Nonlinear optical techniques for improved data capture in fluorescence microscopy and imaging

David S. Bradshaw; Jamie M. Leeder; David L. Andrews

doi:10.1117/12.840946

25 February 2010 Nonlinear optical techniques for improved data capture in fluorescence microscopy and imaging

David S. Bradshaw, Jamie M. Leeder, David L. Andrews

Proceedings Volume 7571, Single Molecule Spectroscopy and Imaging III; 75710B (2010) https://doi.org/10.1117/12.840946
Event: SPIE BiOS, 2010, San Francisco, California, United States

Abstract

Multiphoton fluorescence microscopy is now a well-established technique, currently attracting much interest across all fields of biophysics - especially with regard to enhanced focal resolution. The fundamental mechanism behind the technique, identified and understood through the application of quantum theory, reveals new optical polarization features that can be exploited to increase the information content of images from biological samples. In another development, based on a newly discovered, fundamentally related mechanism, it emerges the passage of off-resonant probe laser pulses may characteristically modify the intensity of single-photon fluorescence, and its associated optical polarization behavior. Here, the probe essentially confers optical nonlinearity on the decay transition, affording a means of optical control over the fluorescent emission. Compared to a catalogue of other laser-based techniques widely used in the life sciences, most suffer limitations reflecting the exploitation of specifically lifetime-associated features; the new optical control mechanism promises to be more generally applicable for the determination of kinetic data. Again, there is a prospect of improving spatial resolution, non-intrusively. It is anticipated that tight directionality can be imposed on single-photon fluorescence emission, expediting the development of new imaging applications. In addition, varying the optical frequency of the probe beam can add another dimension to the experimental parameter space. This affords a means of differentiating between molecular species with strongly overlapping fluorescence spectra, on the basis of their differential nonlinear optical properties. Such techniques significantly extend the scope and the precision of spatial and temporal information accessible from fluorescence studies.

Citation Download Citation

David S. Bradshaw, Jamie M. Leeder, and David L. Andrews "Nonlinear optical techniques for improved data capture in fluorescence microscopy and imaging", Proc. SPIE 7571, Single Molecule Spectroscopy and Imaging III, 75710B (25 February 2010); https://doi.org/10.1117/12.840946

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