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Multi-modal CARS Microscopy Using a Simple Femtosecond Source

Date Do, 14.04.2011 - Do, 14.04.2011
Time 10.15
Speaker Prof. Dr. Albert Stolow, Steacie Institute for Molecular Sciences, National Research Council, Ottawa, Canada
Location Universität Bern, Institut für Angewandte Physik, Gebäude exakte Wissenschaften, Hörsaal B116, Sidlerstrasse 5, 3012 Bern
Program We discuss a simplified CARS microscopy using a simple femtosecond Ti:Sapphire laser source combined with a photonic crystal fibre (PCF). By optimally chirping the fs pump and Stokes laser pulses, we achieve high quality multi-modal imaging (simultaneous CARS, two-photon fluorescence, and second harmonic generation) of live cells and tissues. The tunable Ti:sapphire output provides the pump beam directly, while part of this is converted to the red-shifted Stokes pulse using a PCF having two close-lying zero dispersion wavelengths. This type of PCF gives good power and stability over Stokes shifts ranging from below 1700 cm-1 to over 4000 cm-1. The noise equivalent power of the PCF output at frequencies relevant to live cell microscopy is not a significant issue, as is seen in the high quality images. Chirp as a control parameter permits simultaneous optimization of the spectral resolution and signal levels of these imaging modalities. By matching the chirp rates to create a constant frequency difference between the pump and Stokes pulses, we enhance the CARS spectral resolution (spectral focusing). By simply controlling the time delay between the input pulses, we achieve fast and continuous computer-controlled tuning of the Stokes shift over a broad range, without involving any adjustment of either the fs laser or the PCF. The simultaneous optimization of CARS, two-photon fluorescence and second harmonic generation is achieved by controlling the degree of chirp and involves a trade-off between spectral resolution and signal strength. This trade-off can be optimized under user control to suit the problem at hand. This simplified optical arrangement for multi-modal CARS microscopy [1,2] was recently commercialized by Olympus Corp [3].
In order to develop CARS microscopy applications outside the laboratory (e.g. in hospitals and clinics), the sensitive free space femtosecond laser and optics must be replaced with telecom-stability all-fibre sources. Using an IMRA fibre femtosecond laser as the pump and an Ultra Highly Nonlinear Fibre (suspended core) for Stokes generation, we recently demonstrated that this is feasible [4] and will likely be the route to further commercialization of CARS microscopes. With this very simple source, we demonstrate additional functionalities such as Spectral Scanning FM CARS. This is based on the fact that, in our arrangement, Stokes time delay corresponds to a tuning of the second order (CARS) resonance. Therefore, rapid modulation of this time delay leads to a rapid frequency sweep (FM) which can maintained while simultaneously scanning the pump-Stokes time offset (spectral scan). We also demonstrate time-correlated photon counting Fluorescence Lifetime Imaging (FLIM)-CARS microscopy, again based on this same simple femtosecond source. Live cell viability issues limit light exposure to signal levels corresponding typically to less than one anti-Stokes photon per laser pulse. In such cases, single photon counting becomes profitable, removing the detector electronics amplitude noise and permits the use of the time-correlated lifetime as an additional contrast mechanism.
REFERENCES 1. A.F. Pegoraro, A. Ridsdale, D.J. Moffatt, Y. Jia, J.P. Pezacki & A. Stolow, Optics Express 17, 2984 (2009) 2. A.F. Pegoraro, A. Stolow, A. Ridsdale, D.J. Moffatt, J.P. Pezacki, Y. Jia, Biophotonics 18(8), 36 (2009) 3 FV1000MPE femtoCARS Add-On 4. A.F. Pegoraro, A. Ridsdale, D.J. Moffatt, J.P. Pezacki, A. Stolow, B.K. Thomas, L. Fu, L. Dong, M.E. Fermann, Optics Express 17, 20700 (2009)
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