The integration of Olympus FV3000 confocal laser scanning microscope with the Newport SF-TRU Timing and Recombination Unit in Helsinki is almost complete and the setup is ready for some test measurements plus performance optimization. With the customized non-descanned multiphoton detectors, the microscope has now the capability to measure CARS and SRS signals in both forward and backward scattered direction. The title picture shows a diffraction limited image of 200 nm polysterene beads imaged with SRS at 3054 cm-1, which is a good test for image resolution.
With the fast spectral focusing capability of SF-TRU, the instrument can record at maximum about three spectral points per second in imaging mode. Therefore, the spectral measurement of cyclohexane in the figure below, took about one minute to complete with 1.6 cm-1 step and 128×128 frame size. The inset shows an example frame. The figure also tells the maximum spectral range (about 200 cm-1) which can be measured without changing the laser wavelength.
Spectral focusing is a technique, which allows narrowband Raman excitation with short (fs) broadband pump and Stokes laser pulses. Conventionally, a selective Raman excitation requires long (ps) narrowband laser pulses as the spectral width of transform limited pulses directly defines the Raman excitation bandwidth as exemplified in the figure below for CARS (left part of the figure). Then, to access a different Raman frequency the laser wavelength needs to be changed. In spectral focusing, the broadband pulses are temporarily chirped in such a way that their instantaneous optical frequency changes linearly over time. With equally chirped pump and Stokes pulses, the instantaneous pump-Stokes frequency difference (IFD) is narrowband, and can selectively excite a Raman frequency as represented on the right side of the figure below. Furthermore, the IFD depends on the pump-Stokes pulse overlap, which can be rapidly changed with an optical delay line without changing the laser wavelength.