In 1994 we commissioned a multiphoton imaging system that featured an all solid-state excitation source, a 1047nm Nd:YLF laser that was developed to our specifications by Prof. Allister Ferguson's group at the University of Strathclyde, Scotland, and by Microlase Ltd. a company founded by Prof Ferguson. This was the first all-solid state multiphoton system to be developed (Wokosin et al., 1996. Proc.SPIE 2678, 38). The laser is coupled to the scan head by an optical fiber providing a compact and simple installation. The Nd:YLF laser operates at a wavelength 1047nm which is somewhat longer than can be readily attained by a Ti:sapphire laser. We favor the use of longer wavelengths because of improved viability, and it is possible to obtain images from deeper within samples than with shorter wavelengths. In spite of not being tunable, we have found that we can image most of the commonly used fluorophores such as rhodamine, fluorescein or Texas Red. In addition, we found that the Nd:YLF system can effectively excite blue-emitting fluorophores, such as the DNA label DAPI, by the mechanism of three-photon excitation. The level of three-photon excitation depends on the cube of the excitation power and the square of the pulse width. In order to enhance the performance of our multiphoton system, we commissioned the design and development of an improved pulse compressor from Microlase. This compressor has a lower insertion loss and provides shorter pulses (150 femtosec) than the previous compressor and has facilities for pre-chirping the pulses to compensate for group velocity dispersion in the microscope optics. The new compressor gave us approximately an order of magnitude improvement in three-photon signals.
The multiphoton imaging system was implemented using a modified BioRad 600 scan-head and electronics. We retained the confocal capability and have carried out direct comparisons between confocal and multiphoton imaging on the same sample (Centonze & White 1998. J. Biophysics 75: 2015, Wokosin et. al. IEEE EMBS 20:1707, Squirrel et. al., Nature Biotechnology in press).
This original multiphoton microscope has become the work-horse of the LOCI and has been used regularly in the past four years. We designed and constructed an three gas environmental chamber for the microscope for studies on mammalian cells and embryos. This system envelops the entire Nikon inverted microscope. We found that this arrangement gives better temperature control than a heated stage.
