Quantitative Fluorescence Microscopy Methods To Investigate Molecular Interactions And Dynamics In Living Cells

Biomolecules such as proteins and lipids have vital roles in numerous cellular functions, including biomolecule transport, protein functions, cellular homeostasis and biomembrane integrity. Traditional biochemistry methods do not provide precise information about cellular biomolecule distribution and behavior under native environmental conditions since they are not transferable to live cell samples. Consequently, this can lead to inaccuracies in quantifying biomolecule interactions due to potential complexities arising from the heterogeneity of native biomembranes. To overcome these limitations, minimal invasive microscopic techniques, such as fluorescence fluctuation spectroscopy (FFS) in combination with fluorescence proteins (FPs) and fluorescence lipid analogs, have been developed. FFS techniques and membrane property sensors enable the quantification of various parameters, including concentration, dynamics, oligomerization, and interaction of biomolecules in live cell samples. In this work, several FFS approaches and membrane ...

Fluorescence Microscopy of Living Cells in Culture, Part B

Fluorescence methods play a leading role in the investigation of biological objects. They are the only non-destructive methods for investigating living cells and microorganisms in vivo. Using intrinsic and artificial fluorescence methods provides deep insight into mechanisms underlying physiological and biochemical processes. This book covers a wide range of modern methods involved in experimental biology. It illustrates the use of fluorescence microscopy and spectroscopy, confocal laser scanning microscopy, flow cytometry, delayed fluorescence, pulse-amplitude-modulation fluorometry, and fluorescent dye staining protocols. This book provides an overview of practical and theoretical aspects of fluorescence methods and their successful application in the investigation of static and dynamic processes in living cells and microorganisms.

Biological membranes play a central role in cell structure, shape and functions. However, investigating the membrane bilayer has proved to be difficult due to its highly dynamic and anisotropic structure, which generates steep gradients at the nanometer scale. Due to the decisive impact of recently developed fluorescence-based techniques, tremendous advances have been made in the last few years in our understanding of membrane characteristics and functions. In this context, the present book illustrates some of these major advances by collecting review articles written by highly respected experts. The book is organized in three parts, the first of which deals with membrane probes and model membranes. The second part describes the use of advanced quantitative and high-resolution techniques to explore the properties of biological membranes, illustrating the key progress made regarding membrane organization, dynamics and interactions. The third part is focused on the investigation of membrane proteins using the same techniques, and notably on the membrane receptors that play a central role in signaling pathways and therapeutic strategies. All chapters provide comprehensive information on membranes and their exploration for beginners in the field and advanced researchers alike.

Fluorescence microscopy has proven to be immensely powerful for the study of biological systems at both the cellular and systems biological levels. The ability to specifically label a single molecular species fluorescently has enabled the study of complex cellular structures through the visualization of their constituent components both individually as well as in context of the overall structure. Since the advent of engineered fluorescent proteins (such as GFP) and other proteins capable of being genetically encoded as fusion constructs, the utility of fluorescence microscopy has increased exponentially in terms of the ability to efficiently, specifically label desired molecules while limiting perturbations to the biology under study. With this enhanced ability of visualization came a hand-in-hand evolution of computational techniques to extract quantitative information from microscopy images. In this thesis, I focus on the application of fluorescence imaging at the biophysical level in living cells: analyzing the motion/dynamics of single molecules and complexes, which are small relative to the structures of the cell, in order to elucidate their molecular function and mechanism. The motion of these "particles" within living cells is necessarily related to their functions as well as their interacting partners, which can vary dynamically during their lifetimes. Observation and analysis of this motion using a combination of fluorescence microscopy and robust quantitative analysis allows one to infer these characteristics. Here, I study three diverse biological systems in the context of live-cell fluorescence microscopy and biophysical analysis: 1) the transport of 0-actin mRNA particles in primary mouse neurons, 2) kinetochore motion during cell division, specifically focusing on anaphase dynamics, and 3) the motion of cell-growth-implicated membrane proteins in Bacillus subtilis.

This new edition of Fluorescent Proteins presents current applications of autofluorescent proteins in cell and molecular biology authored by researchers from many of the key laboratories in the field. Starting from a current review of the broad palette of fluorescent proteins available, several chapters focus on key autofluorescent protein variants, including spectral variants, photodynamic variants as well as chimeric FP approaches. Molecular applications are addressed in chapters that detail work with single molecules, approaches to generating protein fusions and biosensors as well as analysis of protein-protein interactions in vivo by FRET, fluorescence polarization and fluorescence cross correlation techniques. A number of approaches to in vivo dynamics are presented, including FRAP, photoactivation, and 4-dimensional microscopy. Behavior of spindle components, membrane proteins, mRNA trafficking as well as analysis of cell types in tissues and in development are detailed and provide models for a wide variety of experimental approaches. In addition, several chapters deal directly with the computational issues involved in processing multidimensional image data and using fluorescent imaging to probe cellular behavior with quantitative modeling. This volume brings together the latest perspective and techniques on fluorescent proteins and will be an invaluable reference in a wide range of laboratories.

A self-contained treatment of the latest fluorescence applications in biotechnology and the life sciences This book focuses specifically on the present applications of fluorescence in molecular and cellular dynamics, biological/medical imaging, proteomics, genomics, and flow cytometry. It raises awareness of the latest scientific approaches and technologies that may help resolve problems relevant for the industry and the community in areas such as public health, food safety, and environmental monitoring. Following an introductory chapter on the basics of fluorescence, the book covers: labeling of cells with fluorescent dyes; genetically encoded fluorescent proteins; nanoparticle fluorescence probes; quantitative analysis of fluorescent images; spectral imaging and unmixing; correlation of light with electron microscopy; fluorescence resonance energy transfer and applications; monitoring molecular dynamics in live cells using fluorescence photo-bleaching; time-resolved fluorescence in microscopy; fluorescence correlation spectroscopy; flow cytometry; fluorescence in diagnostic imaging; fluorescence in clinical diagnoses; immunochemical detection of analytes by using fluorescence; membrane organization; and probing the kinetics of ion pumps via voltage-sensitive fluorescent dyes. With its multidisciplinary approach and excellent balance of research and diagnostic topics, this book is an essential resource for postgraduate students and a broad range of scientists and researchers in biology, physics, chemistry, biotechnology, bioengineering, and medicine.

This comprehensive reference work details the latest developments in fluorescence imaging and related biological quantification. It explores the most recent techniques in this imaging technology through the utilization and incorporation of quantification analysis which makes this book unique. It also covers super resolution microscopy with the introduction of 3D imaging and high resolution fluorescence. Many of the chapter authors are world class experts in this medical imaging technology.

In the last decade, fluorescence microscopy has evolved from a classical “retrospective” microscopy approach into an advanced imaging technique that allows the observation of cellular activities in living cells with increased resolution and dimensions. A bright new future has arrived as the nano era has placed a whole new array of tools in the hands of biophysicists who are keen to go deeper into the intricacies of how biological systems work. Following an introduction to the complex world of optical microscopy, this book covers topics such as the concept of white confocal, nonlinear optical microscopy, fluctuation spectroscopies, site-specific labeling of proteins in living cells, imaging molecular physiology using nanosensors, measuring molecular dynamics, muscle braking and stem cell differentiation.

This book offers a comprehensive selection of essays by leading experts, which covers all aspects of modern imaging, from its application and up-scaling to its development. The chapter content ranges from the basics to the most complex overview of method and protocols. There is ample practical and detailed "how-to" content on important, but rarely addressed topics. This first edition features all-colour-plate chapters, licensed software and a unique, continuously updated website forum.