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We show that structural protein arrays consisting largely of collagen, myosin, and tubulin, and their associated proteins can be imaged in three dimensions with high contrast and resolution by laser-scanning second harmonic generation (SHG) microscopy. SHG is a nonlinear optical scheme and this form of microscopy shares several common advantages with multiphoton excited fluorescence, namely, intrinsic...
Two-photon microscopy has revolutionized life sciences by enabling long-term imaging of living preparations in highly scattering tissue while minimizing photodamage. At the same time, commercial two-photon microscopes are expensive and this has prevented the widespread application of this technique to the biological community. As an alternative to commercial systems, we provide an update of our efforts...
GFP-based fluorescence resonance energy transfer (FRET) probes that visualize local activity-changes of Ras and Rho GTPases in living cells are now available for examining the spatio-temporal regulation of these proteins. This article describes principles and strategies to develop intramolecular FRET probes for Ras- and Rho-family GTPases. The procedure for characterizing candidate probes, and image...
Increasing numbers of small animal models are in use in the field of neuroscience research. Magnetic resonance imaging (MRI) provides an excellent method for non-invasive imaging of the brain. Using three-dimensional (3D) MR sequences allows lesion volumetry, e.g. for the quantification of tumor size.Specialized small-bore animal MRI scanners are available for high-resolution MRI of small rodents’...
The application of novel assays for basic cell research is tightly linked to the development of easy-to-use and versatile tools and protocols for implementing such technologies for a wide range of applications and model species. The bimolecular fluorescence complementation (BiFC) assay is one such novel method for which tools and protocols for its application in plant cell research are still being...
Genetically encoded calcium indicators (GECIs) allow researchers to measure calcium dynamics in specific targeted locations within living cells. Such indicators enable dissection of the spatial and temporal control of calcium signaling processes. Here we review recent progress in the development of GECIs, highlighting which indicators are most appropriate for measuring calcium in specific organelles...
Over the past decade, the number of publications using micro-computed tomography (μCT) imaging in preclinical in vivo studies has risen exponentially. Higher spatial and temporal resolution are the key technical advancements that have allowed researchers to capture increasingly detailed anatomical images of small animals and to monitor the progression of disease in small animal models. The purpose...
Structural imaging research offers excellent translational benefits when non-human primate (NHP) models are employed. In this paper, we will discuss the development of anatomical MR imaging protocols for two important applications of structural imaging in NHPs: studies of genetic variability in brain morphology and longitudinal imaging of fetal brain maturation trends. In contrast with imaging studies...
Computational modeling can be extremely useful in interpreting experimental results. Here we describe how a relatively sophisticated stochastic model for microtubule dynamic instability in the mitotic spindle can be developed starting with straightforward rules and simple programming code. Once this model is developed, the method for comparing simulation results to experimental data must be carefully...
Epigenetics in general and microRNA (miRNA) in particular are an important and growing field of research, and while significant advances in the role of miRNA in a variety of diseases including cancer have been made, the majority of the information on the relationship between miRNA and its putative target proteins have been made on homogenized tissue, which, while useful, provides no information on...
The dynamic nature of cellular processes is emerging as an important modulator of physiological and pathological events. The key event in the life cycle of the human immunodeficiency virus type 1 (HIV-1) is transcription: it controls both viral gene expression and the latent phenotype. The basal transcription machinery and cellular and viral regulatory elements are dynamically recruited to the proviral...
Aptamers are chemical antibodies that bind to their targets with high affinity and specificity. These short stretches of nucleic acids are identified using a repetitive in vitro selection and partitioning technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since the emergence of this technology, many modifications and variations have been introduced to enable the selection...
Atomic force microscopy (AFM) is widely used in the biological sciences. Despite 25years of technical developments, two popular modes of bioAFM, imaging and single molecule force spectroscopy, remain hindered by relatively poor force precision and stability. Recently, we achieved both sub-pN force precision and stability under biologically useful conditions (in liquid at room temperature). Importantly,...
Zebrafish gain increasing popularity as animal model for the study of various aspects of modern cell biology as well as model organism for human diseases. This is owed to the fact that zebrafish represent a cost effective and versatile in vivo alternative to in vitro cell culture systems and to invertebrate- and classic rodent models as they combine many strengths of each of these systems. Zebrafish...
A vast diversity of biological systems, ranging from prokaryotes to multicellular organisms, show cell migration behavior. Many of the basic cellular and molecular concepts in cell migration apply to diverse model organisms. Drosophila, with its vast repertoire of tools for imaging and for manipulation, is one of the favorite organisms to study cell migration. Moreover, distinct Drosophila tissues...
The development and molecular composition of muscle tissue is evolutionarily conserved. Drosophila is a powerful in vivo model system to investigate muscle morphogenesis and function. Here, we provide a short and comprehensive overview of the important developmental steps to build Drosophila body muscle in embryos, larvae and pupae. We describe key methods, including muscle histology, live imaging...
Of all the ways in which complex materials (including many biological systems) can be explored, imaging is perhaps the most powerful because delivering high information content directly. This is particular relevant in aspects of cellular localization where the physical proximity of molecules is crucial in biochemical processes. A great deal of effort in imaging has been spent on enabling chemically...
Mass spectrometry imaging (MSI) was introduced more than five decades ago with secondary ion mass spectrometry (SIMS) and a decade later with laser desorption/ionization (LDI) mass spectrometry (MS). Large biomolecule imaging by matrix-assisted laser desorption/ionization (MALDI) was developed in the 1990s and ambient laser MS a decade ago. Although SIMS has been capable of imaging with a moderate...
Imaging of specific small molecules is particularly challenging using conventional optical microscopy techniques. This has led to the development of alternative imaging modalities, including mass spectrometry (MS)-based methods. This review aims to provide an overview of the technologies, methods and future directions of laser-based mass spectrometry imaging (MSI) of small molecules. In particular...
Due to its selective overexpression in prostate cancer (PCa), the prostate-specific membrane antigen (PSMA) has been recognized as a highly promising target for diagnostic and therapeutic applications. So far, various PSMA ligands have been developed for radiolabeling with radioisotopes such as 68Ga or 18F which can be used for specific visualization and diagnosis of PSMA-expressing PCa. In addition,...
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