Applications of Light Sheet Microscopy Techniques
The ultimate goal of biological imaging was to be able to visualize increasingly physiologically relevant systems. A string of recent technological advances has been drawing such a goal closer for many years and now, with the wide range of sophisticated research techniques available, it has finally become feasible.
An important aspect in observing biological experiments is the impact of the imaging techniques used. Visualization needs to be achieved in a manner that maintains the physiological integrity of the system under study and minimizes perturbations and the introduction of artefacts. One of the most challenging aspects of biological imaging is minimizing or, if possible, avoiding photodamage and phototoxicity. Exposure to light, and especially the high intensities of light needed for fluorescent imaging, can dramatically impact the function and well-being of living cells and organisms. Imaging approaches that minimize exposure of the specimen to light were thus needed.
The answer came in the form of light sheet microscopy, which reduces phototoxic effects by illuminating the specimen in only a single plane at a time.
Light sheet microscopy
Light sheet microscopy is a general name describing a growing family of planar illumination techniques that have revolutionized the optical imaging of biological specimens. It was made possible by separating the illumination and detection optical pathways so that novel methods of less damaging illumination could be adopted.
The light sheet that forms the basis of the technique is formed by laser light shaped into a hyperbolic ‘sheet’ of illumination1. Alternatively, an approximation to a light sheet can be reached using a scanned beam. Detection is performed along a different axis from that of the illumination. The use of separate axes maximizes detection efficiency whilst minimizing artefacts from features that are not in the field of focus. Typically, high-sensitivity light sheet microscopy uses two objectives for illumination and a further two for detection in order to double the amount of light collected in each plane.
The specimen is placed at the intersection of the axis of the illumination and the axis of detection. The excitation of the sample by the light sheet results in the emission of fluorescence, which is detected by high-speed cameras to produce high quality images. The plane of imaging can be easily changed by rotating the sample, thereby providing serial sections of the specimen that can be reconstructed to provide a 3D representation.
Light sheet microscopy solutions
Andor have developed cameras specifically designed to serve as detectors for high-speed light sheet microscopy2. The Neo and Zyla range of sCMOS cameras offer a large field of view and high resolution without compromising read noise or frame rate. In addition, the small pixel size (6.5 micrometers) ensures there is sufficient oversampling of the point spread-function, even for objectives with low magnification. The iXon back-illuminated EMCCD camera platforms provide single photon sensitivity and exceptional speed performance whilst maintaining quantitative stability throughout.
Applications of light sheet microscopy
Light sheet microscopy is a versatile and virtually non-destructive imaging tool. It provides faster imaging speed and higher resolution than other non-destructive imaging techniques, such as magnetic resonance imaging and computerized tomography. It is therefore the ideal technique for imaging sensitive samples or fast dynamic processes.
Using a plane of light to achieve optical sectioning of a living organism or tissue, visualisation of the sub-cellular structure is achieved with high resolution. The use of only a thin sheet of light means it is possible to perform several scans of a particular specimen without risking photobleaching or phototoxicity. Consequently, multiple views may be acquired from different angles and the series of sections used to obtain a 3D reconstruction of tissue structures.
By enabling the rapid imaging of biological samples, including those larger than can be viewed with other microscopy techniques, with high resolution, light sheet microscopy has advanced research in many fields.
It has proved to be a great tool for the study of embryonic development, generating quantifiable data and overall cell and tissue characteristics of morphogenetic processes3. Imaging of whole living embryos preserves many factors regulating development, such as the gradients of signalling molecules, so accurate observation of the precise developmental pathway can be achieved. High-speed image acquisition allowed high resolution visualization of axon guidance and growth cone dynamics during embryonic neuronal development in nematodes, despite considerable fast movement and twitching of the embryos4. Similarly, the entire embryonic nervous system of Drosophila was imaged using light sheet microscopy providing insight into the cellular dynamics of the development of the central and peripheral nervous system5.
Light sheet microscopy is also a valuable tool for rapid, high-resolution quantitative mapping of the structure and function of large biological systems. It has been used to image a clarified thick coronal slab of human brain and the non-isomorphic body shape changes of the hydra6. Similarly, it has facilitated 3D live imaging of cellular and sub-cellular behaviours in multicellular specimens7.
In addition to research applications, light sheet microscopy has the potential to provide rapid analysis of clinical specimens to inform treatment decisions. It has been shown that the technique enables rapid 2D and 3D imaging of intact tissues with the same level of detail as traditional pathology8. For example, it could provide rapid intra-operative assessment of tumour-margin surfaces or volumetric assessment of optically cleared core–needle biopsies.
The future of light sheet microscopy
Light-sheet microscopy is a versatile tool capable of both rapid surface microscopy and deep volumetric microscopy of specimens. The non-destructive nature of the technique means it can be applied to the study of a wide range of living biological systems. Furthermore, it can provide live 3D imaging with only minimal risk of photodamage.
This relatively new imaging methodology has already found numerous applications across both cell biology and developmental biology. With on-going modifications to the basic technique to adapt it to specific research requirements, the application of light-sheet microscopy is set to expand vastly. In addition, the development of visualization tools and pattern recognition software will further improve the interpretation of the data acquired and facilitate the generation of models for human physiology and disease.
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- Andor. https://andor.oxinst.com/products/
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- Glaser AK, et al. Nat Biomed Eng. 2017;1(7):0084.