Simultaneous measurement of position and color of single fluorescent emitters using diffractive optics.

We propose a method for simultaneously measuring the position and emission color of single fluorescent emitters based on the use of a large pitch diffraction grating in the emission light path. The grating produces satellite spots adjacent to the main spot; the relative distance between the spots is a measure for the emission wavelength. We present proof-of-principle experiments on beads and mixtures of quantum dots using a spatial light modulator for making a programmable diffraction grating. A wavelength precision of around 10 nm can be achieved for 1000 signal photons and practical background levels, while maintaining a localization precision of around 10 nm.

[1]  E. Yeung,et al.  High-throughput single-molecule spectroscopy in free solution. , 2000, Analytical chemistry.

[2]  Christian Eggeling,et al.  Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. , 2010, Biophysical journal.

[3]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[4]  Michael W. Davidson,et al.  Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes , 2007, Proceedings of the National Academy of Sciences.

[5]  S. Stallinga,et al.  The lateral and axial localization uncertainty in super-resolution light microscopy. , 2014, Chemphyschem : a European journal of chemical physics and physical chemistry.

[6]  Travis J Gould,et al.  Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. , 2011, Biophysical journal.

[7]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[8]  S. Stallinga,et al.  Accuracy of the gaussian point spread function model in 2D localization microscopy. , 2010, Optics express.

[9]  Christian Eggeling,et al.  Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species. , 2008, Nano letters.

[10]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[11]  David Baddeley,et al.  4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues , 2011, PloS one.

[12]  A. Sergé,et al.  Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes , 2008, Nature Methods.

[13]  D. W. Scott.  On optimal and data based histograms , 1979 .

[14]  G. A. Blab,et al.  Simultaneous wide-field imaging and spectroscopy of localized fluorophores. , 2004, Optics letters.

[15]  Mark Bates,et al.  Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes , 2007, Science.

[16]  Sheng Liu,et al.  Multi-Color Quantum Dot Tracking Using a High-Speed Hyperspectral Line-Scanning Microscope , 2013, PloS one.

[17]  H. Flyvbjerg,et al.  Optimized localization-analysis for single-molecule tracking and super-resolution microscopy , 2010, Nature Methods.