Single Plane Illumination Microscopy

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Single Plane Illumination Microscopy (SPIM) is a non-invasive method of observing an organism and gaining as much biological data from the specimen as possible.

The technique of observing a biological specimen through SPIM is very similar to regular light microscopy, in the sense that the microscope detecting a beam of light being passed through a specimen and producing an image. However, rather than having the specimen on a flat glass slide, SPIM requires the specimen to be placed inside a cuvette whilst suspended in a liquid. A singe plane of light is then passed through the sample with a laser, then a camera detects any fluorescent signals emitted perpendicularly to the plane.

How Single Plane Illumination Microscopy Works

This type of microscopy is used to analyse larger organisms, which are still living. Traditional light microscopy techniques presented challenges when observing larger, multicellular organisms and samples, due to these specimens having light scattering and absorptive qualities. SPIM can generate multi-dimensional, high-resolution images of a large biological sample, by taking advantage of the originally challenging aspects of using light microscopy to analyse the specimen.

SPIM is comprised of 5 sections that each address a different aspect of SPIM: illumination of the specimen, generation of the appropriate light beam, light detection, translation/rotation of the specimen, and finally, the control of the many mechanical parts alongside the collection and processing of the final data.

Once all these aspects have been addressed, a specimen can be collected, placed into a cuvette, and rotationally observed to product the final high-resolution, multi-dimensional image. This image can be used to analyse both the outer surface layers and inner biomolecular structures in multicellular organisms (e.g. a forming embryo) from many different viewpoints.

Uses of Single Plane Illumination Microscopy

This specific microscopy method is becoming an increasingly popular method of analysing developmental biological samples. Using light microscopy samples a few millimetres in diameter are unable to be analysed, whereas SPIM enables the study of biological samples like whole intact animal embryos.

This also means that the three-dimensional structure of an embryo can be observed at a much higher resolution than some other current multi-dimensional methods of microscopy. This can assist in the diagnosis of early-presenting developmental problems when observing the embryo in-vitro – before, for example, implanting said embryo into a womb via in-vitro fertilisation.

Other issues presented by basic light microscopy include the possibility of premature photo-bleaching, as well as background-distortion due to the focus not being precise enough. These factors can affect the observation and subsequent analysis of mitochondrial dynamics, membrane ruffles, filopodia, mitotic chromosomes, intracellular vesicles and other cellular components. Through the use of SPIM, these issues can be avoided. The light beams produced are on a single plane, preventing long-term light exposure bleaching. In addition to this the final images produced no longer have out-of-focus backgrounds due to the images being rotational, high in resolution and multi-dimensional.

Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane

To obtain even higher resolution images – which are required for advancing in-depth research in the life sciences, the quantitative measurement of certain molecular parameters performed by SPIM can be combined with camera-based fluorescence correlation spectroscopy (FCS). This combination of techniques can produce diffusion coefficient images, as well as contiguous particle number(s).

In 2010, the Optical Society of America used this technique to assist them in studying live zebrafish (Danio rerio) embryos. Using this method of microscopy meant that they could observe intricate biomolecular interactions within and between each of the cells, all within a physiologically relevant multi-dimensional environment.