Sub-diffraction-limit imaging using mode multiplexing

Abstract Pixel-by-pixel processed fluorescence difference microscopy is experimentally demonstrated by multiplexing excitation laser beams with Gaussian and donut spot shapes and then demultiplexing the fluorescent signals using lock-in amplifiers. With this scheme, a fixed sample of fluorescent spheres and a slice of mouse brain tissue are imaged with resolutions that exceed the diffraction limit. Compared to previously reported subtraction imaging techniques, this pixel-by-pixel scan can be applied to improve the resolution of a moving sample without introducing subtraction errors. The synchronized signal detection feature makes this method extendible to various applications.

[1]  M. Davidson,et al.  Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination , 2012, Proceedings of the National Academy of Sciences.

[2]  Hiromichi Tsurui,et al.  Sub-diffraction-limit imaging using mode multiplexing , 2015, SPIE Scanning Microscopies.

[3]  Vassilios Sarafis,et al.  Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes. , 2003, Micron.

[4]  Ana Doblas,et al.  Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector. , 2012, Optics letters.

[5]  Michel Piché,et al.  Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging. , 2013, Optics express.

[6]  D. Balding,et al.  HLA Sequence Polymorphism and the Origin of Humans , 2006 .

[7]  X. Zhuang,et al.  Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells , 2010, Cell.

[8]  Satoshi Kawata,et al.  High-resolution confocal microscopy by saturated excitation of fluorescence. , 2007, Physical review letters.

[9]  R. Hochstrasser,et al.  Wide-field subdiffraction imaging by accumulated binding of diffusing probes , 2006, Proceedings of the National Academy of Sciences.

[10]  Katsumi Midorikawa,et al.  Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy , 2012, Biomedical optics express.

[11]  T. Ha,et al.  Single-molecule high-resolution imaging with photobleaching. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Shunichi Sato,et al.  Resolution enhancement of confocal microscopy by subtraction method with vector beams. , 2014, Optics letters.

[13]  Alberto Diaspro,et al.  Strategies to maximize the performance of a STED microscope. , 2012, Optics express.

[14]  Alf Honigmann,et al.  Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution. , 2013, Biophysical journal.

[15]  Xiang Hao,et al.  Breaking the Diffraction Barrier Using Fluorescence Emission Difference Microscopy , 2013, Scientific Reports.

[16]  Haruo Kasai,et al.  Sub-diffraction resolution pump-probe microscopy with shot-noise limited sensitivity using laser diodes. , 2014, Optics express.

[17]  J. Matthew Mauro,et al.  Long-term multiple color imaging of live cells using quantum dot bioconjugates , 2003, Nature Biotechnology.

[18]  Keng C Chou,et al.  Review of Super-Resolution Fluorescence Microscopy for Biology , 2011, Applied spectroscopy.

[19]  P. Selvin,et al.  3D super-resolution imaging with blinking quantum dots. , 2013, Nano letters.

[20]  S. Weiss,et al.  Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI) , 2009, Proceedings of the National Academy of Sciences.

[21]  Kjell Carlsson,et al.  Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique , 1993 .

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

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

[24]  Paul R Selvin,et al.  Single-molecule-based super-resolution images in the presence of multiple fluorophores. , 2011, Nano letters.

[25]  S. S. Gorthi,et al.  Fluorescence imaging of flowing cells using a temporally coded excitation. , 2013, Optics express.

[26]  Xu Liu,et al.  Effects of polarization on the de-excitation dark focal spot in STED microscopy , 2010 .

[27]  S. Hell,et al.  Two-color far-field fluorescence nanoscopy. , 2007, Biophysical journal.

[28]  J. Lichtman,et al.  3D Multicolor Super-Resolution Imaging Offers Improved Accuracy in Neuron Tracing , 2012, PloS one.

[29]  S. Hell.  Far-Field Optical Nanoscopy , 2007, Science.

[30]  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.

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

[32]  S. Hell,et al.  Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms , 2010, PMC biophysics.

[33]  Katharina Landfester,et al.  Characterization via two-color STED microscopy of nanostructured materials synthesized by colloid electrospinning. , 2011, Langmuir : the ACS journal of surfaces and colloids.

[34]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[35]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[36]  R. Heintzmann,et al.  A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non‐conjugate images , 2001, Journal of microscopy.

[37]  Christian Eggeling,et al.  STED microscopy reveals crystal colour centres with nanometric resolution. , 2009 .

[38]  Takayoshi Kobayashi,et al.  Numerical study of the subtraction threshold for fluorescence difference microscopy. , 2014, Optics express.

[39]  Hiromichi Tsurui,et al.  Label-free imaging of melanoma with nonlinear photothermal microscopy. , 2015, Optics letters.