Abstract
We report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto-fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.
Highlights
Scanning microscopy using two-photon excitation of fluorescence is well established
For three-photon excitation of fluorophores with singlephoton absorption in the visible region of the spectrum, wavelengths are needed which are above the range of the ubiquitous Ti:Sapphire laser (Wokosin et al, 1996) and the Cr:Forsterite laser, so, in this work, we have examined excitation at λ = 1500 nm
The bright-field images are shown from time t = 0 min in 5 min increments to 15 min for continuous irradiation with (a) the Ti:Sapphire and (b) the optical parametric oscillator (OPO) system
Summary
Scanning microscopy using two-photon excitation of fluorescence is well established. it is desirable to explore the potential of three-photon processes, which are relatively little studied. Some promising results have already been obtained for three-photon laser scanning microscopy (3PLSM) using third harmonic generation (Barad et al, 1997; Mueller et al, 1998; Squier et al, 1998; Millard et al, 1999; Canioni et al, 2001) For example, by increasing the wavelength to λ = 1260 nm with a femtosecond-pulsed Cr:Forsterite laser, an improved depth of tissue imaging by third harmonic generation has been achieved (Debarre et al, 2006; Sun et al, 2004) and three-photon excitation laser scanning fluorescence has been demonstrated (Matsuda et al, 2006) for a range of samples, albeit at shorter (λ < 1100 nm) wavelengths.
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