Method of generating high temporal and spatial resolution images using scanning microscopes


University of Nevada, Reno researchers Nelson Publicover and John Sutko in the Department of Electrical and Biomedical Engineering and the Department of Pharmacology, respectively, conduct research in the biomedical and medical fields. Their novel method relates to data generating and image containing the temporal and spatial locations of “discrete events” associated with a specimen.

Technology Summary
In conventional methods for pixel-based scanning/sampling systems and image formation, “discrete events” (such as photons or other units of radiant energy) are detected on a specimen, and their counts subsequently binned (accumulated and summed as a group) during each sampling period, and corresponding to a pixel location of an image display. During the pixel sampling method, information is typically lost during the binning process resulting in more discrete events typically detected than would be necessary if the system did not lose spatial and temporal information for individual discrete events. Conventional imaging systems face constraints in scan rate with regard to the quality of the output signal produced; an increased scan rate means fewer photons per pixel per scan are accumulated, which leads to a decrease in the intensity of the pixels and their signal-to-noise ratios. Also an issue, because existing techniques are optically based, they have an inherent limit of resolution known as a diffraction limit. Light passing through a lens to illuminate the sample interferes with itself and creates a ring-shaped diffraction pattern that blurs the image.

Over-illumination is also a major limitation of conventional photon sampling and imaging methods; many fluorescent molecules can only fluoresce a limited number of times, and result in eventual excitation radiation cessation, known as photo-bleaching. When using live samples, emission of photons from the tissue can damage cells, with over-illumination resulting in photo-toxicity.

This novel method applies to any scanned sample that emits photons or other discrete events in response to scanning during a scan period, resulting in an image. The discrete events detected are associated with the location of the origin of the individual events, and are acquired with reference to a scan frame that may be defined as a single instance of a scan pattern. The x- and y-positions can be obtained from position signals indicating the specific site in, or on, the specimen at the exact instant that the event is detected, or can be obtained from time-based signals that indicate the location of the imaged sites. The positions acquired can be stored and viewed statically on different time scales and used for post-hoc processing. This allows for further analysis of the specimen even after it has become either unavailable or unresponsive to further excitation radiation due to photo-bleaching or photo-toxicity. Since multiple discrete events are acquired and statistical approaches used to determine spatial locations of event clusters, measurement is not limited by the diffraction limit, and finer resolution can be achieved than conventional imaging apparatuses.

One feature of the method is the use of probability-density functions (PDF) to form an image, which describe the likelihood that a detected photon or event came from a region in the vicinity of its actual position. Compared to pixel-based images, photon event distribution sampling images display increased signal-to-noise ratios and comparable spatial resolution, and are superior to pixel-based image formation in recognizing the presence of structured (non-random) event distributions at low photon or event counts. As more events are detected, the PDFs will begin to narrow, increasing the certainty and precision of the localization of the site of the origin of clustered events in or on the specimen. Because the PDFs are summed, fewer discrete events are required to associate a distribution with an image feature than conventional pixel-based binning methods. With fewer discrete events necessary, sensitivity of measurement can be improved since less excitation illumination of a specimen is necessary to produce the discrete events. This can eliminate or reduce the over-irradiation of the specimen.

Potential Applications

Microscopy specific applications

  • Single- or multi-photon excitation confocal scanning microscope systems.
  • Near-field scanning optical microscopy (NSOM).
  • Electron microscopy techniques for living biological specimens. For example, the measurement of the release of calcium ion, Ca2+, from intracellular sarcoplasmic reticulum (SR) stores in cardiac cells.
  • Electromagnetic radiation detected using optical or non-optical techniques.
  • The retrofitting of existing commercial/custom-built optical systems

Many other imaging applications are also covered by the patents

UNR is seeking expressions of interest from parties interested in collaborative research to further develop, evaluate, or commercialize this technology.

IP Status
UNR ID#: UNR04-022
Photon Event Distribution Sampling Apparatus and Method
US Patent No.: 7,960,702; 8,031,926; and 8,168,955

Publication: Photon event distribution sampling: an image formation technique for scanning microscopes that permits tracking of sub-diffraction particles with high spatial and temporal resolutions.


Patent Information:
For Information, Contact:
Shannon Sheehan
Manager, Technology Commercialization
University of Nevada, Reno
John Sutko
Nelson Publicover
Joshua Larkin