Superresolved multiphoton microscopy with spatial frequency-modulated imaging

Jeffrey J Field; Keith A Wernsing; Scott R Domingue; Alyssa M Allende Motz; Keith F DeLuca; Dean H Levi; Jennifer G DeLuca; Michael D Young; Jeff A Squier; Randy A Bartels

doi:10.1073/pnas.1602811113

Superresolved multiphoton microscopy with spatial frequency-modulated imaging

Proc Natl Acad Sci U S A. 2016 Jun 14;113(24):6605-10. doi: 10.1073/pnas.1602811113. Epub 2016 May 26.

Authors

Affiliations

¹ Department of Electrical and Computer Engineering, Colorado State University, Fort Collins, CO 80523; Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523; Microscope Imaging Network Foundational Core Facility, Colorado State University, Fort Collins, CO 80523; Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523; jjfield@rams.colostate.edu.
² Department of Electrical and Computer Engineering, Colorado State University, Fort Collins, CO 80523;
³ Department of Physics, Colorado School of Mines, Golden, CO 80401; National Renewable Energy Laboratory, Golden, CO 80401;
⁴ Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523; Microscope Imaging Network Foundational Core Facility, Colorado State University, Fort Collins, CO 80523; Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523;
⁵ National Renewable Energy Laboratory, Golden, CO 80401;
⁶ Department of Physics, Colorado School of Mines, Golden, CO 80401;
⁷ Department of Electrical and Computer Engineering, Colorado State University, Fort Collins, CO 80523; Microscope Imaging Network Foundational Core Facility, Colorado State University, Fort Collins, CO 80523; Institute for Genome Architecture and Function, Colorado State University, Fort Collins, CO 80523; School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523.

Abstract

Superresolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection. We show multimodal superresolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2η below the diffraction limit, where η is the highest power of the nonlinear intensity response. MP-SPIFI can be used to provide enhanced resolution in optically thin media and may provide a solution for superresolved imaging deep in scattering media.

Keywords: harmonic generation; multiphoton microscopy; superresolution.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Microscopy, Fluorescence, Multiphoton / methods*
Models, Theoretical*

Abstract

Publication types

MeSH terms

Grants and funding