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Two-Color, Two-Photon Imaging at Long Excitation Wavelengths Using a Diamond Raman Laser

Published online by Cambridge University Press:  05 August 2016

Johanna Trägårdh Open the ORCID record for Johanna Trägårdh [Opens in a new window] ,
Michelle Murtagh ,
Gillian Robb ,
Maddy Parsons ,
Jipeng Lin ,
David J. Spence  and
Gail McConnell
Johanna Trägårdh*
Affiliation:
Centre for Biophotonics, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK
Michelle Murtagh
Affiliation:
Centre for Biophotonics, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK MQ Photonics, Department of Physics and Astronomy, Macquarie University, NSW 2109, Australia
Gillian Robb
Affiliation:
Centre for Biophotonics, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK
Maddy Parsons
Affiliation:
Randall Division of Cell and Molecular Biophysics, King’s College London, Guy’s Campus, London, SE11UL, UK
Jipeng Lin
Affiliation:
MQ Photonics, Department of Physics and Astronomy, Macquarie University, NSW 2109, Australia
David J. Spence
Affiliation:
MQ Photonics, Department of Physics and Astronomy, Macquarie University, NSW 2109, Australia
Gail McConnell
Affiliation:
Centre for Biophotonics, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK
*
*Corresponding author. [email protected]
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Abstract

We demonstrate that the second-Stokes output from a diamond Raman laser, pumped by a femtosecond Ti:Sapphire laser, can be used to efficiently excite red-emitting dyes by two-photon excitation at 1,080 nm and beyond. We image HeLa cells expressing red fluorescent protein, as well as dyes such as Texas Red and Mitotracker Red. We demonstrate the potential for simultaneous two-color, two-photon imaging with this laser by using the residual pump beam for excitation of a green-emitting dye. We demonstrate this for the combination of Alexa Fluor 488 and Alexa Fluor 568. Because the Raman laser extends the wavelength range of the Ti:Sapphire laser, resulting in a laser system tunable to 680–1,200 nm, it can be used for two-photon excitation of a large variety and combination of dyes.

Keywords

nonlinear microscopyfluorescence microscopyRaman lasersfluorescent proteinstwo-photon microscopy
Type
Technique and Instrumentation Development
Information
Microscopy and Microanalysis , Volume 22 , Issue 4 , August 2016 , pp. 803 - 807
Copyright
© Microscopy Society of America 2016 

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