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A New Generation, Fast 3D Fluorescence Microscope using Wavefront Coding Optics

Published online by Cambridge University Press:  02 July 2020

Carol J. Cogswell ,
Matthew R. Arnison ,
Edward R. Dowski ,
Sara C. Tuckert  and
W. Thomas Catheyt
Carol J. Cogswell
Affiliation:
School of Physics andUniversity of Sydney, NSW2006, Australia-, [email protected] Australian Key Centre for Microscopy and Microanalysis, University of Sydney, NSW2006, Australia-, [email protected]
Matthew R. Arnison
Affiliation:
School of Physics andUniversity of Sydney, NSW2006, Australia-, [email protected]
Edward R. Dowski
Affiliation:
Optoelectronic Computing Systems Center, University of Colorado, Boulder, CO
Sara C. Tuckert
Affiliation:
Optoelectronic Computing Systems Center, University of Colorado, Boulder, CO
W. Thomas Catheyt
Affiliation:
Optoelectronic Computing Systems Center, University of Colorado, Boulder, CO
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We are developing a “new-generation” fluorescence microscope that will allow very fast (milliseconds) acquisition of fully three-dimensional (3D) images for a wide spectrum of biological applications. This new system will overcome the slow image acquisition constraint of existing confocal and widefield deconvolution microscopes (the two most commonly used instruments for 3D fluorescence imaging) that has prevented them from being used for investigations of live-cell dynamics in three dimensions. Our new microscope incorporates the innovative techniques of optical wavefront coding, pioneered by W. T. Cathey and E. R. Dowski, University of Colorado. With this new system, as compared to the normal sequential plane-by-plane image acquisition requirement of confocal and widefield microscopes, we need acquire only a single CCD camera image to obtain an equivalent extended-depth-of-focus (EDF) rendering of a thick specimen, and a minimum of only two images for a 3D stereo view that has full depth.

Our microscope system uses a special-purpose optical element to uniformly “code” the information from all planes throughout the specimen volume onto a single CCD camera image. Specimen-independent digital processing is then used to “decode” this raw image. In effect, the coded raw image is blurred by a special type of aberration which produces an image that is nearly independent of focus. The system then uses a fast, non-iterative, digital filtering algorithm to remove this special blur so that a large volume of the specimen image appears sharply focused all at once.

Type
Recent Advances in Confocal Microscopy
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 466 - 467
Copyright
Copyright © Microscopy Society of America

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References

1)Dowski, E. R. and Cathey, W. T., Applied Optics 34 (1995) 1859.CrossRefGoogle Scholar
2)Wach, H. B., Dowski, E. R. and Cathey, W. T., Applied Optics 37 (1998)CrossRefGoogle Scholar
3)Bradburn, S. C., Dowski, E. R. and Cathey, W. T., Applied Optics 36 (1997) 9157.CrossRefGoogle Scholar