The recent explosion in the availability and variety of fluorescent proteins, new organic dyes and quantum dots has been a driving force in the growing use of Total Internal Reflection Fluorescence Microscopy (TIRFM). TIRFM only illuminates molecules that are within a thin volume near the coverslip surface of a specimen and not those deeper in solution. This allows for an unparalleled signal-to-noise ratio and tremendous resolution. In the March issue of Cold Spring Harbor Protocols, Samara Reck-Peterson, Nathan Derr and Nico Stuurman present Imaging Single Molecules Using Total Internal Reflection Fluorescence Microscopy (TIRFM), which includes an overview of the theory behind TIRFM, considerations for TIRFM setup and purification/labeling of proteins, and a discussion of new techniques for imaging single molecules with super-resolution localization. In addition, the group offers step-by-step protocols for Determining Single-Molecule Intensity as a Function of Power Density and Imaging Single Molecular Motor Motility with TIRFM. An example of TIRFM imaging of single dynein molecules labeled with TMR (green) moving along axonemal microtubules labeled with Cy5 (red) can be seen here.

Cold Spring Harbor Protocols is hosting the movie figures that accompany the new lab manual, Live Cell Imaging, Second Edition, edited by Robert Goldman, Jason Swedlow and David Spector, . These movies are freely accessible to all, and worth a look if you’re interested in seeing the state of the art in time lapse imaging.

Live cell imaging techniques are driving a revolution in biological research. Instead of viewing dead tissues and cells fixed at a particular stage of activity, scientists can now visualize dynamic changes as they happen, permitting a better understanding of complete processes. The revolution has been fueled by the implementation of genetically encoded fluorescent proteins, the subject of the 2008 Nobel Prize in Chemistry.

The diverse array of applications benefiting from fluorescent proteins ranges from markers targeted at organelles and protein fusions designed to monitor intracellular dynamics to reporters of transcriptional regulation and in vivo probes for whole-body imaging and detection of cancer. Fluorescent proteins have enabled the creation of highly specific biosensors to monitor a wide range of intracellular phenomena, including pH and metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. In the December issue of Cold Spring Harbor Protocols, David Piston and colleagues present Fluorescent Protein Tracking and Detection: Fluorescent Protein Structure and Color Variants, a comprehensive overview of the wide variety of fluorescent proteins that are currently available. The article features more than twenty movies of different fluorescent proteins in action and is a great primer for planning imaging experiments. As one of December’s featured articles, it is freely available to subscribers and non-subscribers alike.

In addition, the same authors have also contributed Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells. This article provides some general tips for the practical aspects of using and imaging enhanced green fluorescent protein (EGFP) and newer members of the color palette, as well as quantitative imaging of fluorescent proteins and imaging of several fluorescent proteins at the same time. Finally, an overview is provided for the different types of biosensors that have been derived from flourescent proteins.

Both articles are adapted from the spectacular new manual, Live Cell Imaging: A Laboratory Manual, Second Edition which is due out by month’s end.

CSH Protocols December Cover

Light carries momentum, so an object that reflects or refracts a beam of light experiences a force. This force is very small, but still strong enough to manipulate objects, such as a polystyrene bead. Using light focused through a lens, beads can be “trapped” near the focus. These optical traps, or “optical tweezers” have become an important tool that allows researchers to manipulate individual molecules or molecular complexes. High-resolution optical trapping techniques can now detect movements on the scale of a single base pair of DNA, 3.4 angstroms. The October issue of Cold Spring Harbor Protocols includes a series of articles detailing the concepts behind optical trapping, the components of an optical trapping system and the single-molecule experiments in which they are used. Carlos Bustamante, Yann Chemla, and Jeffrey Moffitt provide an introduction to High Resolution Dual-Trap Optical Tweezers with Differential Detection, and subsequent articles on Managing Environmental Noise, Instrument Design, Data Collection and Instrument Calibration, Minimizing the Influence of Measurement Noise and Alignment of Instrument Components.

As new imaging methods are developed and our knowledge of neural development deepens, methods for growing neuronal cells in culture where they are readily manipulated and observed become more and more important. In the August issue of Cold Spring Harbor Protocols, Amy MacDermott and colleagues from Columbia University provide a series of protocols describing neuronal culture techniques that are particularly useful for studying synapse formation and interconnection.

Preparation of Coverslips for Neuronal Cultures describes setting up glass coverslips for three different types of cell culture: Mass Culture, which results in an extensive and widespread network of synapses, Microisland Culture, which allows easier identification of synaptically connected neurons, and Macroisland Culture, which strikes a balance between the better survival rates and better identification of synapses offered by the two methods above.

Survival of CNS neurons in culture is usually greatly improved if the neurons are plated on top of a confluent astrocyte layer, as is described in Dissection, Plating, and Maintenance of Cortical Astrocyte Cultures. Under these conditions, neurons attach more firmly and develop a more easily identifiable bi-dimensional neuritic tree than in culture conditions where primary cells are plated on top of collagen, laminin or other cell-free substrates.

Once established the astrocyte layer can be used to in the Dissection, Plating, and Maintenance of Dorsal Horn Neuron Cultures, which are suitable for electrophysiological, molecular and immunocytochemical studies. Dorsal horn neurons can be grown by themselves, or co-cultured with embryonic dorsal root ganglion neurons as described in Dissection, Plating, and Maintenance of Dorsal Root Ganglion Neurons for Monoculture and for Coculture with Dorsal Horn Neurons.

Microbial populations have traditionally been studied in carefully controlled, laboratory-grown cultures. New metagenomic approaches are being developed to study these organisms in environmental or medical samples. The July issue of Cold Spring Harbor Protocols presents a method developed by Holger Daims from the University of Vienna for quantifying populations of microorganisms in a variety of naturally occurring conditions such as plankton samples or biofilms. Use of Fluorescence In Situ Hybridization and the daime Image Analysis Program for the Cultivation-Independent Quantification of Microorganisms in Environmental and Medical Samples combines fluorescent in situ hybridization using rRNA-targeted probes with digital image analysis. The results show an organism’s “biovolume fraction” in a given sample; this indicates the share of biochemical reaction space occupied by the quantified population and can be more relevant ecologically than absolute cell numbers. Like all of our featured articles, this protocol is freely available to subscribers and non-subscribers alike.

Micropatterning methods are rapidly becoming standard approaches for investigating cellular behaviors such as growth and migration. Adhesive Micropatterns for Cells: A Microcontact Printing Protocol from Matthieu Piel and colleagues at the Institut Curie offers a simple, fast, and efficient method for generating micropatterns for cellular studies. Employing an elastomeric stamp to print proteins on the substrate of choice, this technique does not require much of the expensive equipment and technical expertise needed for most micropatterning methods, making it easier to implement in biology laboratories. The authors have provided a movie that illustrates the technique step-by-step as part of the protocol. The article is a featured protocol for July, and like all our featured articles, it is freely available to subscribers and non-subscribers alike.

The May issue of Cold Spring Harbor Protocols is out and it contains a set of articles detailing the use of adenovirus vectors for gene transfer. Genetically modified adenoviruses serve as one of the most versatile and efficient gene delivery systems in use today. Laboratories throughout the world use adenoviruses for the delivery of DNA to cells for basic science and for gene therapy applications. Unlike most other vectors, adenoviruses can infect post-mitotic cells, which makes them particularly useful as vectors for gene delivery into cells like neurons.

In one of May’s featured articles, Robin Parks and colleagues from the Ottawa Health Research Institute provide Construction and Characterization of Adenovirus Vectors, a set of detailed instructions for the generation, propagation, purification, and characterization of adenovirus vectors. Like all of our featured articles, the protocol is freely accessible to subscribers and non-subscribers alike.

In addition, the May issue also contains a set of methods for Cell and Tissue Targeting from David Curiel and colleagues. Transfecting specific cells in a mixed population can be a difficult process. Adenovirus vectors are well-characterized, so they are excellent candidates for modification for targeting to specific cell types. The protocols here describe the creation of adenovirus vectors that enable targeting at the level of binding and entry in targeted cells through primary and/or secondary receptors (transduction), and protein expression of the transgene in the targeted cells (transcription/translation). The articles are:
Construction of Adenovirus Vectors with RGD-Modified Fiber for Transductional Targeting
Construction of Fusion Proteins for Transductional Targeting
and
Construction of Adenovirus Vectors for Transcriptional Targeting

Along with new cutting-edge methods, Cold Spring Harbor Protocols is home to an in-depth library of basic laboratory methods. The just-released April issue features articles that exemplify the attention to lab standard techniques.

Dye Loading with Patch Pipettes from Arthur Konnerth and colleagues from the Institut fur Physiologie der Ludwig-Maximilians-Universitat Munchen, describes the loading of individual cells with fluorescent probes via patch pipettes. This method allows for combined electrophysiological and optical measurements at a quantitative level. The patch-clamp methodology has been successful for single-cell dye labeling in cultured neurons, brain slices, and in vivo preparations. A wide range of dyes can be loaded using this method, including probes for morphological reconstruction, ion-sensitive indicator dyes for monitoring second-messenger cascades, and dye-labeled proteins for fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP) studies. The most widespread application of this technique has been for Ca2+ imaging.

Like all our featured articles, Dye Loading with Patch Pipettes is freely available to subscribers and non-subscribers alike.

February’s issue of Cold Spring Harbor Protocols contains a Topic Introduction on the subject of Photoactivation by Graham Ellis-Davies from Drexel University:

“Specific molecular interactions control cellular function. The photorelease of caged compounds (nucleotides, neurotransmitters, peptides, second messengers, proteins, etc.) can be used to control these interactions in living cells. Caged compounds are biological effector molecules whose active functionality has been chemically masked with a photoremovable protecting group. Illumination produces a concentration jump from the caged molecule. This article discusses the basic principles underlying photoactivation, the properties of caging chromophores and commercially available caged compounds, and practical considerations for their effective use.”

Our collection of protocols includes a wide variety of applications using photoactivation including the following:
Design, Synthesis, and Characterization of Caged Compounds
Introduction of Caged Peptide/Protein into Cells Using Microinjection
Introduction of Caged Peptide/Protein into Cells Using Bead Loading
Photoactivation-Based Labeling and In Vivo Tracking of RNA Molecules in the Nucleus
Inorganic Caged Compounds: Uncaging with Visible Light
Chemical Two-Photon Uncaging
Infrared-Guided Laser Stimulation of Neurons in Brain Slices
Photoactivation Cell Labeling for Cell Tracing in Avian Development