Digital optical and scanning probe microscopy for biocells inspection and manipulation

This paper was presented at the 3rd Micro and Nano Flows Conference (MNF2011), which was held at the Makedonia Palace Hotel, Thessaloniki in Greece. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, Aristotle University of Thessaloniki, University of Thessaly, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute.Nanobiotechnology and scanning probe microscopy (SPM) are new fields that are of interest to modern medical science. Nowadays atomic force microscopy (AFM) is widely used in medical-biological researches from the all variety of SPM. Without special methods of preparation AFM gives an opportunity to investigate the morphology of the surface of different biological objects with nanoresolution. Also this method allows to analyze the physical and mechanical properties at micro- and nanoscale. Our experimental complex with the functions of scanning probe and optical microscopy is intended for different materials investigation including biological cells. A special optical system makes it possible to visualize the objects position of the probe in the microscale. AFM is used for visualization and identification of the local adhesion and viscoelastic properties of biological cells. Dynamic laser speckle (DLS) is used for real-time monitoring of cells motility in living tissues. Another opportunity of this complex is some manipulation with the cell by means of the applied load variation. This technique greatly enhances the possibilities and opens a new field of experiments in cell biology. The purpose of this work is to show the possibility of AFM and DLS for studies of biological cells, namely measurement of the general cells motility in living tissues, the elastic modulus of the single cell membrane, as well as to identify the forces causing damage of the membrane.This study is partially funded by the National Academy of Sciences and Foundation for Basic Research of Belarus with grants and projects “ЭЭ 1.6.1”, T11MC-023, T10Р-029

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Dynamics of Natural Killer cell receptor revealed by quantitative analysis of photoswitchable protein

Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies which rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report a novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, following a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 +- 0.06 micron^2/s) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse and remains surprisingly stable there. Unexpectedly however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected inter-synaptic exchange of protein.Comment: 25 pages, 5 figure

Index to NASA Tech Briefs, 1975

This index contains abstracts and four indexes--subject, personal author, originating Center, and Tech Brief number--for 1975 Tech Briefs

Off-axis electron holography of bacterial cells and magnetic nanoparticles in liquid

The mapping of electrostatic potentials and magnetic fields in liquids usingelectron holography has been considered to be unrealistic. Here, we showthat hydrated cells ofMagnetospirillum magneticumstrain AMB-1 and assem-blies of magnetic nanoparticles can be studied using off-axis electronholography in a fluid cell specimen holder within the transmission electronmicroscope. Considering that the holographic object and reference waveboth pass through liquid, the recorded electron holograms show sufficientinterference fringe contrast to permit reconstruction of the phase shift ofthe electron wave and mapping of the magnetic induction from bacterialmagnetite nanocrystals. We assess the challenges of performingin situmagne-tization reversal experiments using a fluid cell specimen holder, discussapproaches for improving spatial resolution and specimen stability, and outlinefuture perspectives for studying scientific phenomena, ranging from interpar-ticle interactions in liquids and electrical double layers at solid–liquidinterfaces to biomineralization and the mapping of electrostatic potentialsassociated with protein aggregation and folding

Aerospace Medicine and Biology: A continuing bibliography (supplement 160)

This bibliography lists 166 reports, articles, and other documents introduced into the NASA scientific and technical information system in October 1976

Fluctuations in active membranes

Active contributions to fluctuations are a direct consequence of metabolic energy consumption in living cells. Such metabolic processes continuously create active forces, which deform the membrane to control motility, proliferation as well as homeostasis. Membrane fluctuations contain therefore valuable information on the nature of active forces, but classical analysis of membrane fluctuations has been primarily centered on purely thermal driving. This chapter provides an overview of relevant experimental and theoretical approaches to measure, analyze and model active membrane fluctuations. In the focus of the discussion remains the intrinsic problem that the sole fluctuation analysis may not be sufficient to separate active from thermal contributions, since the presence of activity may modify membrane mechanical properties themselves. By combining independent measurements of spontaneous fluctuations and mechanical response, it is possible to directly quantify time and energy-scales of the active contributions, allowing for a refinement of current theoretical descriptions of active membranes.Comment: 38 pages, 9 figures, book chapte

Applications of AFM in pharmaceutical sciences

Atomic force microscopy (AFM) is a high-resolution imaging technique that uses a small probe (tip and cantilever) to provide topographical information on surfaces in air or in liquid media. By pushing the tip into the surface or by pulling it away, nanomechanical data such as compliance (stiffness, Young’s Modulus) or adhesion, respectively, may be obtained and can also be presented visually in the form of maps displayed alongside topography images. This chapter outlines the principles of operation of AFM, describing some of the important imaging modes and then focuses on the use of the technique for pharmaceutical research. Areas include tablet coating and dissolution, crystal growth and polymorphism, particles and fibres, nanomedicine, nanotoxicology, drug-protein and protein-protein interactions, live cells, bacterial biofilms and viruses. Specific examples include mapping of ligand-receptor binding on cell surfaces, studies of protein-protein interactions to provide kinetic information and the potential of AFM to be used as an early diagnostic tool for cancer and other diseases. Many of these reported investigations are from 2011-2014, both from the literature and a few selected studies from the authors’ laboratories