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Light sheet microscopy is essential when imaging large, sensitive, living specimens that are easily damaged by light exposure.
As a fluorescence imaging technique, it utilizes a planar sheet of light in order to illuminate the live sample and excite fluorophores within a specific focal volume. The excitation light can either be created using a cylindrical lens or virtually generated using a scanning beam or dithering beam array.
The latter technique is normally referred to as digitally scanned laser light sheet microscopy (DSLM), while the former is called selective plane illumination microscopy, or SPIM.
Detection occurs along an axis perpendicular to that of illumination so that only the section being observed is lit, rather than the entire specimen or any out-of-focus features. This planar illumination technique produces optical sectioning, or clear images of multiple focal planes within the sample.
Furthermore, using the perpendicular axis reduces the amount of photo-damage, photo-bleaching, and phototoxicity to the specimen so that imaging can occur long-term.
It makes light sheet microscopy more ideal compared to processes like wide-field fluorescence microscopy that can damage your sample more rapidly and reduce long-term viability.
Optical sectioning offers a wide field of view, increased contrast, faster image acquisition speeds, and high axial resolution and spatial resolution. These capabilities support a variety of applications, including the real-time, three-dimensional imaging monitoring of neural responses in live organisms, and enable the 3D reconstruction of a 2D image by computationally combining the image data from a stack of images.
Light sheet microscopy’s principle benefits apply across multiple fields, including drug discovery, neurobiology, developmental biology, embryology, plant biology, and more.
Being able to 3D image everything from large, live organisms, tissue explants, to 3D cell cultures and single cells is crucial in many different areas of research.
This is especially true in developmental biology, where many model organisms are used to understand human development, such as fruit fly embryos (Drosophila melanogaster), an excellent model system for understanding the basic biology underlying our own embryonic development.
Another example of these organisms is the zebrafish, which is primarily used to study vascular development and disease.
Light sheet imaging has been pivotal in 3D examination, avoiding disruption or damage of the organism’s development cycle.
It is first important to mention that there is no perfectly planar light sheet. The sheet is only approximated and given over a range of values using different methods such as static planar sheets or scanned beams that approximate a light sheet over time. Some examples of these methods include:
This optical imaging technique is relatively older, and uses a singular cylindrical lens to project the light sheet into the sample, allowing illumination to be expanded across a single axis. This is also known as selective plane illumination microscopy or SPIM.
Compared to a traditional confocal microscope, acquisition rates using this method are much faster by several orders of magnitude.
Also called a digital sheet, this principle involves scanning a light sheet in lateral directions and then stitching the images together. This creates a larger composite 3D image, and is beneficial for observing moving samples.
The downside of this method is that it scans in both the axial and lateral directions, meaning longer acquisition times and more exposure to light.
Both of the aforementioned techniques can be used in addition to multiphoton excitation. Two photon light sheet microscopy uses a rare phenomenon where two photons are absorbed in a single quantum event. This happens at longer wavelengths that are optimal for imaging deep tissues in high resolution.
This type of excitation coupled with Bessel beam/airy beam light sheets helps reduce unfavorable aspects of those illumination patterns, including out-of-focus excitation/illumination.
This technique was first developed by the lab of Alexander Rohrbach. Bessel beams are used to create digitally scanned light sheets and are formed by shaping the illumination light with an axicon, a specialized type of lens which has a conical surface.
A Bessel beam is a non-diffracting wave that does not “spread out” as light usually does. If a Bessel beam is obstructed, it can “self-heal” and reform further down the axis. These aspects combine to enable enhanced imaging at greater depths within biological specimens. Airy beams function in the same vein, as they are also both non-propagating and “self-healing.”
This type of light sheet fluorescence microscopy is used to accurately observe live cells for longer periods of time without affecting or damaging their behavior. It allows for high speed acquisition and subcellular resolution through the use of a structured light pattern rather than a thicker, non-uniform sheet of light.
It uses the same fundamentals as a Bessel beam microscope, but aims to reduce illumination out of the depth of field of the specimen by creating a lattice of Bessel beams.
LSFM has become the technique of choice to image dynamic processes Because of reduced light dosage and, hence, lower phototoxicity and photobleaching.
To properly analyze complex dynamics within these datasets, image quality has to be improved, either during acquisition or through post-processing, to remove artifacts coming from stripe patterns or to increase signal contrast and penetration depth.
Because LSFM datasets have excellent axial resolution, dynamic range, and low signal to noise, there don’t generally require any preprocessing routines. However, there are limits to image quality.
One of them includes stripe patterns that exist due to the absorption of light on the surface of the sample. The stripes create shadows that impede the use of segmentation tools used to track various cell characteristics.
There are different approaches to handle stripe pattern removal. One examples include post-acquisition processing tools, such as variational stationary noise removal or multiview fusion, which allow the correction of artifacts up to some extent. These methods do require powerful workstations and long processing times, however.
Another example is a double-sided illumination system, which allows researchers to pivot the light sheet illumination angle and create longer integration times at the camera to obtain an average image, remove stripes from shadows during acquisition, and reduce image processing time.
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