The ultimate aim of biological imaging is to be in a position to visualize systems that are increasingly physiologically relevant. A series of technological advances over the past two decades brought such an aim closer, and the broad array of advanced research methods available means it is now possible to visualize many systems.
A crucial aspect of visualizing biological experiments is the effect of the imaging techniques that are used. It is necessary to achieve visualization such that it preserves the physiological integrity of the system being studied and reduces perturbations and the introduction of artifacts.
One of the most difficult aspects of biological imaging is reducing or, perhaps, eliminating phototoxicity and photodamage. Exposure to light, specifically the high intensities of light required for fluorescent imaging, could have a drastic effect on the function and well-being of organisms and living cells. Hence, there was a need for imaging techniques that reduce the exposure of the specimen to light.
The solution was light sheet microscopy, which minimizes phototoxic effects by illuminating the specimen in only a single plane at a given instant of time.
Light Sheet Microscopy
Light sheet microscopy is a common term that describes an expanding class of planar illumination methods that have totally transformed the optical imaging of biological specimens. It was rendered feasible by isolating the illumination and detection optical pathways to enable the use of innovative techniques of less damaging illumination.
The light sheet that is the foundation for the method is formed by laser light shaped into a hyperbolic “sheet” of illumination.1 As an alternative, an approximation to a light sheet can be achieved using a scanned beam. Detection is carried out along an axis that is different from that of the axis of illumination.
The use of separate axes enhances the detection efficiency and reduces the artifacts from features that are not in the field of focus. In general, high-sensitivity light sheet microscopy uses two objectives for illumination and another two for detection. This doubles the amount of light collected in every plane.
The specimen is positioned at the intersection of the axis of detection and the axis of illumination. When the sample is excited by the light sheet, it leads to the emission of fluorescence, which can be detected by high-speed cameras to generate high-quality images. The sample can be rotated to easily change the imaging plane, thus providing serial sections of the specimen that can be reconstructed to offer a 3D representation.
Light Sheet Microscopy Solutions
The cameras designed by Andor are specifically aimed at serving as detectors for high-speed light sheet microscopy.2 The Neo and Zyla range of sCMOS cameras provide high resolution and a large field of view without compromising on the frame rate or read noise.
The small pixel size (6.5 μm) guarantees there is adequate oversampling of the pointspread-function, even for objectives that have low magnification. The iXon back-illuminated EMCCD camera platforms offer exceptional speed performance and single photon sensitivity, apart from maintaining quantitative stability throughout.
Applications of Light Sheet Microscopy
Light sheet microscopy is a virtually non-destructive and valuable imaging tool. It offers higher resolution and faster imaging speed compared to other non-destructive imaging methods, such as computerized tomography and magnetic resonance imaging. Hence, it is the perfect method for imaging sensitive samples or rapid dynamic processes.
The high-resolution visualization of the sub-cellular structure is realized by using a plane of light to enable optical sectioning of a living organism or tissue. The use of just a thin sheet of light indicates that it is feasible to carry out multiple scans of a particular specimen without leading to phototoxicity or photobleaching. As a result, multiple views can be obtained from different angles and the series of sections applied to achieve a 3D reconstruction of tissue structures.
Light sheet microscopy has advanced research in various fields by facilitating the rapid imaging of biological samples, including those larger than can be viewed with other microscopy methods, with higher resolution.
It has been shown to be an optimal tool for the analysis of embryonic development, producing quantifiable data and qualitative cell and tissue characteristics of morphogenetic processes.3 Various factors that regulate development such as the gradients of signaling molecules can be preserved through the imaging of whole living embryos, thereby achieving accurate observation of the precise developmental pathway.
High-speed image acquisition enabled high-resolution visualization of axon guidance and growth cone dynamics at the time of embryonic neuronal development in nematodes, in spite of considerable fast movement and twitching of the embryos.4
Likewise, light sheet microscopy was used to image the complete embryonic nervous system of Drosophila, thus enabling the cellular dynamics of the development of the central and peripheral nervous system to be understood.5
Light sheet microscopy is also a versatile tool for performing rapid, high-resolution quantitative mapping of the structure and function of large biological systems. It can be used to image the non-isomorphic variations in the body shape of the hydra as well as a clarified thick coronal slab of human brain.6 Likewise, it has enabled 3D live imaging of cellular and sub-cellular functions in multicellular specimens.7
Apart from research applications, light sheet microscopy has the ability to facilitate rapid analysis of clinical specimens to inform treatment decisions. It has been demonstrated that the method allows rapid 2D and 3D imaging of intact tissues with the same level of detail as conventional pathology.8 For instance, it can enable quicker intra-operative evaluation of tumor-margin surfaces or volumetric assessment of optically cleared core-needle biopsies.
The Future of Light Sheet Microscopy
Light sheet microscopy is a valuable tool with the potential to perform not only rapid surface microscopy but also deep volumetric microscopy of specimens. Thanks to its non-destructive nature, it can be used to study a broad array of living biological systems. Moreover, it has the ability to allow live 3D imaging with only minimal risk of photodamage.
This comparatively new imaging technique has already been applied for numerous applications across both developmental biology and cell biology. The on-going modifications to the fundamental technology to adapt it to particular research needs are expected to largely increase the application of light sheet microscopy.
Furthermore, the interpretation of the obtained data will be further enhanced by the development of visualization tools and pattern recognition software, thereby enabling the generation of models for human physiology and disease.
References and Further Reading
- Santi PA. J Histochem Cytochem. 2011; 59(2):129–138.
- Andor. https://andor.oxinst.com/products/
- Icha J, et al. J Vis Exp. 2016;110:53966.
- Wu Y, et al. PNAS, USA, 2011; 108(43):17708‑17713.
- Tomer R, et al. Nat Methods. 2012;9(7):755‑63.
- Migliori B, et al. BMC Biol. 2018;16:57.
- Fu Q, et al. Nat Commun. 2016;7:11088.
- Glaser AK, et al. Nat Biomed Eng. 2017;1(7):0084.
This information has been sourced, reviewed and adapted from materials provided by Andor Technology Ltd.
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