Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

BackgroundMultiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.ResultsHere we describe an MPM system incorporating two, independently controlled sources of two-photon excitation whose wavelengths are adjusted to maximally excite one dye while minimally exciting the other. We report increased signal-to-noise ratios and decreased false positive emission bleed-through using this novel multiple-excitation MPM (ME-MPM) compared to conventional single-excitation MPM (SE-MPM) in a variety of multi-color imaging applications.ConclusionsSimilar to the tremendous gain in popularity of CLSM after the introduction of multi-color imaging, we anticipate that the ME-MPM system will further increase the popularity of MPM. In addition, ME-MPM provides an excellent tool to more rapidly design and optimize pairs of fluorescence probes for multi-color two-photon imaging, such as CFP/YFP or GFP/DsRed for CLSM.

[1]  Winfried Denk,et al.  On the fundamental imaging-depth limit in two-photon microscopy , 2004, SPIE Photonics Europe.

[2]  S. Hell,et al.  Enhancing fluorescence brightness: effect of reverse intersystem crossing studied by fluorescence fluctuation spectroscopy. , 2008, Chemphyschem : a European journal of chemical physics and physical chemistry.

[3]  John White,et al.  Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability , 1999, Nature Biotechnology.

[4]  P. Schwille,et al.  Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

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

[6]  J G Parnavelas,et al.  Neuronal migration in the developing cerebral cortex: observations based on real-time imaging. , 2003, Cerebral cortex.

[7]  David A Boas,et al.  Multi-photon microscopy with a low-cost and highly efficient Cr:LiCAF laser. , 2008, Optics express.

[8]  V. Centonze,et al.  Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. , 1998, Biophysical journal.

[9]  M. Drobizhev,et al.  Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. , 2009, The journal of physical chemistry. B.

[10]  Atsushi Miyawaki,et al.  Fluorescence imaging using a fluorescent protein with a large Stokes shift. , 2008, Methods.

[11]  W. Webb,et al.  Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[12]  W. Webb,et al.  Two-Photon Fluorescence Excitation Cross Sections of Biomolecular Probes from 690 to 960 nm. , 1998, Applied optics.

[13]  F. Bestvater,et al.  Two‐photon fluorescence absorption and emission spectra of dyes relevant for cell imaging , 2002, Journal of microscopy.

[14]  D. A. Lee,et al.  Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death. , 2003, American journal of physiology. Cell physiology.

[15]  D. Piston.  Imaging living cells and tissues by two-photon excitation microscopy. , 1999, Trends in cell biology.

[16]  Winfried Denk,et al.  On the fundamental imaging-depth limit in two-photon microscopy , 2006 .

[17]  R. Webb,et al.  Theoretical basis of confocal microscopy. , 1999, Methods in enzymology.

[18]  R. Haugland,et al.  Alexa Dyes, a Series of New Fluorescent Dyes that Yield Exceptionally Bright, Photostable Conjugates , 1999, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[19]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[20]  Tetsuichiro Saito.  In vivo electroporation in the embryonic mouse central nervous system , 2006, Nature Protocols.

[21]  M. Drobizhev,et al.  Two-photon absorption standards in the 550-1600 nm excitation wavelength range. , 2008, Optics express.

[22]  Scott E Fraser,et al.  Multiphoton excitation spectra in biological samples. , 2003, Journal of biomedical optics.

[23]  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.