Ultrahigh-resolution imaging of biological systems based on standing-wave total internal reflection microscopy

Peter T. C. So

doi:10.1117/12.426783

10 May 2001 Ultrahigh-resolution imaging of biological systems based on standing-wave total internal reflection microscopy

Peter T. C. So

Author Affiliations +

Proceedings Volume 4260, Optical Diagnostics of Living Cells IV; (2001) https://doi.org/10.1117/12.426783
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

Light microscopy is widely used in biomedical research. Its power derives from the ability to study living biological systems on the microscopic scale. Since its inception over three centuries ago, there has been little improvement in the lateral resolution of optical microscopy. Today, electron microscopy is one of the few methods capable of imaging on length scales below 100nm. Although electron microscopy is a powerful high resolution imaging technique, it is restricted to study fixed specimens. Clearly, there is a need for developing an in vivo optical technique with significantly improved lateral resolution. We have recently developed a new technique, Standing Wave Total Internal Reflection Microscopy (SW-TIRM), that can reach a resolution of about 50 nm. However, the theoretical basis for resolution enhancement in standing-wave total internal reflection microscopy has not been completely examined. SW- TIRM technique relies on the formation of an excitation field containing super-diffraction limited spatial frequency components. While the fluorescence generated at the object planes contains high frequency information of the object distribution, this information is lost at the image plane where detection optics act as a low pass filter. From the perspectives of point spread function engineering, one can show that if this excitation field is translatable experimentally, the high frequency information can be extracted from a set of images where the excitation fields have different displacement vectors. We have developed algorithms for this task that combine this image set to generate a composite image with an effective point spread function that is equal to the product of the excitation field and the Fraunhofer point spread function.

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Peter T. C. So "Ultrahigh-resolution imaging of biological systems based on standing-wave total internal reflection microscopy", Proc. SPIE 4260, Optical Diagnostics of Living Cells IV, (10 May 2001); https://doi.org/10.1117/12.426783

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KEYWORDS

Point spread functions

Image resolution

Microscopy

Composites

Imaging systems

Optical microscopy

Algorithm development

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