title: Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser.
abstract: We have studied the wavelength dependence of the two-photon excitation efficiency for a number of common UV excitable fluorescent dyes; the nuclear stains DAPI, Hoechst and SYTOX Green, chitin- and cellulose-staining dye Calcofluor White and Alexa Fluor 350, in the visible and near-infrared wavelength range (540-800 nm). For several of the dyes, we observe a substantial increase in the fluorescence emission intensity for shorter excitation wavelengths than the 680 nm which is the shortest wavelength usually available for two-photon microscopy. We also find that although the rate of photo-bleaching increases at shorter wavelengths, it is still possible to acquire many images with higher fluorescence intensity. This is particularly useful for applications where the aim is to image the structure, rather than monitoring changes in emission intensity over extended periods of time. We measure the excitation spectrum when the dyes are used to stain biological specimens to get a more accurate representation of the spectrum of the dye in a cell environment as compared to solution-based measurements.
authors.affiliation: Centre for Biophotonics, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.
authors.forename: J
authors.initials: J
authors.lastname: Trägårdh
authors.affiliation: Centre for Biophotonics, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.
authors.forename: G
authors.initials: G
authors.lastname: Robb
authors.affiliation: Centre for Biophotonics, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.
authors.forename: R
authors.initials: R
authors.lastname: Amor
authors.affiliation: Centre for Biophotonics, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.|MRC Laboratory of Molecular Biology, Cambridge, U.K.
authors.forename: W B
authors.initials: WB
authors.lastname: Amos
authors.affiliation: Centre for Biophotonics, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.
authors.forename: J
authors.initials: J
authors.lastname: Dempster
authors.affiliation: Centre for Biophotonics, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, U.K.
authors.forename: G
authors.initials: G
authors.lastname: McConnell
mesh_terms: D000537:Aluminum Oxide; D001557:Benzenesulfonates / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes; D007211:Indoles / Q000737:chemistry; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry; D017785:Photons; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
chemical_list: D001557:Benzenesulfonates; D005456:Fluorescent Dyes; D007211:Indoles; D009930:Organic Chemicals; C402795:SYTOX Green; C007293:DAPI; C007061:C.I. Fluorescent Brightening Agent 28; D000537:Aluminum Oxide
keywords: 3T3 cells; Fluorescent dyes; microscopy; multiphoton microscopy