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Sources come of age for multiphoton microscopy

Girkin, J.M. (2005) Sources come of age for multiphoton microscopy. Biophotonics International, 12. pp. 44-49. ISSN 1081-8693

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In conventional confocal microscopes a visible light source is required that is well matched to the absorption of a specific fluorophore. Generally it will be in the UV to green portion of the spectrum, with high beam quality, and with the option of short pulse operation for the rapidly growing use of fluorescent lifetime imaging (FLIM) methods. Until recently the only suitable source was an argon laser with all the complications of high maintenance and cooling. Blue laser diodes are a promising alternative in some cases [1], however their use is currently limited to shorter wavelength fluorophores. We have been exploring the use of the next generation of semiconductor laser sources - optically pumped surface emitting lasers. These devices most commonly operate in the near infrared [2], and frequency doubled sources are now commercially available. Recently, with improved growth techniques, we have developed such so-called vertical external cavity surface emitting lasers (VECSELs) that operate in the red with high efficiency, producing up to 0.4 W at 674nm [3]. The semiconductor structure basically consists of a VCSEL grown without the top mirror or electrical contacts. In one configuration the cavity is then constructed using bulk optical components in the conventional manner which opens up the opportunity for wavelength tuning, or short pulse production using active mode-locking techniques providing a source suitable for FLIM. Alternatively the heat spreader on the surface of the semiconductor can be coated to act as the end mirror of the cavity producing a compact source with multi-watt output powers. In a recent development using a diffractive optical element (either passive or active) to divide the pump source, multiple lasers have been produced from a single chip [4], with potential applications for rapid multi-point scanning with high efficiency. In an alternative approach for producing visible sources, photonic crystal fibres have been used to generate visible light from mode-locked Ti:Sapphire lasers to enable time resolved imaging using a confocal rather than multiphoton imaging systems [5]. Ultra-short pulse, easy to operate and low cost sources have also been developed for multiphoton imaging applications [6].