We demonstrate fluorescence excitation at multiple planes in a laser-scanning microscope by using the standing wave from a mirror placed close to the specimen. We have observed precise modulation of the standing waves close to a mirror, with a frequency proportional to the Stokes shift, corresponding to a moiré pattern between the excitation and emission standing-wave fields. We use standing-wave excitation to plot the exact contour maps of the red blood cell membrane, with an axial resolution of ≈90 nm. The method may prove useful in the study of diseases which involve the surface membrane of red blood cells.
Multi-photon excitation (MPE) imaging is dominated by the Ti:Sapphire laser as the source for excitation. However, it is
limited when considering 3PE of common fluorophores and efficient 2PE of UV dyes which require wavelengths beyond
the range of the Ti:Sapphire. Two ultra-short pulsed sources are presented as alternatives: a novel optical parametric
oscillator (OPO) geometry (1400–1600nm) and the sum-frequency mixing of an OPO and Yb-doped fibre laser,
providing a tunable output (626-635nm).
For long wavelengths, we report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical
parametric oscillator (OPO) with signal wavelength output at 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 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using
a femtosecond pulsed Ti:Sapphire laser at 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 minutes but
3PLSM showed little or no interference with cell function after 15 minutes. The 1500 nm OPO was thus shown to be a
practical laser source for live cell imaging.
For short wavelengths, we report the use of an all-solid-state ultra-short pulsed source specifically for two-photon
microscopy at wavelengths shorter than those of the conventional Ti:Sapphire laser. Our approach involved sumfrequency
mixing of the output from the long-wavelength OPO described above with residual pump radiation to generate
fs-pulsed output in the red spectral region. We demonstrated the performance of our ultra-short pulsed system using
fluorescently labelled and autofluorescent tissue, and compared with conventional Ti:Sapphire excitation. We observed a
more than 3-fold increase in fluorescence signal intensity using our visible laser source in comparison with the
Ti:Sapphire laser for two-photon excitation at equal illumination powers of 22 mW or less.