Light-sheet microscopy facilitates rapid, high-contrast, volumetric imaging with minimal sample exposure. However, the rapid divergence of a traditional Gaussian light sheet restricts the field of view (FOV) that provides innate subcellular resolution. We show that the Airy beam innately yields high contrast and resolution up to a tenfold larger FOV. In contrast to the Bessel beam, which also provides an increased FOV, the Airy beam's characteristic asymmetric excitation pattern results in all fluorescence contributing positively to the contrast, enabling a step change for light-sheet microscopy.

A conventional microscope produces a sharp image from just a single object-plane. This is often a limitation, notably in cell biology. We present a microscope attachment which records sharp images from several object-planes simultaneously. The key concept is to introduce a distorted diffraction grating into the optical system, establishing an order-dependent focussing power in order to generate several images, each arising from a different object-plane. We exploit this multiplane imaging not just for bio-imaging but also for nano-particle tracking, achieving approximately 10 nm z position resolution by parameterising the images with an image sharpness metric.

We present an experimental realization of spiraling and snaking zero-order Bessel beams; light modes designed to deviate from straight-line propagation. We show that these modes can be generated with a tunable lateral deviation, amplitude and axial periodicity using a dynamic adaptive optical element, namely a spatial light modulator. We demonstrate that such beams can elude obstructions placed on the optical axis. We discuss their applications for micromanipulation and within lab-on-a-chip systems.

Light sheet microscopy is a powerful method for three-dimensional imaging of large biological specimens. However, its imaging ability is greatly diminished by sample scattering and aberrations. Optical clearing, Bessel light modes, and background rejection have been employed in attempts to circumvent these deleterious effects. We present an in situ wavefront correction that offers a major advance by creating an “optimal” light sheet within a turbid sample. Crucially, we show that no tissue clearing or specialized sample preparation is required, and clear improvements in image quality and depth resolution are demonstrated both in Gaussian and Bessel beam-based light sheet modalities.

We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.