The use of exotic optical modes is becoming increasingly widespread in microscopy. Particularly, propagation-invariant beams, such as Airy and Bessel beams and optical lattices, have been particularly useful in light-sheet fluorescence microscopy (LSFM) as they enable high-resolution imaging over a large field-of-view (FOV), possess a resistance to the deleterious effects of specimen induced light scattering, and can potentially reduce photo-toxicity.
Although these propagation-invariant beams can resist the effects of light scattering to some degree, and there has been some interest in adaptive-optical methods to correct for beam aberrations when they cannot, scattering and absorption of the illuminating light-sheet limit the penetration of LSFM into tissues and results in non-uniform intensity across the FOV.
A new degree of control over the intensity evolution of propagation-invariant beams can overcome beam losses across the FOV, restoring uniform illumination intensity and therefore image quality. This concept is compatible with all types of propagation-invariant beams and is characterised in the context of light-sheet image quality.
Another property to control is the wavelength of light used. Optical transmission through tissue is greatly improved at longer wavelengths into the near-infrared due to reduced Rayleigh scattering and two-photon excitation has proved beneficial for imaging at greater depth in LSFM. Three-photon excitation has already been demonstrated as a powerful tool to increase tissue penetration in deep brain confocal microscopy, and when combined with beam shaping can also be a powerful illumination strategy for LSFM.
Recent progress in shaping optical fields for LSFM will be presented.
Optical approaches have broadened their impact in recent years with innovations in both wide-field and super- resolution imaging, which now underpin biological and medical sciences. Whilst these advances have been remarkable, to date, the ongoing challenge in optical imaging is to penetrate deeper. TRAFIX is an innovative approach that combines temporal focusing illumination with single-pixel detection to obtain wide-field multi- photon images of fluorescent microscopic samples deep through scattering media without correction. It has been shown that it can image through biological samples such as rat brain or human colon tissue up to a depth of seven scattering mean-free-path lengths. Comparisons of TRAFIX with standard point-scanning two-photon microscopy show that the former can yield a five-fold higher signal-to-background ratio while significantly reducing photobleaching of the specimen. Here, we show the first preliminary demonstration of TRAFIX with three-photon excitation imaging dielectric beads. We discuss the advantages of the TRAFIX approach combined with compressive sensing for biomedicine.