Projects per year
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.
|Publication status||Published - Apr 2014|
|Event||Focus on Microscopy, FOM 2014 - Sydney, Australia|
Duration: 13 Apr 2014 → 16 Apr 2014
|Conference||Focus on Microscopy, FOM 2014|
|Period||13/04/14 → 16/04/14|
- multi-photon microscopy
- UV excitable dyes
McConnell, G. & Dempster, J.
13/02/13 → 12/02/20
1/01/11 → 28/02/15
Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laserTrägårdh, J., Robb, G., Amor, R., Amos, W. B., Dempster, J. & McConnell, G., 2015, In: Journal of Microscopy. 9 p.
Research output: Contribution to journal › Article › peer-reviewOpen AccessFile16 Citations (Scopus)109 Downloads (Pure)