Region where adult blood stem cells arise identified with time-lapse imaging

David Traver (right) and Neil Chi used zebrafish, grown in tanks at UCSD, to track blood-forming stem cells. Courtesy of UC San Diego.

Biologists at the University of California-San Diego have used time-lapse imaging to identify the specific region in vertebrates where adult blood stem cells arise during embryonic development. The discovery could lead to safer and more effective stem cell therapies for patients with leukemia, multiple myeloma, anemia and other diseases of the blood or bone marrow.

The researchers imaged the process in which primitive embryonic tissues first produce the parent stem cells that produce all adult blood cells. These cells--called haematopoietic stem cells (HSCs)--are responsible for the benefits of bone marrow transplants.

Currently, transplantation involves infusing donor stem cells into a patient's bone marrow. The procedure hopefully generates new healthy blood cells, but it also comes with serious risks such as immune rejection. Generating the stem cells using the patient's own precursor cells might overcome the risk and complication of bone marrow transplants, but requires understanding the molecular processes of haematopoietic stem cells.

The researchers used fluorescent reporter transgenes, confocal time-lapse microscopy, and flow cytometry to study zebrafish, demonstrating that haematopoietic stem cells arise directly from cells lining the floor of the dorsal aorta. They imaged the process in living embryos and also documented the steps in of haematopoietic stem cell formation.

In a related paper, researchers from Erasmus Medical Center in Rotterdam, The Netherlands imaged haematopoietic stem cells emerging from the aortic endothelium in a mouse embryo.

Click here to see a video on the UC San Diego work.

Research papers:
Haematopoietic stem cells derive directly from aortic endothelium during development

Julien Y. Bertrand, Neil C. Chi, Buyung Santoso, Shutian Teng, Didier Y. R. Stainier & David Traver doi:10.1038/nature08738

In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium
Jean-Charles Boisset, Wiggert van Cappellen, Charlotte Andrieu-Soler, Niels Galjart, Elaine Dzierzak & Catherine Robin doi:10.1038/nature08764

Fluorescent reporter allows imaging of single cells infected with hepatitis C

Hepatitis C virus (HCV) infects 2 to 3 percent of the world population. The recent development of methods to culture the virus may lead to antiviral therapies, but better imaging methods are needed to understand the infection process. In the February issue of Nature Biotechnology researchers at Rockefeller University, MIT and other institutions report on a cell-based fluorescent reporter that allows imaging of live or fixed individual HCV-infected cells.

They constructed the reporter by fusing a fluorescent protein--either EGFP, mCherry, or Tag RFP--to the C-terminal region of a known substrate of the HCV NS3-4A protease. They were able to use this reporter to distinguish between infected and uninfected cells and to image previously unseen events such as viral propagation and host response. The researchers used a Nikon Eclipse TE300 for wide-field fluorescence imaging, a Zeiss inverted Axiovert 200 laser scanning confocal microscope for imaging fixed samples, the Axiovert 200 with a Perkin-Elmer spinning-disk confocal head for imaging live samples, and a Olympus IX71 inverted microscope for time-lapse imaging.

The reporter system doesn’t require genetic modification of the viral genome and worked for all the HCV genomes they tested, which gives it potential for use in identifying new infectious isolates directly from patient samples, according to the researchers. They were also able to combine the HCV reporter with a fluorescent marker of cellular stress to correlate viral and host events.

Research Paper:
Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system, Nature Biotechnology 28, 167 - 171 (2010) 
doi:10.1038/nbt.1604

Flat top stage for live cell studies

Prior has released the H117P2IX flat top stage for the Olympus IX series of inverted microscopes. It is ideal for high-precision biomedical and material science scanning operations, and is especially useful for prolonged live cell studies. The newest version of the H117P2IX stage maximizes nosepiece access for correction collar adjustment by using miniaturized drive boxes. Drive components are mounted below the top plate, providing easy access for micromanipulators, environmental chambers and robotic loaders. The stage allows scanning with a broad range of sample holders, including microtitre plates, slide holders, petri dishes, well plates, flasks, haemocytometers and metallurgical sample holders. Stages can be driven by the Prior series of motor controllers or compatible systems in existing OEM configurations.
For more information visit www.prior.com.

Data management software for cellular imaging

PerkinElmer Inc. launched the Columbus 2.0 platform at the High Content Analysis East conference in Boston this week. The high content screening (HCS) data management software is designed for cellular imaging and analysis and provides a central repository to import, export, and manage cell image data. The latest edition has faster interfacing between images and data management and is fully web-enabled, meaning it can be used without installing software. One installation can serve an entire laboratory, department or even organization.

It is fully compliant with the Open Microscopy Environment (OME) and uses the OMERO server, developed by the OME, which enables it to support a wide range of file formats, allowing flexible, rapid and high performance analysis of images regardless of their origin. The software comes in two versions: Columbus Gallery, which provides data archiving, management, and visualization; and Columbus Conductor, which comes with the Gallery functions but also allows analyzing or reanalyzing of HCS data from PerkinElmer's Opera or Operetta imaging readers image data from other instruments using Acapella image analysis software.

More information here.

An automatic supply of water for water-immersion microscopy

Water immersion objectives are important for abberation-free focusing into live cell samples. However, the fact that water evaporates creates a challenge for long-term experiments. The new Water Immersion Micro Dispenser from Leica Microsystems can help over come this challenge. It can be retrofitted to water immersion objectives, and it automatically pumps water from a small water reservoir magnetically attached to the center of the objective nosepiece. 

The correction ring stays accessible and the full free working distance of the water objective is maintained. The design of the water cap keeps the water droplet in place even when the stage is moved. The water is dispensed at the right temperature and thus does not require heating. A protective collar around the objective prevents any water from getting into the microscope. The Water Immersion Micro Dispenser is designed for the Leica DMI6000 B inverted microscope, which is a component of confocal as well as high-end widefield systems.

Microscopy images reveal how B cells are activated

Researchers at Sydney's Garvan Institute of Medical Research and the University of California San Francisco have captured microscopy images that reveal some important new details of how the immune system works.

The researchers were interested in knowing what drives the immune system's B cell activation and where it occurs. B cells produce antibodies in response to a specific invaders’ antigens. These antibodies help fight the invader at that time as well as in the future. Scientists have not understood how these B cells get exposed to the invader’s antigens if macrophages and dendritic cells are constantly destroying the invaders.

The researchers studied these immune interactions in a living mouse’s lymph nodes--where invaders are brought to be destroyed by macrophages and dendritic cells. Intravital and multiphoton microscopy images revealed highly specialized 'subcapsular sinus (SCS) macrophages' embedded in the lining of the lymph nodes’ sinus. The images showed the heads of these macrophages capturing the antigen on one side of the lining and their tails delivering it to B cells on the other side of the lining. In other words, the intact antigen passes through the subcapsular sinus almost like it is on a conveyer belt. 

This finding helps to clarify other groups’ observations of unusual macrophages in the subcapsular sinus and B cells residing nearby, and it clarifies how and where the B cells become activated. View videos of findings are here.

Research paper: Tri Giang Phan, Jesse A Green, Elizabeth E Gray, Ying Xu & Jason G Cyster, Nature Immunology, Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation. Published online: June 7, 2009, doi:10.1038/ni.1745.

Image courtesy of the U.S. National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) Program.

New fluorescent protein constructs for labeling organelles

Organelle Lights reagents from Molecular Probes (Invitrogen) now come in new colors for labeling lysosomes, mitochondria, endoplasmic reticulum (ER), and endosomes (Lyosomes-GFP, Mito-RFP, ER-RFP, and endosomes-RFP). The prepackaged fluorescent protein constructs are fused with signal peptides and come ready-to-use. The reagents consist of suspensions of baculovirus carrying an expression construct with a fluorescent protein fused to either a cellular protein or a localization peptide. To use them for imaging, add the virus suspension to your cells and incubate overnight. The reagents can be used with other stains in live or fixed cells and let you avoid potentially harmful cell treatments.

More information on this product line here. 

Microscopy for high content analysis

GE Healthcare has introduced the IN Cell Analyzer 2000 cell imaging system for high content analysis. Although this isn’t a traditional microscope, you’ll find a microscope inside the box. The system can handle a variety of experiments, acquiring images fast enough for screening applications while including tools for investigative microscopy. It can image organelles, cells, tissues, and whole organisms. Its ability to capture an entire well in one image makes it easier to catch rare events. The system includes objectives ranging from 2X-100X and six imaging restoration modes. A manual microscope mode lets you see the real time effects of adjusting settings, and a selected sample area can be quickly previewed at any available magnification before starting image acquisition. 
The instrument is being shown at the Society for Biomolecular Sciences 15th Annual Conference and Exhibition in Lille, France this week. 

More information here. 

Sit back and watch cells dance

Make sure you turn up the volume when you watch the cells dance on this video from Dr. Simon Vipoir of Improvision (a PerkinElmer company).  You'll see cell types from many different areas of life science including neurones, neutrophils, liver cells and islets, T cells, skin cells, whole maggots, and drosophila embryos. 

The images were acquired and/or analysed using Improvision (a PerkinElmer company) imaging products. You may know this company's Volocity 3-D (and 4-D) imaging software. 

You can see more by subscribing to Dr. Vipoir's channel on YouTube. More information on the software is here. 

Invitrogen releases new tools for imaging biofilms and cells

Imaging biofilms
Biofilms can be difficult to image, but the FilmTracer fluorescent stains from Invitogren may help. These stains are optimized and validated specifically for imaging biofilms, and the product line includes various stains for imaging different components of a biofilm. They can be used with biofilm samples grown in the lab or collected from the environment (rocks, pipes, or catheters). Combine them with confocal laser scanning microscopy for the best 3-D images.

Alexa Fluor 488 conjugated primary antibodies
Want to study tight junctions and the cytoskeleton but tired of the cost and extra time involved in secondary antibody detection? Invitrogen has eliminated that extra step with Alexa Fluor 488 conjugates of antibodies against occludin, claudin-1, claudin-4, claudin-5, and α–tubulin. 

More info here.