Three-dimensional image formation under single-photon ultra-short pulsed illumination.

The major thrust of modern day fluorescence laser-scanning microscopy have been towards achieving better and better depth resolution embodied by the invention and subsequent development of confocal and multi-photon microscopic techniques. However, each method bears its own limitations: in having sufficient background fluorescence and photo-damage resulting from out-of-focus illumination for the former, while low multi-photon absorption cross-sections of common fluorophores for the latter. Here we show how the intelligent choice of single-photon ultrashort pulsed illumination can circumvent all these shortcomings by exemplifying the tiny spatial stretch of an ultrashort pulse. Besides achieving a novel way of optical sectioning, this new method offers improved signal-to-noise ratio as well as reduced photo-damage which are crucial for live cell imaging under prolonged exposure to light.