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Posts Tagged ‘Superreolution Microscopy’

Superresolution Microscopy

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Lens-Free 3-D Microscope Sharp Enough for Pathology

LOS ANGELES, Dec. 18, 2014 —

http://www.photonics.com/Article.aspx?AID=57019&refer=TechMicroscopy&utm_source=TechMicroscopy_2015_10_13

A computational lens-free, holographic on-chip microscope could provide a faster and cheaper means of diagnosing cancer and other diseases at the cellular level.

Developed by researchers at the University of California, Los Angeles, the compact system illuminates tissue or blood samples with a laser or LED, while a sensor records the pattern of shadows created by the sample.

The device processes these patterns as a series of holograms using the transport-of-intensity equation, multiheight iterative phase retrieval and rotational field transformations. Computer algorithms correct for imaging artifacts and enhance contrast in the reconstructed 3-D images, which can be focused to any depth within the field of view even after image capture.
A tissue sample image created by a new chip-based, lens-free microscope. Images courtesy of Aydogan Ozcan/UCLA.
With a field of view several hundred times larger than that of an optical microscope, the lens-free device could considerably speed up diagnostic imaging. It is also much smaller than conventional microscopes.

“While mobile health care has expanded rapidly with the growth of consumer electronics — cellphones in particular — pathology is still, by and large, constrained to advanced clinical laboratory settings,” said professor Dr. Aydogan Ozcan. “Accompanied by advances in its graphical user interface, this platform could scale up for use in clinical, biomedical, scientific, educational and citizen-science applications, among others.”

The researchers tested the device using Pap smears that indicated cervical cancer, tissue specimens containing cancerous breast cells and blood samples containing sickle cell anemia.

In a blind test, a board-certified pathologist analyzed sets of specimen images created by the lens-free technology and by conventional microscopes. The pathologist’s diagnoses using the lens-free microscopic images were accurate 99 percent of the time, the researchers said.

“By providing high-resolution images of large-area pathology samples with 3-D digital focus adjustment, lens-free on-chip microscopy can be useful in resource-limited and point-of-care settings,” the researchers wrote in Science Translational Medicine (doi: 10.1126/scitranslmed.3009850).

Funding for the project came from the Presidential Early Career Award for Scientists and Engineers, the National Science Foundation, the National Institutes of Health, the U.S. Army Research Office, the Office of Naval Research and the Howard Hughes Medical Institute.

Smartphone DNA measurements

Ozcan’s lab also recently demonstrated an optomechanical attachment that enables smartphones to perform fluorescence microscopy measurements on DNA.

The 3-D-printed device augments the phone’s camera by creating a high-contrast darkfield imaging setup with an inexpensive external lens, thin-film interference filters, a miniature dovetail stage and a laser diode for oblique excitation of fluorescent labels. The molecules are labeled and stretched on disposable chips that fit into the smartphone attachment.
A smartphone add-on enables imaging of DNA in the field.
The device also includes an app that transmits data to a server at UCLA, which measures the lengths of the individual DNA molecules.

This project was funded by the National Science Foundation. The results were published in ACS Nano (doi: 10.1021/nn505821y).

For more infromation, visit www.ucla.edu.

Expanded Tissues Show Confocal Microscopes More Detail

CAMBRIDGE, Mass., Jan. 26, 2015 —

http://www.photonics.com/Article.aspx?AID=57140&refer=TechMicroscopy&utm_source=TechMicroscopy_2015_10_13

Nobel Prize-winning superresolution microscopy techniques circumvent the diffraction limit of light to image the smallest details of cells. But there is another way.

Researchers at MIT have developed a method for making biological tissue samples physically larger, rendering their nanoscale features visible to conventional confocal microscopes.

Using inexpensive, commercially available chemicals and microscopes commonly found in research labs, the technique could give more scientists access to 3-D superresolution imaging.

“Instead of acquiring a new microscope to take images with nanoscale resolution, you can take the images on a regular microscope,” said MIT professor Dr. Edward Boyden. “You physically make the sample bigger, rather than trying to magnify the rays of light that are emitted by the sample.”

The process involves meshes of sodium polyacrylate, the superabsorbent chemical used in disposable diapers. When exposed to water, these meshes expand, and the cellular structures around them expand too.

Cells in rodent brain slices, as well as cells grown in vitro, are first fixed in formaldehyde and then gently stripped of their fatty membranes before being labeled with fluorescent markers.

A precursor is then added and heated to form the polyacrylate gel. Proteins that hold the specimen together are digested, allowing it to expand uniformly. Finally, the sample is washed in salt-free water to trigger the expansion.

Even though the proteins have been broken apart, the original location of each fluorescent label stays the same relative to the overall structure of the tissue because it is anchored to the polyacrylate gel by antibodies.
Scientists modified the superabsorbant diaper compound sodium polyacrylate to enlarge brain tissue and image it in 3-D using fluorescent tags and confocal microscopes. Courtesy of the Boyden Lab/MIT.
Samples were imaged before expansion using superresolution microscopes, and imaged afterward using confocal microscopes. Expansion gave the confocal microscopes an effective 70-nm lateral resolution — sharp enough to resolve details of the cell protein complexes, the spaces between rows of skeletal microtubule filaments and the two sides of synapses.

Trade-offs

The diffraction limit means standard microscopes can’t resolve objects smaller than about 250 nm. “Unfortunately, in biology that’s right where things get interesting,” Boyden said.

Three inventors of superresolution microscopy won the 2014 Nobel Prize in chemistry. Superresolution techniques, however, have their own limitation: They work best with small, thin samples, and take a long time to image large samples. They can also be hampered by optical scattering in thick samples.

“If you want to map the brain, or understand how cancer cells are organized in a metastasizing tumor, or how immune cells are configured in an autoimmune attack, you have to look at a large piece of tissue with nanoscale precision,” Boyden said.

The MIT technique allowed imaging of samples approximately 500 × 200 × 100 µm in volume.

“The other methods currently have better resolution, but are harder to use, or slower,” said graduate student Paul Tillberg. “The benefits of our method are the ease of use and, more importantly, compatibility with large volumes, which is challenging with existing technologies.”

Funding came from the National Institutes of Health, National Science Foundation, New York Stem Cell Foundation, Jeremy and Joyce Wertheimer and the Fannie and John Hertz Foundation.

The research was published in Science (doi: 10.1126/science.1260088).

For more information, visit www.mit.edu.

Microscope Takes 3-D Images From Inside Moving Subjects

NEW YORK, Jan. 19, 2015 —

http://www.photonics.com/Article.aspx?AID=57106&refer=TechMicroscopy&utm_source=TechMicroscopy_2015_10_13

A new kind of microscope enables rapid 3-D imaging of living and moving samples, potentially offering advantages over laser-scanning confocal, two-photon and light-sheet microscopy.

Developed by Columbia University professor Dr. Elizabeth Hillman and graduate student Matthew Bouchard, swept confocally aligned planar excitation (SCAPE) microscopy involves simplified equipment and does not require sample mounting or translation. The microscope scans a sheet of light through the sample, making it unnecessary to position the sample or the microscope’s single objective.

“The ability to perform real-time, 3-D imaging at cellular resolution in behaving organisms is a new frontier for biomedical and neuroscience research,” Hillman said. “With SCAPE, we can now image complex, living things, such as neurons firing in the rodent brain, crawling fruit fly larvae and single cells in the zebrafish heart while the heart is actually beating spontaneously.”
SCAPE yields data equivalent to conventional light-sheet microscopy, but using a single, stationary objective lens; no sample translation; and high-speed 3-D imaging. This unique configuration permitted volumetric imaging of cortical dendrites in the awake, behaving mouse brain. Courtesy of Elizabeth Hillman/Columbia Engineering.
Conventional light-sheet microscopes use two orthogonal objectives and require that samples be in a fixed position. Confocal and two-photon microscopes can image a single plane within a living sample, but cannot generate 3-D images quickly enough to capture events like neurons firing.

SCAPE does have one drawback: Using a 488-nm laser, it cannot penetrate tissue as deeply as two-photon microscopy.

The new technique could be combined with optogenetics and other tissue manipulations, the researchers said. It could also be used for imaging cellular replication, function and motion in intact tissues, 3-D cell cultures and engineered tissue constructs; as well as imaging 3-D dynamics in microfluidics and flow-cell cytometry systems.

Hillman next plans to explore clinical applications of SCAPE, such as video-rate 3-D microendoscopy and intrasurgical imaging.

Funding for the project came from the National Institutes of Health, Human Frontier Science Program, Wallace H. Coulter Foundation, Dana Foundation and the U.S. Department of Defense.

The research was published in Nature Photonics (doi:10.1038/nphoton.2014.323).

For more information, visit www.engineering.columbia.edu.

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