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Microscopic Analysis of Chromatin Localization and Dynamics in C. elegans

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell ...

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Single-Particle Tracking for Studying the Dynamic Properties of Genomic Regions in Live Cells

The appropriate functioning of living cells depends on a variety of dynamic processes that necessitate delicate motion, transportation, association, and disassociation in time and space. Different dynamic patterns such as directed motion, normal diffusion, and restricted dif ...

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Imaging Nascent RNA Dynamics in Dictyostelium

Dictyostelium cells have great utility for live imaging of single gene transcriptional dynamics. The cells allow efficient molecular genetics, for targeting of RNA reporters and fluorescent proteins to individual, defined loci. Dictyostelium cells share many signalling, chr ...

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Single mRNP Tracking in Living Mammalian Cells

The translocation of single mRNPs (mRNA–protein complexes) from the nucleus to the cytoplasm through the nuclear pore complex (NPC) is an important basic cellular process. Originally, in order to visualize this process, single mRNP export was examined using electron microscopy (EM) in f ...

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Nuclear Trafficking and Export of Single, Native mRNPs in Chironomus tentans Salivary Gland Cells

Real-time observation of single molecules or biological nanoparticles with high spatial resolution in living cells provides detailed insights into the dynamics of cellular processes. The salivary gland cells of Chironomus tentans are a well-established model system to study the ...

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Tracking Nuclear Poly(A) RNA Movement Within and Among Speckle Nuclear Bodies and the Surrounding Nucleoplasm

The movement of polyadenylated RNA transcripts (poly(A) RNA) through speckles in the nucleus can be detected and studied using fluorescence correlation microscopy (FCM) and photoactivation RNA tracking techniques. Speckles, sometimes called interchromatin granule clust ...

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Measuring Transcription Dynamics in Living Cells Using Fluctuation Analysis

Single-cell studies of gene regulation suggest that transcription dynamics play a fundamental role in determining expression heterogeneity within a population. In addition, the three-dimensional organization of the nucleus seems to both reflect and influence expression pa ...

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Single-Molecule Resolution Fluorescent In Situ Hybridization (smFISH) in the Yeast S. cerevisiae

Regulating gene expression is a major task for all cellular systems. RNA production and degradation plays a critical role in this process and accurately measuring cellular mRNA levels is essential to understanding gene expression regulation. Classical biochemical assays that stu ...

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Live-Cell Imaging Combined with Immunofluorescence, RNA, or DNA FISH to Study the Nuclear Dynamics and Expression of the X-Inactivation Center

Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic s ...

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High-Throughput Fluorescence-Based Screen to Identify Factors Involved in Nuclear Receptor Recruitment to Response Elements

The glucocorticoid receptor is an inducible transcription factor which plays important roles in many �physiological processes. Upon activation, GR interacts with regulatory elements and modulates the expression of genes. Although GR is widely expressed in multiple tissues, its ...

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Imaging Non-fluorescent Nanoparticles in Living Cells with Wavelength-Dependent Differential Interference Contrast Microscopy and Planar Illumination

Optical microscopy is a simple yet robust strategy to study live cellular processes. By changing the wavelength of the illumination light, different non-fluorescent nanoparticle probes can be identified and tracked dynamically inside crowded living cells with either differen ...

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Laser Scanning Cytometry: Principles and ApplicationsAn Update

Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the par ...

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Laser Capture Microdissection for Protein and NanoString RNA Analysis

Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of inter ...

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Mica Functionalization for Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy

Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy s ...

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Viewing Dynamic Interactions of Proteins and a Model Lipid Membrane with Atomic Force Microscopy

The information covered in this chapter will present a model homogenous membrane preparation technique and dynamic imaging procedure that can be successfully applied to more than one type of lipid study and atomic force microscope (AFM) instrument setup. The basic procedural steps have ...

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

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 cantil ...

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Porosome: The Secretory NanoMachine in Cells

Cells synthesize and store within membranous sacs products such as hormones, growth factors, neurotransmitters, or digestive enzymes, for release on demand. As recently as just 15 years ago, it was believed that during cell secretion, membrane-bound secretory vesicles completely me ...

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Atomic Force Microscopy Functional Imaging on Vascular Endothelial Cells

One of the challenging tasks in molecular cell biology is to identify and localize specific binding sites on biological samples with high spatial accuracy (in order of several nm). During the past 5 years, simultaneous topography and recognition imaging (TREC) has become a powerful AFM-bas ...

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A Novel Combined Imaging/Morphometrical Method for the Analysis of Human Sural Nerve Biopsies for Clinical Diagnosis

Nerve Morphometry is one tool employed in the clinical assessment of peripheral sural nerve pathological abnormalities. A new method is presented in this chapter incorporating an unbiased approach to quantitative sural nerve evaluation. Using conventional epoxy embedded nerv ...

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Stereology and Morphometry of Lung Tissue

This chapter deals with the stereological quantification of structural characteristics of the lung. The aim of design-based stereological methods is the unbiased and efficient estimation of structural features without making any assumptions on the underlying nature of the biol ...

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