Efficient excitation of blue emitting dyes by two-photon microscopy at visible wavelengths

Johanna Trägårdh, Gillian Robb, Rumelo Amor, John Dempster, Gail McConnell

Research output: Contribution to conferencePoster

Abstract

An important advantage of multi-photon microscopy over more traditional single-photon imaging techniques is the elimination of high-energy laser sources that can cause significant photo-damage to delicate biological specimens. This is particularly acute when short wavelength excitation is required.
A number of papers (including [1,2]) have investigated the optimum two-photon excitation wavelengths for short-wavelength excitable molecules and have reported that for several fluorochromes the two-photon cross-section increases rapidly at shorter wavelengths. However, most papers thus far have used a Ti:Sapphire laser, which has a wavelength tuning range of 680-1080nm, and therefore few (and mainly cuvette based) data of two-photon excitation efficiency is available for excitation wavelengths shorter than 680nm.
Using a new wavelength tunable optical parametric oscillator and frequency mixing system (Coherent Chameleon OPOVis), we have studied the wavelength dependence of two-photon excitation efficiency of a number of common UV excitable dyes, such as the nuclear stain DAPI, starch staining dye Calcofluor White and Alexa 350, in the wavelength range 530nm to 770nm. We will report details of the optical system and biomedical specimens used to make our measurements, together with a summary of our results.
One key finding is that DAPI, a very commonly used nuclear marker, when excited at 590nm, results in a 7 times increase in the fluorescence signal output when compared to excitation at 680nm with the Ti:Sapphire laser. 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.
LanguageEnglish
PagesP2-D/02
Publication statusPublished - Apr 2014
EventFocus on Microscopy, FOM 2014 - Sydney, Australia
Duration: 13 Apr 201416 Apr 2014

Conference

ConferenceFocus on Microscopy, FOM 2014
CountryAustralia
CitySydney
Period13/04/1416/04/14

Fingerprint

Photons
Microscopy
Coloring Agents
Lasers
Aluminum Oxide
Fluorescence
Optical Devices
Lizards
Fluorescent Dyes
Starch
Staining and Labeling

Keywords

  • multi-photon microscopy
  • UV excitable dyes

Cite this

Trägårdh, J., Robb, G., Amor, R., Dempster, J., & McConnell, G. (2014). Efficient excitation of blue emitting dyes by two-photon microscopy at visible wavelengths. P2-D/02. Poster session presented at Focus on Microscopy, FOM 2014, Sydney, Australia.
Trägårdh, Johanna ; Robb, Gillian ; Amor, Rumelo ; Dempster, John ; McConnell, Gail. / Efficient excitation of blue emitting dyes by two-photon microscopy at visible wavelengths. Poster session presented at Focus on Microscopy, FOM 2014, Sydney, Australia.
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Trägårdh, J, Robb, G, Amor, R, Dempster, J & McConnell, G 2014, 'Efficient excitation of blue emitting dyes by two-photon microscopy at visible wavelengths' Focus on Microscopy, FOM 2014, Sydney, Australia, 13/04/14 - 16/04/14, pp. P2-D/02.

Efficient excitation of blue emitting dyes by two-photon microscopy at visible wavelengths. / Trägårdh, Johanna; Robb, Gillian; Amor, Rumelo; Dempster, John; McConnell, Gail.

2014. P2-D/02 Poster session presented at Focus on Microscopy, FOM 2014, Sydney, Australia.

Research output: Contribution to conferencePoster

TY - CONF

T1 - Efficient excitation of blue emitting dyes by two-photon microscopy at visible wavelengths

AU - Trägårdh, Johanna

AU - Robb, Gillian

AU - Amor, Rumelo

AU - Dempster, John

AU - McConnell, Gail

PY - 2014/4

Y1 - 2014/4

N2 - An important advantage of multi-photon microscopy over more traditional single-photon imaging techniques is the elimination of high-energy laser sources that can cause significant photo-damage to delicate biological specimens. This is particularly acute when short wavelength excitation is required.A number of papers (including [1,2]) have investigated the optimum two-photon excitation wavelengths for short-wavelength excitable molecules and have reported that for several fluorochromes the two-photon cross-section increases rapidly at shorter wavelengths. However, most papers thus far have used a Ti:Sapphire laser, which has a wavelength tuning range of 680-1080nm, and therefore few (and mainly cuvette based) data of two-photon excitation efficiency is available for excitation wavelengths shorter than 680nm.Using a new wavelength tunable optical parametric oscillator and frequency mixing system (Coherent Chameleon OPOVis), we have studied the wavelength dependence of two-photon excitation efficiency of a number of common UV excitable dyes, such as the nuclear stain DAPI, starch staining dye Calcofluor White and Alexa 350, in the wavelength range 530nm to 770nm. We will report details of the optical system and biomedical specimens used to make our measurements, together with a summary of our results.One key finding is that DAPI, a very commonly used nuclear marker, when excited at 590nm, results in a 7 times increase in the fluorescence signal output when compared to excitation at 680nm with the Ti:Sapphire laser. 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.

AB - An important advantage of multi-photon microscopy over more traditional single-photon imaging techniques is the elimination of high-energy laser sources that can cause significant photo-damage to delicate biological specimens. This is particularly acute when short wavelength excitation is required.A number of papers (including [1,2]) have investigated the optimum two-photon excitation wavelengths for short-wavelength excitable molecules and have reported that for several fluorochromes the two-photon cross-section increases rapidly at shorter wavelengths. However, most papers thus far have used a Ti:Sapphire laser, which has a wavelength tuning range of 680-1080nm, and therefore few (and mainly cuvette based) data of two-photon excitation efficiency is available for excitation wavelengths shorter than 680nm.Using a new wavelength tunable optical parametric oscillator and frequency mixing system (Coherent Chameleon OPOVis), we have studied the wavelength dependence of two-photon excitation efficiency of a number of common UV excitable dyes, such as the nuclear stain DAPI, starch staining dye Calcofluor White and Alexa 350, in the wavelength range 530nm to 770nm. We will report details of the optical system and biomedical specimens used to make our measurements, together with a summary of our results.One key finding is that DAPI, a very commonly used nuclear marker, when excited at 590nm, results in a 7 times increase in the fluorescence signal output when compared to excitation at 680nm with the Ti:Sapphire laser. 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.

KW - multi-photon microscopy

KW - UV excitable dyes

UR - http://www.focusonmicroscopy.org/2014/index.html

M3 - Poster

SP - P2-D/02

ER -

Trägårdh J, Robb G, Amor R, Dempster J, McConnell G. Efficient excitation of blue emitting dyes by two-photon microscopy at visible wavelengths. 2014. Poster session presented at Focus on Microscopy, FOM 2014, Sydney, Australia.