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Self-Interference Fluorescence Microscopy

Members: Oguzhan Avci, Anna K. Swan, M. Selim Ünlü

 

Alumni: Irsadi Aksun, Julia Lemoine Barsky, Katherine Calabro, Brynmor J Davis, Mehmet Dogan, Aysun Gokoglu, Bennett B. Goldberg, Joseph Thomas Greenspun, Stephen Bradley Ippolito, Lev Moiseev, Margo R. Monroe, Alexander Robert Mott, Raviv Perahia, Tane Remington, Joshua D. Spitzberg, Philipp Stefan Spuhler, Ayca Yalcin, Xirui Zhang

 

Principles of Spectral Self Interference

For many years scientists have recognized that fluorophores are often quenched when they are placed upon surfaces. Mechanisms of energy transfer, standing wave nodes in the excitation field, and destructive interference in the emission all lead to significant reductions in fluorescence. Ten years ago, Fromherz and co-workers noted that the intensity of the total fluorescence oscillates as a function of the fluorophore height above a reflecting substrate. Building upon work performed many years ago by Drexhage they utilized a convolution of the excitation standing wave and emission interference to sensitively locate the vertical position of the fluorophore in close proximity to a reflecting surface. Because the fluorophore is within ~λ of the reflecting surface, the entire spectrum of the emission is quenched or enhanced as the light undergoes constructive and destructive interference as a function of the vertical distance. Fromherz's method is based on measuring the intensity of the fluorophore for several known silicon dioxide heights in order to determine the height of the fluorophore above the dioxide layer.

Spectral self-interference fluorescence microscopy is different in that the separation between the fluorophore and the reflecting substrate is much greater, on the order of 10-15 wavelengths. The longer path length difference between the direct and reflected light means only a small change in wavelength is needed to go between constructive and destructive interference. The result is spectral oscillations, or fringes, in the spectrum - a unique signature of the height of the emitter. Small height differences produce shifts in the fringes and changes in the period of oscillation (the latter are less noticeable). If the fluorescent markers are at a prescribed distance from the surface, the resulting spectrum can be calculated. Inversely, the distance above the mirror can be determined solely from the oscillations within the spectrum. Unlike fluorescence interference-contrast microscopy, it should be noted that as the height information is encoded in the spectrum, our approach is independent of the fluorophore density, emission intensity, and the excitation field strength.

Green's Function Formalism

In order to analyze the spectral oscillations and to be able to determine the orientation and vertical position of a fluorophore, the radiation pattern of the fluorophore should be known. We are implementing an accurate and reliable way of finding the radiation intensity of an emitter by using Green's functions for the stratified media. In this formalism, the electric field Green's function is written in terms of vector potential Green's functions and scalar potential Green's functions. Traditionally, these functions are represented by the Sommerfeld integrals in the spatial domain, and by closed-form expressions in the spectral domain. Green's functions in spectral domain can be found analytically and spatial domain Green's functions are written in terms of the spectral domain Green's functions using the Sommerfeld integral:

where G and are Green's functions in the spatial and spectral domains respectively, H0(2) is the Hankel function of the second kind, and SIP is the Sommerfeld integration path. The above Sommerfeld integral cannot be integrated easily to find the spatial domain Green's function. However, when the spectral domain Green's functions are approximated in terms of complex exponentials using a technique called generalized pencil of function (GPOF) method, the analytical evaluation of the Sommerfeld integral is possible by the Sommerfeld identity:

As a final step, electric field is written in terms of spatial Green's functions and the radiation intensity is found using the electric field at any observation point. In this formalism, the information about the reflections from the layer interfaces is encoded in the spectral domain Green's functions that depend on the geometry and optical properties of the medium. This method is a reliable and accurate method as all the plane wave components originating from the source is taken into consideration by the nature of the formalism.

Measurement of DNA conformation and orientation using SSFM

The SSFM technique maps the spectral oscillation by a fluorophore located on a layered reflection surface into a precise position determination. Analysis of the spectral oscillations (using a grating spectrometer) due to the self-interference from the direct and reflected emission yields the vertical position of that fluorophore with sub-nanometer accuracy. Assuming known orientation, e.g. randomly oriented DNA anchored to the oxide surface, it is possible to induce conformation off the DNA (single stranded vs. double stranded) on the surface based on the fluorophore – surface distance. Assuming known conformation, e.g. double stranded DNA with contour lengths shorter than the persistence length, it is possible to induce the orientation of the DNA immobilized on the surface. (Moiseev PNAS 2005). SSFM is also useful for nanometer scale characterization of other molecules, for example the characterization of a surface bound polymer or the different binding locations and orientations of DNA to a 3D binding platform (Yalcin Anal Chem, 2009).

Fig. 1: Example of SSFM Measurement Response and Setup

Fig. 2: Image Copyright 2009 ACS Publications

Publications

E. Cevik, G. G. Daaboul, X. Zhang, S. M. Scherr, N. Lortlar Ünlü, J. H. Connor, and M. S. Ünlü, "DNA-Directed Antibody Immobilization for Enhanced Detection of Single Viral Pathogens," Analytical Chemistry, Vol. 87, No. 20, October 2015, pp. 10505-10512, DOI:10.1021/acs.analchem.5b02702

X. Zhang, G. G. Daaboul, P. S. Spuhler, D. S. Freedman, A. Yurt, S. Ahn, O. Avci, and M. S. Ünlü, "Nanoscale Characterization of DNA Conformation Using Dual-color Fluorescence Axial Localization and Label-free Biosensing," Analyst, 13 October 2014

X. Zhang, "Dual-spectral Interferometric Sensor for Quantitative Study of Protein-DNA Interactions," Ph.D. Dissertation, August 2014

X. Zhang, P. S. Spuhler, D. S. Freedman, and M. S. Ünlü, "Spectral Self-Interference Fluorescence Microscopy to Study Conformation of Biomolecules with Nanometer Accuracy," Nanoscale Spectroscopy with Applications, November 2013

X. Zhang, G. G. Daaboul, P. S. Spuhler, D. S. Freedman, A. Yurt, S. Ahn, and M. S. Ünlü, "Quantification of DNA hybridization by nanoscale axial localization and label-free biosensing," BMES 2013 Annual Meeting , 22-25 October 2013

S. Ahn, D. D. Sevenler, and M. R. Monroe, "Biosensors - Innovations in nanobiosensing and biophotonics at Boston University," Boston University, August 2013

X. Zhang, P. S. Spuhler, L. Sola, M. Chiari, and M. S. Ünlü, "A high speed micro-arrayed biosensing platform for the detection of specific DNA-protein interactions," Biosensors, 2012, Cancun, Mexico, 15-18 May 2012

G. di Carlo, F. Damin, M. S. Ünlü, P. S. Spuhler, and M. Chiari, "Synthesis and conformational characterization of functional di-block copolymer brushes for microarray technology," Surface Science, Vol. 258, 1 February 2012, pp. 3750-3756

P. S. Spuhler, "HIGH-THROUGHPUT DETECTION OF DNA ORIENTATION AND CONFORMATION FOR CHARACTERIZATION OF PROTEIN-DNA INTERACTIONS ," Ph.D. Dissertation, October 2011

P. S. Spuhler, L. Sola, X. Zhang, M. R. Monroe, J. T. Greenspun, M. Chiari, and M. S. Ünlü, "Precise Control of DNA Orientation for Improved Functionality in Protein Binding Microarrays," International Conference on Optical MEMS & Nanophotonics, August 2011

X. Zhang, P. S. Spuhler, L. Sola, M. S. Ünlü, and M. Chiari, "A high-throughput biosensor for detection and quantification of protein induced DNA bending," International Conference on Optical MEMS & Nanophotonics, 8 August 2011, pp. 229-230

M. Dogan, I. Aksun, A. K. Swan, B. B. Goldberg, and M. S. Ünlü, "Closed-form representations of field components of fluorescent emitters in layered media," Journal of the Optical Society of America A, Vol. 26, No. 6, June 2009, pp. 1458-1466

A. Yalcin, F. Damin, E. Ozkumur, G. di Carlo, L. Sola, M. S. Ünlü, and M. Chiari, "Nanoscale Determination of a Polymeric Coating for Microarray Applications," 23rd International Symposium on MicroScale Bioseparations (MSB) 2009, February 2009

A. Yalcin, E. Ozkumur, B. B. Goldberg, and M. S. Ünlü, "High lateral resolution spectral self-interference fluorescence microscopy using annular apertures," Photonics West 2009 - BIOS, January 2009

M. Dogan, "Interference Techniques in Fluorescence Microscopy," Ph.D. Dissertation, January 2009

A. Yalcin, F. Damin, E. Ozkumur, G. di Carlo, B. B. Goldberg, M. Chiari, and M. S. Ünlü, "Direct Observation of Conformation of a Polymeric Coating with Implications in Microarray Applications," Analytical Chemistry, Vol. 81, 2009, pp. 625-630

A. Yalcin, F. Damin, E. Ozkumur, G. di Carlo, B. B. Goldberg, M. Chiari, and M. S. Ünlü, "Nanoscale Determination of Conformation of a Polymeric Coating on Layered Surfaces," AVS 55th International Symposium and Exhibition, October 2008

J. R. Dupuis, and M. S. Ünlü, "Time-Domain Surface Profile Imaging via HS-FTS," Optics Letters, Vol. 33, June 2008, pp. 1368-1370

J. R. Dupuis, J. Needham, E. Ozkumur, D. A. Bergstein, B. B. Goldberg, J. R. Engel, D. L. Carlson, and M. S. Ünlü, "Hyperspectral Fourier transform spectrometer for reflection spectroscopy and spectral self-interference fluorescence microscopy," Applied Optics, Vol. 47, 20 March 2008, pp. 1223-1234

M. Dogan, A. Yalcin, S. Jain, M. B. Goldberg, A. K. Swan, M. S. Ünlü, and B. B. Goldberg, "Spectral Self-Interference Fluorescence Microscopy for Subcellular Imaging," IEEE Journal of Selected Topics in Quantum Electronics, Vol. 14, No. 1, January/February 2008

A. Yalcin, F. Damin, E. Ozkumur, G. di Carlo, B. B. Goldberg, M. S. Ünlü, and M. Chiari, "Characterization of A Polymeric Coating for Microarray Applications using Spectral Self-Interference Fluorescence Microscopy," Photonics West 2008 - BIOS, 2008

M. S. Ünlü, E. Ozkumur, J. Needham, D. A. Bergstein, B. B. Goldberg, A. Yalcin, P. S. Spuhler, R. Irani, and C. DeLisi, "Applications of Optical Resonance to Biological Imaging and Label-free Protein Microarrays (invited paper)," EMBC08, Vancouver, Canada, August 20-24, 2008, 2008

M. Dogan, B. B. Goldberg, S. Jain, M. B. Goldberg, A. K. Swan, and M. S. Ünlü, "Probing Bacterial Surfaces Using 4Pi Spectral Self-interference Fluorescence Microscopy," Proceedings of CLEO/QELS 2008, 2008

M. Dogan, A. K. Swan, M. S. Ünlü, and B. B. Goldberg, "Nanometer Scale Axial Localization of Fluorescent Emitters for Cellular Imaging," Proceedings of IEEE Lasers and Electro-Optics Society 2007 Annual Meeting, October 2007

A. Yalcin, F. Damin, E. Ozkumur, G. di Carlo, B. B. Goldberg, M. Chiari, and M. S. Ünlü, "Characterization of Swelling of A Polymeric Coating for DNA Microarray Applications Using Spectral Self-Interference Fluorescence Microscopy," Proceedings of IEEE Lasers and Electro-Optics Society 2007 Annual Meeting, October 2007

B. J. Davis, P. S. Carney, A. K. Swan, M. S. Ünlü, W. C. Karl, and B. B. Goldberg, "Fluorescence Imaging With Nanometer Precision Using Spectral Self-Interference Microscopy ," 2007 IEEE ICONIC Conference, 27 June 2007

A. N. Vamivakas, S. B. Ippolito, A. K. Swan, M. S. Ünlü, M. Dogan, E. R. Behringer, and B. B. Goldberg, "Phase-sensitive detection of dipole radiation in a fiber-based high numerical aperture optical system ," Optics Letters, Vol. 32, No. 8, 15 April 2007, pp. 970-972

M. Dogan, P. Dröge, A. K. Swan, M. S. Ünlü, and B. B. Goldberg, "Probing DNA-IHF Interactions on Surfaces Using Optical Interference Techniques," Bulletin of APS Meeting, March 2007

B. J. Davis, A. K. Swan, M. S. Ünlü, W. C. Karl, B. B. Goldberg, J. C. Schotland, and P. S. Carney, "Spectral self-interference microscopy for low-signal nanoscale axial imaging," Journal of the Optical Society of America, Vol. 24, 2007, pp. 3587-3599

B. J. Davis, M. Dogan, B. B. Goldberg, W. C. Karl, M. S. Ünlü, and A. K. Swan, "4Pi spectral self-interference microscopy," Journal of the Optical Society of America A, Vol. 24, 2007, pp. 3762-3771

M. Dogan, B. B. Goldberg, A. K. Swan, and M. S. Ünlü, "4Pi Spectral Self-interference Fluorescence Microscopy," OSA Frontiers in Optics 2006/Laser Science XXII, Rochester, New York, October 8-12, 2006., 8 October 2006

M. S. Ünlü, and B. B. Goldberg, "Applications of Optical Resonance to Biological Sensing and Imaging (invited)," OSA Frontiers in Optics 2006/Laser Science XXII, Rochester, New York, October 8-12, 2006., 8-12 October 2006

B. J. Davis, "Analysis of Multi-Channel Microscopy: Spectral Self-Interference, Multi-Detector Confocal and 4Pi Systems," Ph.D. Dissertation, May 2006

L. Moiseev, M. S. Ünlü, A. K. Swan, B. B. Goldberg, and C. R. Cantor, "DNA Conformation on Surfaces Measured by Fluorescence Self-Interference," Proceedings of the National Academy of Science, Vol. 103, 21 February 2006, pp. 2623-2628

M. S. Ünlü, and B. B. Goldberg, "Applications of Optical Resonance to Biological Sensing and Imaging (invited)," OSA Frontiers in Optics 2006/Laser Science XXII, Rochester, New York, October 8-12, 2006., 2006

M. S. Ünlü, "Applications of microresonators: from photodetectors to biological sensing and imaging (Keynote Presentation)," European Optical Society Topical Meeting: Optical Microsystems, 15 September 2005

B. J. Davis, W. C. Karl, B. B. Goldberg, A. K. Swan, and M. S. Ünlü, "Using out-of-focus light to improve image acquistion time in confocal microscopy," Proceedings of SPIE: Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XII, Vol. 5701, March 2005

L. Moiseev, A. K. Swan, M. S. Ünlü, B. B. Goldberg, and C. R. Cantor, "Spectral Self-Interference on Fluorescently Labeled DNA Monolayers," Proceedings of IEEE Lasers and Electro-Optics Society 2004 Annual Meeting, November 2004

L. Moiseev, C. R. Cantor, I. Aksun, M. Dogan, B. B. Goldberg, A. K. Swan, and M. S. Ünlü, "Spectral self-interference fluorescence microscopy," Journal of Applied Physics, Vol. 96, No. 9, 1 November 2004, pp. 5311-5315

B. J. Davis, W. C. Karl, A. K. Swan, M. S. Ünlü, and B. B. Goldberg, "Capabilities and limitations of pupil-plane filters for superresolution and image enhancement," Optics Express, Vol. 12, No. 17, August 2004, pp. 4150

B. J. Davis, W. C. Karl, B. B. Goldberg, A. K. Swan, and M. S. Ünlü, "Sampling below the Nyquist rate in interferometric fluorescence microscopy with multi-wavelength measurements to remove aliasing," Proceedings of IEEE 11th Digital Signal Processing Workshop 2004, August 2004

B. J. Davis, W. C. Karl, A. K. Swan, B. B. Goldberg, M. S. Ünlü, and M. B. Goldberg, "Reconstruction of objects with a limited number of non-zero components in fluorescence microscopy," Proceedings of SPIE: Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XI, Vol. 5324, July 2004

B. B. Goldberg, A. K. Swan, L. Moiseev, M. Dogan, W. C. Karl, B. J. Davis, C. R. Cantor, S. B. Ippolito, S. A. Thorne, M. G. Eraslan, Z. Liu, M. B. Goldberg, and Y. Leblebici, "Seeing inside chips and cells: High-resolution subsurface imaging of integrated circuits, quantum dots and subcellular structures," Proceedings of CLEO/QELS 2004, May 2004

M. Dogan, I. Aksun, M. S. Ünlü, A. K. Swan, and B. B. Goldberg, "A novel analytical model for field calculations of arbitrary oriented radiating dipoles near stratified dielectric interfaces," Bulletin of APS Meeting, March 2004

L. Moiseev, C. R. Cantor, A. K. Swan, B. B. Goldberg, and M. S. Ünlü, "Biological applications of spectral self-interference," Proceedings of SPIE: Nanobiophotonics and Biomedical Applications, Vol. 5331, January 2004, pp. 36-43

D. J. Dean, and B. J. Korte, "A New Federal Institute Focuses on Biomedical Imaging & Bioengineering," Optics & Photonics News, October 2003, pp. 38-42

A. K. Swan, L. Moiseev, C. R. Cantor, B. J. Davis, S. B. Ippolito, W. C. Karl, B. B. Goldberg, and M. S. Ünlü, "Towards nanoscale optical resolution in fluorescence microscopy," IEEE Journal of Selected Topics in Quantum Electronics, Vol. 9, No. 2, March/April 2003, pp. 294-300

A. K. Swan, L. Moiseev, Y. Tong, S. H. Lipoff, W. C. Karl, B. B. Goldberg, and M. S. Ünlü, "High resolution spectral self-interference fluorescence microscopy," Proceedings of SPIE: Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX, Vol. 4621, May 2002, pp. 77-85

H. A. Jones-Bey, "Self-interference method yields sensitive spectral microscopy," Laser Focus World, March 2002

A. K. Swan, M. S. Ünlü, Y. Tong, B. B. Goldberg, L. Moiseev, and C. R. Cantor, "Self-Interference Fluorescent Emission Microscopy - 5nm Vertical Resolution," Post Conference Proceedings of CLEO, 6-11 May 2001, pp. 360-361


Collaborators

Prof. W. Clem Karl , Boston University, ECE Department
Prof. Irsadi Aksun , KOC University, Electrical and Electronics Engineering Department
Prof. Charles R. Cantor, Boston University, Department of Biomedical Engineering


Support

NSF Division of Biological Infrastructure Grant Ref # : DBI-0138425


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