real-time imaging on molecular-length scales are providing researchers with powerful strategies for characterizing molecular and cellular structures and dynamics. Combinatorial techniques that integrate two or more distinct imaging modalities are particularly compelling as they provide a means for overcoming the limitations of the individual modalities and, when applied simultaneously, enable the collection of rich multi-modal datasets. Almost since its inception, scanning probe microscopy has closely associated with optical microscopy. This is particularly evident in the fields of cellular and molecular biophysics where researchers are taking full advantage of these real-time, in situ, tools to acquire three-dimensional molecular-scale topographical images with nanometer resolution, while simultaneously characterizing their structure and interactions though conventional optical microscopy. The ability to apply mechanical or optical stimuli provides an additional experimental dimension that has shown tremendous promise for examining dynamic events on sub-cellular length scales. In this chapter, we describe recent efforts in developing these integrated platforms, the methodology for, and inherent challenges in, performing coupled imaging experiments, and the potential and future opportunities of these research tools for the fields of molecular and cellular biophysics with a specific emphasis on the application of these coupled approaches for the characterization of interactions occurring at membrane interfaces.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-62703-137-0_24

Real-Time Analysis of G Protein-Coupled Receptor Signaling in Live Cells

Sun, 05 Apr 2026 22:00:49 +0000

Seven transmembrane-spanning receptors, widely referred to as G protein-coupled receptors (GPCRs), mediate a broad spectrum of extracellular signals at the plasma membrane through G proteins, thereby modulating a variety of biological processes. In addition to G proteins, they also interact with a number of other cytoplasmic proteins. Thus, methods to understand GPCR signaling and their interactions with intracellular proteins in real time in live cells are of importance. Recent developments in microscopy methods and the availability of fluorescent proteins facilitated the development of techniques to unravel these interactions more precisely. This chapter describes the methodology for sequential capturing of images of membrane and cytoplasmic proteins fused to different fluorescence probes to understand GPCR interaction with cytosolic proteins and their colocalization.

Source: http://www.springerprotocols.com/Abstract/doi/10.1385/1-59745-048-0:159

Delivery of Molecular Beacons for Live-Cell Imaging and Analysis of RNA

Sun, 05 Apr 2026 22:00:49 +0000

Over the past decade, a variety of oligonucleotide-based probes have been developed that allow for direct visualization of RNA molecules in living cells. Of these, molecular beacons have garnered a particularly high degree of interest due to their simple yet exquisite unimolecular stem-loop design that allows for the efficient conversion of target recognition into a specific fluorescent signal. As a result of their favorable fluorescent enhancement and their high specificity, molecular beacons have been used for a wide range of applications, including the monitoring of RNA expression and localization in living cells, cancer cell detection, and the study of viral infections. In this chapter we describe a general methodology that can be followed for the imaging and analysis of RNA in living cells using molecular beacons. Several commonly employed methods for delivering molecular beacons into the cytosol are discussed including toxin-based cell membrane permeabilization, microinjection, and microporation. Strategies for acquiring ratiometric measurements are also described.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-61779-005-8_10

Gene Loss-of-Function and Live Imaging in Chick Embryos

Sun, 05 Apr 2026 22:00:49 +0000

Planar cell polarity (PCP) is the coordinate organization of cells within the plane of a tissue. PCP is essential for tissue function, such as for proper hearing in the vertebrate ear or for accurate vision in the Drosophila eye. Using the chick embryo, we have recently shown that during early muscle formation, the first formed muscle fibres utilize the PCP pathway to orient parallel to a WNT11 source present in the medial border of somites. Our results further establish that WNT11 acts as a directional cue to regulate this process. To perform this study, two major techniques have been utilized, the gene loss-of-function using a vector-based shRNAmir expression and confocal videomicroscopy of fluorescent gene reporters targeted in specific cell subpopulations by in vivo electroporation. Here we describe the two techniques.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-61779-510-7_9