Flexible Light-Sheet Generation by Field Synthesis

Abstract

Light-sheet microscopy has revolutionized biological imaging by affording rapid and efficient volumetric imaging while minimizing the applied light-dosage and thus phototoxicity. However, due to beam divergence, there is a trade-off between the light-sheet thickness and the useful field-of-view for imaging: the confocal parameter, which approximates the propagation length of a light-sheet, shrinks nonlinearly with the size of the beam waist, and consequently, modest axial resolution is achieved when imaging large specimens. To overcome this trade-off, propagation invariant beams have been employed in light-sheet generation, such as Bessel or Airy beams. These beams maintain their cross-sectional profile over extended distances and overcome the beam divergence of Gaussian beams. To generate a light-sheet, a propagation invariant beam is scanned laterally to create a time averaged sheet of light. Such setups require dedicated optical trains, which increase the cost and complexity of a light-sheet microscope. We have recently discovered and proven the ‘Field Synthesis’ theorem, which describes a universal mechanism for light-sheet generation. Field synthesis predicts that any scanned light-sheet can be synthesized by an incoherent superposition of 1D intensity patterns. We demonstrate that in practice, this results in far less complex optical systems and much higher light-efficiency compared to previous setups using propagation invariant beams. Through careful comparisons, we show that through Field Synthesis we can re-create the time averaged intensity of Bessel and Lattice light-sheets. However, in contrast to scanned Bessel beams, we achieve 100% spatial duty cycle, which results in drastically reduced photobleaching. Compared to lattice light-sheet microscopy, Field Synthesis is achromatic and can hence perform simultaneous multicolor imaging. We present careful comparisons of each illumination mode and demonstrate biological imaging applications of light-sheets produced by Field Synthesis.