Editorial Feature

A Guide to Lattice Light Sheet Microscopy

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Lattice light sheet microscopy is a newer light sheet microscopy technique that is primarily used in biological settings. It is a method that uses sheets of light in a lattice conformation to take images and videos of a sample by causing them to fluoresce. Compared to other methods, it is a technique that is low in both photobleaching and phototoxic effects, and this is one of the main reasons—alongside being able to image live samples—why it is mainly used in a biological setting. In this article, we explain what lattice light sheet microscopy is and how it works.

What is Lattice Light Sheet Microscopy?

Lattice light sheet microscopy is a modified and improved version of light sheet fluorescence microscopy. Fluorescence light sheet microscopy is a technique that uses the light from a laser to illuminate a sample and only illuminates a small section of a sample at any one time. Upon illumination, the sample fluoresces, and this is picked up by the detector. Lattice light sheet microscopy is like its predecessor, but it uses a series of light beams in a lattice configuration to image a sample of interest.

To create a lattice pattern, thin light beams are required, and this translates to the sample being exposed to a lesser amount of light—which in turn reduces the degree of phototoxicity that the sample is subjected to and reduces the possibility of photobleaching on the image. Lattice light sheet microscopy also images the sample in planes, so the sample is never exposed to too much light at any one time, and this is another reason why phototoxic and photobleaching effects are minimized. It is a technique that is often used in a biological setting to take 3D images of cells by reconstructing the images taken of each plane into a single image, to making continuous fluorescence movies of a sample, and for characterizing biological phenomena. It is also an imaging technique that can be used on live biological samples.

Lattice light sheet microscopy is a technique that also has a very high resolution and is much better than other comparative techniques, such as confocal microscopy and two-photon microscopes. Some of the other benefits of this technique include high contrast, high acquisition speed, adjustable imaging parameters, images and videos with multiple colors, the ability to video samples for hours at a time, and the ability to use different imaging modes to suit the specific imaging application.

How Lattice Light Sheet Microscopy Works

Whilst the internal workings of lattice light sheet microscopy are similar to light sheet fluorescence microscopy, it also incorporates principles from Bessel beam microscopy and structured illumination microscopy (SIM).

In lattice light sheet microscopy, the illumination of the sample occurs perpendicular to where the image is detected. The first part to the imaging process is the generation of the lattice light sheet itself, which is done in multiple stages. The first part of the light sheet generation process involves using two pairs of cylindrical lenses to stretch the linearly polarized circular input beam to form a sheet that is then projected on to a spatial light modulator. The first pair stretches the light beam along the x-axis and the second pair along the z-axis. The spatial light modulator varies the waveform of the light beam so that any unwanted diffraction is eliminated, and this leads to the generation of an optical lattice similar to that of Bessel beams. The next stage employs an annular mask—an opaque optical mask which contains a transparent annulus—to further reduce the diffraction of the light beam and lengthen the light sheet. The final stage is to either the light laterally (x-direction) so that the image is recorded in the z-plane.

The light sheet then takes an image, or a real-time video, of the sample, but images lateral slices and builds up a 3D picture from the resulting images. For videos, the sample is continuously scanned, whereas the sample is only scanned once for images. The images are captured using an electron multiplying charge coupled device (EMCCD) camera.

But there are some variations to the imaging modes. First off, in the mode known as sheet scan, the light sheet and the objectives are moved together to generate a 3D image. This differs to sample scan mode where the sample is moved but the light sheet and the objectives remain stationary. The sample scan mode is often used for larger samples. The imaging mode that is the most distinct from the conventional imaging modes is the structured illumination microscopy mode. In this mode, the light sheet is stepped along multiple phase steps in the x-axis and a grid pattern of light is superimposed on the sample and rotated at every phase step. This method can be done in either sheet scan or sample scan mode and uses computer algorithms to reconstruct the images from each step and produce an image that is beyond the diffraction limit of the objectives in the microscope.

Sources and Further Reading

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Liam Critchley

Written by

Liam Critchley

Liam Critchley is a writer and journalist who specializes in Chemistry and Nanotechnology, with a MChem in Chemistry and Nanotechnology and M.Sc. Research in Chemical Engineering.

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