May 6 2014
Light sheet fluorescence microscopy (LSFM), also known as selective plane illumination microscopy (SPIM), is a non-destructive, versatile imaging tool, which operates as a microscope and microtome, using a plane of light to optically section and view tissues and living organisms with remarkable sub-cellular resolution.
LSFM basically operates by illuminating a sample from the side in the focal plane of a detection path. The illumination and detection paths are perpendicular to each other. The sample is placed at the intersection of the axis of the illumination and the detection paths. The light sheet excites the sample, and the emitted fluorescence is collected by detection optics.
In 2012, LSFMs became available commercially. The award-winning Lightsheet Z.1 from Zeiss, a specialist in the optical and opto-electronic sector, is an example of a multiview light sheet fluorescence microscope. It is designed to record the development of large, living samples and image them in an unobtrusive way.
Benefits of Light Sheet Microscopy
LSFMs are photon efficient, as they generate photons only in the focal plane and not in other layers. Multi-color fluorescence imaging is possible with LSFMs.
(Image source Carl Zeiss)
Advanced camera technology, such as EMCCD and sCMOS, enables fast and sensitive detection. When Neo sCMOS technology is combined with the LSFM method, the complete development of the nervous system can be viewed with excellent temporal resolution. LSFMs provide high imaging speed, as well as high signal-to-noise ratio.
By rotating the sample, this innovative tool can image virtually any plane - with multiple views gathered from different angles. They produce clear serial sections that can be used for 3D reconstruction of tissue structures.
They are ideal for imaging deep within transparent tissues, or within whole organisms. Good penetration is achievable even in scattering tissues. As tissues are exposed to only a thin plane of light, photobleaching and phototoxicity are comparatively less than in confocal, wide-field fluorescence, or multiphoton microscopy. Hence it is possible to conduct far more scans per specimen.
Applications of Light Sheet Microscopy
As LSFMs have only been commercially available since 2012, few fields, such as material macrography, life sciences, photomicrography and developmental biology, have begun exploring the technology closely.
In developmental biology, this technology enables prolonged observations of embryonal development in large organisms. It is possible to view live in-toto-imaging of organs with minimal phototoxicity.
In life sciences, it is used in applications such as live imaging of fast dynamic processes in single cells. When combined with tissue clearing techniques, this technology enables easy and quick imaging of large samples, with superior coverage and high spatial resolution.
Samples up to the size of entire mammalian brains can be recorded and quantitatively analyzed in an efficient manner.
In material macrography, LSFM tools are used to visualize the surface of minerals and metals.
LSFM strikes the right balance between confocal/multiphoton microscopes, and magnetic-based, low-resolution microscopes. Although new in the market, LSFM tools are being developed and improved by scientists in laboratories at a rapid pace, due to the numerous advantages they offer. Scientists are aiming to provide higher resolution and faster imaging times by applying structured illumination, stimulated emission depletion, and two-photon instrument modifications.
Other modifications, such as using whole-tissue immunohistochemical antibody methods and fluorophore-labeled molecules, are also being introduced so that tissue structures can be labeled, and automatic segmentation is feasible.