Selective Plane Illumination Microscopy (SPIM) is a scientific imaging method that blends the speed of wide-field imaging techniques, with optical sectioning and low photobleaching. Structured SPIM (SSPIM) takes SPIM a step further by supplying an open-source, beam-shaping resource that can produce digital patterns for a number of illumination beams.
The defining quality of SPIM is planar illumination of a sample from the side, which results in a thin slice of the sample being illuminated. This approach mitigates potential photodamage while providing optical sectioning. Since the image is generated in a two-dimensional manner, this technique can be done faster than point-scanned microscopy, which looks at only one point at any given time.
SPIM has quickly become popular for volumetric imaging due to three major features: The minimization of photodamage, high-quality optical sectioning and high speed of acquisition rates.
Producing a Sheet of Light
There are two primary ways to produce a sheet of light for microscopy. A 'static' sheet approach involves the use of a cylindrical lens to focus into the thickness of the sheet, allowing light to spread across the sheet width.
A 'digital' or 'scanned' sheet approach involves the use of a galvo mirror to track a beam of light over the field of view during successive exposures of an imaging camera.
Each method has benefits and drawbacks. Static sheets produce minimal photodamage and are less difficult to produce. However, illumination intensity generally isn’t constant over the entire field. Digital sheets have a uniform intensity and offer a “stop motion” effect that is helpful for shifting samples.
The optimal light sheet is very thin, with intensity restricted to the focal plane of the objective, while still long enough to cover the complete field of view of an imaging device. Unfortunately, light diverges, and therefore the slimmer the sheet, the smaller the area. The length over which the sheet is fairly thin is known as the confocal length and frequently scales quadratically with sheet thickness. The confocal length is usually coordinated to the sample size or field of view.
To gather data in three dimensions, the sheet of light must be passed throughout the sample, or the sample could be moved to produce a stack of pictures. The light sheet can be shifted through the use of a galvo mirror, and the focal plane for the objective must be moved in conjunction. While the acquisition speed is bound by the camera speed and brightness, piezo stages are favored for rapid volumetric imaging for the reason that can return to the primary position more rapidly than motorized stages. Motorized stages are favored for sizeable or flat samples.
SSPIM
The infiltration of the light into the test specimen is afflicted with variations in the refractive index inside the sample itself. The dispersal of the light inside the test sample causes shadows, which are known as a ‘ghost image’ that generate undesirable artifacts over the imaging field of view. A pivoting technique is applied to reduce this unwanted effect. It involves the static light sheet being scanned at various angles within each plane by way of a scanning mirror. Additionally, this issue can be mitigated by using various illumination beams, which can be scanned over the field of view. Other imaging qualities of SPIM can be enhanced by more state-of-the-art, non-diffracting beams.
The various kinds of illumination beams utilized for SPIM applications can be easily produced by digital beam shaping processes by way of a spatial light modulator (SLM). To be able to affect beam shaping, the SLM need some type of digital pattern or mask. SSPIM can effectively produce various digital patterns for beam shaping. SSPIM enables the creation of a variety of beams that have been used eff3ectively in SPIM analyses.
Sources and Further Reading
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