Ultrafast Fiber Lasers for Multiphoton Microscopy

Introduction to multiphoton microscopy and laser technology

Figure: Two-photon microscopy images using the FemtoFiber ultra 920 for fluorescence excitation. a, Image of a human stem cell expressing GFP attached to its actin network. b, Human stem cell with ATTO594 labeling of the actin network and DAPI labeling of the cell nucleus. All images have been recorded by the research group of Prof. Thomas Hellerer at the University of Applied Sciences in Munich.

Multi-photon fluorescence microscopy has become a key technology in biological imaging enabling three-dimensional, noninvasive studies of biological tissue on the submicron scale. The contrast mechanism in multi-photon microscopy is based on the excitation of fluorophores by 2 or more photons, typically in the infrared spectral range. Upon excitation the fluorophores relax back by emitting a photon in the visible which is detected. In contrast to conventional linear fluorescence microscopy, where the molecules are excited with a single photon in the visible, the nonlinear character and the excitation wavelength in multi-photon microscopy offer several advantages: (i) the reduced absorption of infrared photons leads to a larger probing depth and enables in-vivo imaging due to lower absorption damage. (ii) Since the fluorescence excitation is limited to the focal plane of the microscope, no spatial filtering is required.

Important laser requirements in multi-photon microscopy!

Multi-photon microscopy is a nonlinear microscopy technique and as such the image brightness and quality dramatically depends on the peak-power of the excitation laser. The peak-power is determined by the laser power, pulse duration, repetition rate, and most importantly also by the temporal pulse quality. Optimizing the pulse duration and the laser power inside the multi-photon microscope is also a critical aspect in multi-photon microscopy. Typically, this calls for elaborate and large optical setups that include optics for pulse compression (group-delay dispersion (GDD) compensation) to ensure a minimum pulse duration at the sample and fast optical modulators for power control and beam blanking.

TOPTICA's FemtoFiber ultra 920 has been developed based on these requirements. With more than sufficient output power, femtosecond pulses, and our unique Clean-Pulse Technology, the FemtoFiber ultra 920 enables ultimate peak-power and thus image brightness in two-photon microscopy. Turn-key and intuitive operation, fully-integrated dispersion compensatation, and build-in power control make the system extremely user-friendly and allows you to focus on your research!

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