Design of an epi fluorescence microscope with multiplex CARS for marker free analysis

An epifluorescence microscope setup has been developed and tested featuring Raman and multiplex-CARS capabilities. Samples were stored in 96 multi well plates on a sample stage with triaxial piezo drive stages for sample scanning. Raman excitation was achieved by a 785 nm laser with up to 100 mW output power and below 1 pm wavelength bandwidth (FWHM). Multiplex CARS was excited by a laser system providing 1064 nm primary pulses with 1 ns duration and broadband "white"anti-Stokes radiation (1100 nm ... 1700 nm) generated by a non-linear optical fiber. Repetition rates around 30 kHz resulted in an average excitation power of up to 200 mW. For both excitation principles the signal was captured by a Raman spectrograph (Ocean Insight QEPRO) designed for 790 nm ... 1070 nm spectral range, resolution below 1 nm and a thermoelectrically cooled detector for a good signal-to-noise ratio (SNR) to detect weak RAMAN and CARS signals. First measurements have been performed on selected samples and promising results have been achieved.