Multiphoton microscopy has become an ubiquitous research tool for neuroscience, disease studies, embryology and immunology. Refinements in non-linear imaging techniques have gone hand-in-hand with improvements in tuning range, power, pulse management and other features of femtosecond lasers specifically designed for these applications. While potential clinical applications require lasers with multiple outputs able to support multimodal imaging, recent developments in neuronal imaging like 3-photon microscopy or optogenetic activation require higher energies per pulse and longer wavelengths than conventional multiphoton microscopy. Here we describe the design and characteristics of laser sources able to address all these applications, staring with the more established ultrafast laser technology based on Titanium Sapphire lasers and their OPOs and covering recently developed sources like Ytterbium fiber mode-locked lasers and amplifiers with their tunable wavelength extensions.
In the last few years Multiphoton Excitation Microscopy witnessed a mutation from tool for imaging cellular structures in living animals deeper than other high-resolution techniques, into an instrument for monitoring functionality and even stimulating or inhibiting inter-cellular signalling. This paradigm shift has been enabled primarily by the development of genetically encoded probes like Ca indicators (GECI) and Opsins for optogenetics inhibition and stimulation. These developments will hopefully enable the understanding of how local network of hundreds or thousands of neurons operate in response to actual tasks or induced stimuli. Imaging, monitoring signals and activating neurons, all on a millisecond time scale, requires new laser tools providing a combination of wavelengths, higher powers and operating regimes different from the ones traditionally used for classic multiphoton imaging. The other key development in multiphoton techniques relates to potential diagnostic and clinical applications where non-linear imaging could provide all optical marker-free replacement of H and E techniques and even intra-operative guidance for procedures like cancer surgery. These developments will eventually drive the development of specialized laser sources where compact size, ease of use, beam delivery and cost are primary concerns. In this talk we will discuss recent laser product developments targeting the various applications of multiphoton imaging, as fiber lasers and other new type of lasers gradually gain popularity and their own space, side-by-side or as an alternative to conventional titanium sapphire femtosecond lasers.
We report on multiphoton imaging of biological samples performed with continuum infrared source generated
in photonic crystal fibers (PCFs). We studied the spectra generated in PCFs with dispersion profiles designed
to maximize the power density in the 700-1000 nm region, where the two-photon absorption cross sections of
the most common dyes lie. Pumping in normal dispersion region, with <140 femtosecond pulses delivered by a
tunable Ti:Sa laser (Chameleon Ultra II by Coherent Inc.), results in a limitation of nonlinear broadening up to a
mean power density above 2 mW/nm. Axial and lateral resolution obtained with a scanning multiphoton system
has been measureed to be near the theoretical limit. The possibility of simultaneous two-photon excitation of
different dyes in the same sample and high image resolution are demonstrated at tens of microns in depth.
Signal-to-noise ratio and general performances are found to be comparable with those of a single wavelength
system, used for comparison.
We describe the realization and characterization of a broadband, high power density and fully spectrally controllable
source, suitable for multiphoton imaging of biological samples. We used a photonic crystal fiber (PCF)
with selected dispersive and non-linear properties, in order to generate, when pumped with <140 femtosecond
pulses delivered by a tunable Ti:Sa laser (Chameleon Ultra II by Coherent Inc.), a smooth continuum in the
700nm-950nm region, with average power density grater than 2mW/nm. Time distribution of the generated
spectrum has been measured with autocorrelation technique. Axial and lateral resolution obtained with a scanning
multiphoton system has been determined to be near the theoretical limit. The possibility of two-photon
excitation of different dyes in the same sample and high image resolution are demonstrated at tens of microns
in depth. Future developments and different applications are also discussed.
Water-cooled ion lasers have been commercially available for 25 years. Since the introduction of the metal-ceramic plasma tube technology 10 years ago, a considerable amount of research and development activity at Coherent has been devoted to improving the operating life and reliability of this kind of tube. The efficient generation of laser radiation imposes stringent requirements on the discharge parameters and, consequently, on the plasma tube itself. We have developed various methods to analyze the discharge environment, test the effectiveness of new materials and tube designs, and control the manufacturing process. The combined use of these methods allows the production of tubes with lifetimes that can exceed 10,000 hours in the visible wavelengths and 5,000 hours in the ultraviolet.