BioOptics BreakthRoughs

DNA sequencing finds virus responsible for transplant deaths

In the first application of high-throughput DNA sequencing technology to investigate an infectious disease outbreak, scientists from Columbia University Mailman School of Public Health, the Victorian Infectious Diseases Reference Laboratory (Melbourne, Australia), the Centers for Disease Control, and 454 Life Sciences link the discovery of a new arenavirus to the deaths of three transplant recipients who received organs from a single donor in Victoria, Australia, in April 2007.

After failing to implicate an agent using other methods, RNA from the transplanted liver and kidneys was analyzed using rapid sequencing technology established by 454 Life Sciences (the Genome Sequencer FLX System, which utilizes a CCD camera in its optical subsystem) and bioinformatics algorithms developed at Columbia. Examination of tens or thousands of sequences yielded 14 that resembled arenaviruses at the protein level. The team then cultured the virus, characterized it by electron microscopy, and developed specific molecular and antibody assays for infection. The presence of virus in multiple organs, IgM antibodies in the organ donor, and increasing titer of antibody in a recipient were used to implicate the virus. The arenavirus lymphocytic choriomeningitis virus has been implicated in a small number of cases of disease transmission by organ transplantation; however, the newly discovered virus is sufficiently different that it could not be detected using existing screening methods.

Photoacoustics improve tumor and disease detection and treatment

Detection and treatment of tumors, diseased blood vessels, and other soft-tissue conditions could be significantly improved, thanks to a photoacoustic imaging system being developed at University College London (UCL), with funding from the Engineering and Physical Sciences Research Council (EPSRC). The system uses extremely short pulses of low-energy laser light to stimulate the emission of ultrasonic acoustic waves (1 to 50 MHz) from the tissue area being examined (see figure). These waves are then converted into high-resolution 3-D images of tissue structure.

This method can be used to reveal disease in types of tissue that are more difficult to image using techniques based on x-rays or conventional ultrasound. For example, the prototype instrument has been specifically designed to image very small blood vessels (with diameters measured in tens or hundreds of microns) relatively close to the surface. Information generated about the distribution and density of these microvessels can in turn provide valuable data about skin tumors, vascular lesions, burns, other soft tissue damage, and even how well an area of tissue has responded to plastic surgery following an operation.

“This new system offers the prospect of safe, noninvasive medical imaging of unprecedented quality,” says Paul Beard, who leads UCL’s Photoacoustic Imaging Group. “It also has the potential to be an extremely versatile, relatively inexpensive, and even portable imaging option.”

Multiphoton microscopy aids development of antivirus vaccines 

Scientists from the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH; Bethesda, MD), are using multiphoton microscopy to gain a better understanding of how the immune system operates during a viral infection. Focusing on mouse lymph nodes, the researchers discovered that immune cells confront viruses just inside of the lymph node and not deep within these organs as previously thought. The study, led by Jonathan Yewdell, chief of the NIAID Cellular Biology Section and his NIAID colleague, Heather Hickman, is described in a report online in Nature Immunology (Jan. 13, 2008).

The results are significant, the authors say, because they observed in detail the interaction of viruses and immune cells inside a living organism. Combining expertise from disciplines such as imaging, immunology, virology and other specialties, the scientists first extracted and then purified “killer” T cells from mice. The scientists labeled the T cells with a fluorescent marker, injected them back into the mice, and then infected the animals with vaccinia virus, the virus used to make smallpox vaccine, engineered to express a brilliantly colored protein.

Using a multiphoton microscope, the scientists were able to look into the lymph nodes of the infected mice and see that the viruses had infected cells just inside the lymph-node surface, triggering a swarm of T cells. These virus-specific T cells form an elaborate and dynamic communications network that activates them to divide and travel to the site of viral infection, where they kill virus-infected cells. According to the NIAID team, pinpointing where in the lymph-node immune cells fight the virus should help efforts to design effective antivirus vaccines.

Frequency-comb spectroscopy detects disease via breath analysis

A team of scientists at JILA, a joint institute of the National Institute of Standards and Technology (NIST; Boulder, CO) and the University of Colorado at Boulder, has found that cavity-enhanced direct optical-frequency-comb spectroscopy may one day allow doctors to screen people for certain diseases simply by sampling their breath. According to Jun Ye, who led the research, optical-comb spectroscopy is powerful enough to sort through all the molecules in human breath but is also sensitive enough to find rare molecules that may be markers of specific diseases. Just as bad breath may indicate dental problems, excess methylamine can be used to detect liver and kidney disease, ammonia on the breath may be a sign of renal failure, elevated acetone levels in the breath can indicate diabetes, and nitric oxide levels can be used to diagnose asthma. When many breath molecules are detected simultaneously, highly reliable and disease-specific information can be collected.

In the experiments performed by Ye and his colleagues, optical-frequency-comb spectroscopy was used to analyze the breath of several student volunteers. The researchers showed that they could detect trace signatures of gasses like ammonia, carbon monoxide, and methane on the students’ breath. In one of these measurements, they detected carbon monoxide in a student smoker and found that it was five times higher when compared to a nonsmoking student.

TERS enables genetic code to be read directly from RNA

Volker Deckert and his team at the Institute for Analytical Sciences (Dortmund, Germany) have developed a method that could provide a way to directly sequence DNA. Their process, tip-enhanced Raman spectroscopy (TERS), is based on a combination of Raman spectroscopy and atomic-force microscopy. The researchers successfully analyzed DNA’s closest relative, RNA.

In direct sequencing the letters of the genetic code are read directly, as if with a magnifying glass. A DNA or RNA strand has a diameter of only 2 nm, so powerful magnification is required. An atomic-force microscope achieves this degree of magnification. Steered by the microscope, a tiny, silvered glass tip moves over the RNA strand. A laser beam focused on the tip excites the section of the strand being examined and starts it vibrating. The information can be used to derive the sequence of the RNA. If this method can be extended to DNA, it could revolutionize the decoding of genetic information.

3-D stability analysis predicts optofluidic interactions

Building on previous work in optofluidics using polymer waveguides, engineers from Cornell University (Ithaca, NY) have devised a comprehensive model for particle-trapping stability analysis of dielectric particles in an evanescent field of low-index and high-index solid-core waveguide structures integrated with microfluidics (see www.laserfocusworld.com/articles/314424). Their approach, which is outlined in detail in Nanotechnology (January 2008), could have important implications for the design of lab-on-a-chip devices.

Models for predicting propulsive velocities and trapping forces within a static fluidic environment are not new. However, developing a practical optofluidic transport system requires understanding the conditions that bring a particle to a waveguide trap and remain stably trapped within the evanescent field. David Erickson and colleagues in Cornell’s School of Chemical and Biomolecular Engineering and Sibley School of Mechanical and Aerospace Engineering used three-dimensional finite-element-based simulations to determine the electromagnetic and hydrodynamic field variables for two different waveguide systems: silicon (1550 nm) and polymer (1064 nm). A trapping stability number was obtained by comparing the work required to remove a particle from the waveguide with available random thermal energy. These forces were then correlated to controllable experimental parameters such as particle size, fluid velocity, and channel height and a series of trapping stability diagrams was produced, detailing the conditions under which optofluidic transport is possible.

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