Ybridization of laser-induced spectrofluorescence analysis (lifs), matrix-assisted laser desorption / ionization mass spectrometry (maldi), fluorescence recovery after photobleaching (frap) anf fluorescence loss in photobleaching (flip) microtechnics

Abstract

Abstract. Novel MALDI MS + FLIP approaches for verifying continuity of membranous structures and measurements of nucleus-cytoplasm exchange rates are proposed. Novel approaches for the measurements of lateral diffusion / molecular mobility and bindings using MALDI + FRAP hybridization are proposed. FRAP (Fluorescence Recovery After Photobleaching) is a method for the diffusion kinetics measurements in living cells using fluorescence microscopy which allows to estimate quantitatively the two dimensional lateral diffusion in molecularly thin film containing fluorescent-labeled probes, or for single cell examination (i.e. the study of lateral mobility of cellular molecules). Fluorescence Loss in Photobleaching (FLIP) is a microscopic technique predominantly performed using laser scanning microscopy (e.g. for tagged protein local photobleaching by short, intensive laser excitation on CLSM platform) used for the studies on molecular mobility inside the cells and membranes. MALDI (Matrix-Assisted Laser Desorption / Ionization) is a soft ionization technique used in mass spectrometry, allowing for the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragmented when ionized by more conventional ionization methods (according to encyclopedic definition). The above laser-based technique is readily compatible with MALDI using the same laser beam for MALDI and FLIP. IntroductionFRAP (Fluorescence Recovery After Photobleaching) is a method for the diffusion kinetics (and also, correctly, reaction-diffusion kinetics) measurements in living cells using fluorescence microscopy (Zlatanov, 1987;Lopez, 1988;Swaisgood, 1989; Anders, 1990;Nagy, 1990; Bryers, 1998;Salomé, 1998;Tinland, 1998;Higgs, 2000;Reits, 2001;Carrero, 2003;Cha 2004;Joubert, 2004;Fukano, 2004;James, 2004;Sprague, 2005;Lele, 2006;Abbaci, 2007;Febo-Ayala, 2007;Travascio, 2007;Dushek, 2008;Kang, 2008;McNally, 2008;Tolentino, 2008;Lambert, 2009;Travascio, 2009; Hagman, 2010;Mueller, 2010;Mai, 2011;Wachsmuth, 2014;Yapp, 2016) which allows to estimate quantitatively the two dimensional lateral diffusion in molecularly thin film containing fluorescent-labeled probes, or for single cell examination (i.e. the study of lateral mobility of cellular molecules). Similar, though less common techniques (also referred to as FRAP) have been developed for the three dimensional diffusion and molecular binding investigation inside the cell. Modern confocal scanning laser microscopes (CSLMs) (Hardingham, 2000;Gribbon, 2001; Loerke,2005;Seiffert, 2005;Braeckmans, 2007;Campbell, 2007;Maung, 2007;Mazza, 2008;Tsibidis, 2008;Kang, 2010;Roberti, 2011; Kang, 2012;De Los Santos, 2015;Lemcke, 2016) are equipped with the system for local photobleaching in selected regions (Figure 1). But currently there are no completely satisfactory methods for molecular-chemical characterization of diffusion / binding kinetics for FRAP using MALDI (Matrix-Assiste...

Target Chip Based Single-Cell Biotyping and Telemetric Bioluminescence Lensless Microscopy of the Buried Sandwich-Slides as a Novel Way for Measurement, Mapping and Molecular Imaging of Biodegradation/Biofouling of Plastic Surfaces in Real Soils

“…Nevertheless, being pacifists and opponents of an offensive fratricidal war, we believe that our work in this direction would bring much greater benefit to civilization (and fully refuse to work on all topics that are not related to civilian science in the Russian Federation). Unfinished works and advances of our work in this direction are given in the old papers [71][72][73][74][75][76][77]. The priority of this know-how in non-English publications has been fixed since 2012 [78][79][80].…”