Lattice light-sheet microscopy

Lattice light-sheet microscopy is a modified version of light sheet fluorescence microscopy that increases image acquisition speed while decreasing damage to cells caused by phototoxicity. This is achieved by using a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images which can provide information about dynamic biological processes. Lattice light-sheet microscopy is a modified version of light sheet fluorescence microscopy that increases image acquisition speed while decreasing damage to cells caused by phototoxicity. This is achieved by using a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images which can provide information about dynamic biological processes. It was developed in the early 2010s by Eric Betzig. According to the Washington Post, Eric Betzig believes that this development will have a greater impact than the work that earned him the 2014 Nobel Prize in Chemistry for 'the development of super-resolved fluorescence microscopy'. Lattice light sheet microscopy is a novel combination of techniques from Light sheet fluorescence microscopy, Bessel beam microscopy, and Super-resolution microscopy (specifically structured illumination microscopy, SIM). In lattice light sheet microscopy, very similarly to light sheet microscopy, the illumination of the sample occurs perpendicular to the image detection. Initially the light sheet is formed by stretching the linearly polarized circular input beam with a pair of cylindrical lenses along the x axis and then compressing it with an additional pair of lenses along the z axis. This modification creates a thin sheet of light that is then projected onto a binary ferroelectric spatial light modulator (SLM). The SLM is a device that spatially varies the waveform of a beam of light. The light that is reflected back from the SLM is used to eliminate unwanted diffraction. Diffraction is eliminated by the transform lens that creates a Fraunhofer diffraction pattern from the reflected light at an opaque mask containing a transparent annulus. Optical lattices are two or three dimensional interference patterns, which here are produced by the transparent annular ring. The mask is conjugate to x and z galvanometers. This quality of the microscope is important for the dithered mode of operation, where the light sheet must be oscillated within the x axis. The lattice light-sheet microscope has two modes of operation: In the dithered mode, the light sheet is rapidly scanned along the x axis and only one image is recorded per Z plane, at normal diffraction limited resolutions. The second mode of operation is the structured illumination microscopy mode (SIM). SIM is a technique where a grid pattern of excitation light is superimposed on the sample and rotated in steps between the capture of each image. These images are then processed via an algorithm to produce a reconstructed image past the limit of diffraction that is built into our optical instruments. Lattice light sheet microscopy can be viewed as an improvement of Bessel beam light sheet microscopes in terms of axial resolution (also termed resolution in z). In Bessel beam light sheet microscopes, a non-diffracting Bessel beam is first created then dithered in the x direction in order to produce a sheet. However, the lobes of a Bessel functions carry as much energy as the central spot, resulting in illumination out of the depth of field of the observation objective. Lattice light sheet microscopy aims at reducing the intensity of the outer lobes of the Bessel functions by destructive interference. To do so, a two-dimensional lattice of regularly spaced Bessel beams is created. Then, destructive interference can be triggered by carefully tuning the spacing between the beams (that is, the period of the lattice). Practically, the lattice of interfering Bessel beams is engineered by a spatial light modulator (SLM), a liquid-crystal device whose individual pixels can be switched on and off in order to display a binary pattern. Due to the matrix nature of the SLM, the generated pattern contains many unwanted frequencies. Thus, these are filtered out by the means of an annulus placed in a plane conjugated with the back focal plane of the objective (Fourier domain). Finally, to obtain a uniform intensity at the sample rather than a lattice, the sheet is dithered using a galvanometer oscillating in the x direction.