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Dual-Color Auto-Calibration Scanning-Angle Evanescent Field Microscope

Ames Laboratory

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Technology Marketing Summary

Iowa State University and Ames Laboratory researchers have developed a new microscope that can be used for live cell imaging as well as for examining single molecule dynamics.  The new microscope with an automatic high-precision calibration procedure has been tested under laboratory conditions and is available for demonstration. The entire auto calibration procedure is demonstrated to be complete within minutes and incident angles in the full range (from subcritical angles to nearly 90º) with intervals as small as 0.02º were identified.


Total internal reflection fluorescence microscopy (TIRFM) is a mode of fluorescence microscopy that has been widely used for live-cell imaging at the interface between a biological sample and a cover slip or tissue culture well.  TIRFM is based on the induction of an evanescent wave in the liquid adjacent to the interface, which is created when reflected light penetrates the interface, propagates parallel to the surface of the plane of incidence, and decays exponentially with distance.  There are two basic TIRFM systems: prism-based and objective based.  Prism-based systems are preferable since they have lower costs, wider range of incident angles, less excitation light scattering, and higher accuracy in the incident angle determination.  However, the prism-based method has geographical constraints on sample manipulation–it is difficult to recalibrate the system manually for all incident angles–and because image reconstruction can be difficult.   To overcome these drawbacks, ISU and Ames Laboratory researchers have developed an innovative dual-color auto-calibration scanning-angle evanescent field microscope that is easier to operate and more reproducible than existing approaches.  This microscope has utility for live-cell imaging to examine cellular organization and dynamic processes that occur in the  cell/ substrate contact regions.  A computer-controlled automatic high-precision calibration procedure is used to find the incident angles in the full range, and this microscope is able to achieve better axial resolution than currently available systems.

• Permits high axial resolution (5-10 nm)
• Provides quick and automatic creation of an evanescent field for any incident angle in the full range
• Enables dual-color auto-calibration and scanning capability
• Enables dual-color auto-calibration and scanning capability
• Allows rapid re-calibration of new samples
• Enables fine adjustment of the optical trapping forces created by the evanescent field


More Information

This is a commercial-ready technology.  More information available at

Patents and Patent Applications
ID Number
Title and Abstract
Primary Lab
Patent 9,012,872
Auto-calibrated scanning-angle prism-type total internal reflection microscopy for nanometer-precision axial position determination and optional variable-illumination-depth pseudo total internal reflection microscopy
A method, apparatus, and system for improved VA-TIRFM microscopy. The method comprises automatically controlled calibration of one or more laser sources by precise control of presentation of each laser relative a sample for small incremental changes of incident angle over a range of critical TIR angles. The calibration then allows precise scanning of the sample for any of those calibrated angles for higher and more accurate resolution, and better reconstruction of the scans for super resolution reconstruction of the sample. Optionally the system can be controlled for incident angles of the excitation laser at sub-critical angles for pseudo TIRFM. Optionally both above-critical angle and sub critical angle measurements can be accomplished with the same system.
Technology Status
Technology IDDevelopment StageAvailabilityPublishedLast Updated

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To: Jay Bjerke<>