Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser.

Imaging multiple fluorescent proteins (FPs) by two-photon microscopy has numerous applications for studying biological processes in thick and live samples. Here we demonstrate a setup utilizing a single broadband laser and a phase-only pulse-shaper to achieve imaging of three FPs (mAmetrine, TagRFPt, and mKate2) in live mammalian cells. Phase-shaping to achieve selective excitation of the FPs in combination with post-imaging linear unmixing enables clean separation of the fluorescence signal of each FP. This setup also benefits from low overall cost and simple optical alignment, enabling easy adaptation in a regular biomedical research laboratory.

[1]  H. Nishimura,et al.  Seven-color Fluorescence Imaging of Tissue Samples Based on Fourier Spectroscopy and Singular Value Decomposition , 2000, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[2]  Hans Clevers,et al.  Intestinal Crypt Homeostasis Results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells , 2010, Cell.

[3]  D. Shcherbo,et al.  Bright far-red fluorescent protein for whole-body imaging , 2007, Nature Methods.

[4]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[5]  J. Swanson,et al.  Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells , 2007, The Journal of cell biology.

[6]  Joachim Goedhart,et al.  Bright monomeric red fluorescent protein with an extended fluorescence lifetime , 2007, Nature Methods.

[7]  Jeffrey Wyckoff,et al.  Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging , 2011, Nature Protocols.

[8]  A. Miyawaki,et al.  Two-photon dual-color imaging using fluorescent proteins , 2008, Nature Methods.

[9]  Yaron Silberberg,et al.  Coherent quantum control of two-photon transitions by a femtosecond laser pulse , 1998, Nature.

[10]  Vladislav V Verkhusha,et al.  Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy , 2012, Journal of biomedical optics.

[11]  J. Boardman.  Inversion Of Imaging Spectrometry Data Using Singular Value Decomposition , 1989, 12th Canadian Symposium on Remote Sensing Geoscience and Remote Sensing Symposium,.

[12]  M. Leptin,et al.  Fibroblast growth factor signalling controls successive cell behaviours during mesoderm layer formation in Drosophila , 2011, Development.

[13]  Marcos Dantus,et al.  Multiphoton Intrapulse Interference. 1. Control of Multiphoton Processes in Condensed Phases , 2002 .

[14]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[15]  T. Werner,et al.  Bmc Cell Biology Identification of a Novel Rev-interacting Cellular Protein , 2004 .

[16]  Serge Charpak,et al.  Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo , 2009, PloS one.

[17]  Charles P. Lin,et al.  Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy , 2012, Biomedical optics express.

[18]  R. W. Draft,et al.  Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system , 2007, Nature.

[19]  Thomas Weinacht,et al.  Interference without an interferometer: a different approach to measuring, compressing, and shaping ultrashort laser pulses , 2008 .

[20]  W. Denk,et al.  Two-photon excitation in functional biological imaging. , 1996, Journal of biomedical optics.

[21]  Eric R Tkaczyk,et al.  Multiphoton flow cytometry strategies and applications , 2011, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[22]  G. Bearman,et al.  Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy. , 2001, Journal of biomedical optics.

[23]  Lloyd M. Davis,et al.  Extension of multidimensional microscopy to ultrasensitive applications with maximum-likelihood analysis , 2007, SPIE BiOS.

[24]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[25]  Marcos Dantus,et al.  Quantitative investigation of the multiphoton intrapulse interference phase scan method for simultaneous phase measurement and compensation of femtosecond laser pulses , 2006 .

[26]  M. Dantus,et al.  Two-photon fluorescence excitation spectroscopy by pulse shaping ultrabroad-bandwidth femtosecond laser pulses. , 2010, Applied optics.

[27]  Marcos Dantus,et al.  Systematic control of nonlinear optical processes using optimally shaped femtosecond pulses. , 2005, Chemphyschem : a European journal of chemical physics and physical chemistry.

[28]  Guillaume Labroille,et al.  Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses. , 2009, Optics express.

[29]  T. Norris,et al.  Control of Two-photon Fluorescence of Common Dyes and Conjugated Dyes , 2009, Journal of Fluorescence.

[30]  Michael Z. Lin,et al.  Improving the photostability of bright monomeric orange and red fluorescent proteins , 2008, Nature Methods.

[31]  Emmanuel Beaurepaire,et al.  Use of coherent control for selective two-photon fluorescence microscopy in live organisms. , 2006, Optics express.

[32]  A. Weiner.  Femtosecond pulse shaping using spatial light modulators , 2000 .

[33]  Marcos Dantus,et al.  Photobleaching and photoenhancement of endogenous fluorescence observed in two-photon microscopy with broadband laser sources , 2010 .

[34]  Jeff W Lichtman,et al.  Imaging axonal transport of mitochondria in vivo , 2007, Nature Methods.

[35]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[36]  J. Lippincott-Schwartz,et al.  Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. , 2009, Trends in cell biology.

[37]  D. McEwen,et al.  Single Molecule Imaging Reveals Differences in Microtubule Track Selection Between Kinesin Motors , 2009, PLoS biology.

[38]  A. Miyawaki,et al.  Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses. , 2009, Optics express.

[39]  Yuval Garini,et al.  Spectral imaging: Principles and applications , 2006, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[40]  Dawen Cai,et al.  A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection , 2009, PLoS pathogens.

[41]  Guillaume Labroille,et al.  Multicolor two-photon tissue imaging by wavelength mixing , 2012, Nature Methods.

[42]  Marcos Dantus,et al.  Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging. , 2006, Nanomedicine : nanotechnology, biology, and medicine.

[43]  I. Weissman,et al.  Coronary arteries form by developmental reprogramming of venous cells , 2010, Nature.

[44]  Atsushi Miyawaki,et al.  A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy , 2006, Nature Biotechnology.

[45]  M. Dantus,et al.  Multiphoton Intrapulse Interference 3: Probing Microscopic Chemical Environments , 2004 .

[46]  Marcos Dantus,et al.  Selective two-photon microscopy with shaped femtosecond pulses. , 2003, Optics express.

[47]  R. Tsien,et al.  The Fluorescent Toolbox for Assessing Protein Location and Function , 2006, Science.

[48]  Yasushi Hiraoka,et al.  Multispectral imaging fluorescence microscopy for living cells. , 2002, Cell structure and function.

[49]  S W Hell,et al.  Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. , 1999, Biophysical journal.

[50]  Robert E Campbell,et al.  Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors , 2008, Nature Methods.

[51]  Jeffrey Wyckoff,et al.  Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy , 2005, BMC biotechnology.

[52]  M. Dantus,et al.  Multiphoton intrapulse interference 6; binary phase shaping. , 2004, Optics express.

[53]  D. Moore.  Optimal coherent control of sensitivity and selectivity in spectrochemical analysis , 2009, Analytical and bioanalytical chemistry.

[54]  M. Drobizhev,et al.  Two-photon absorption properties of fluorescent proteins , 2011, Nature Methods.

[55]  Mikhail Drobizhev,et al.  A new approach to dual-color two-photon microscopy with fluorescent proteins , 2010, BMC biotechnology.

[56]  M. Dantus,et al.  Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses , 2003 .

[57]  R. Pepperkok,et al.  Spectral imaging and its applications in live cell microscopy , 2003, FEBS letters.