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The fluorescence microscopy technique known as light sheet fluorescence microscopy (LSFM) has a high speed, good optical sectioning capabilities, and an intermediate-to-high optical resolution.
In contrast to epifluorescence microscopy, only a little slice of the sample is illuminated perpendicular to the direction of observation (often a few hundred nanometers to a few micrometres).
A laser light-sheet, or laser beam that is exclusively focused in one direction, is used for illumination (e.g. using a cylindrical lens).
In a second technique, the lightsheet is produced by scanning a circular beam in a single direction. This technique lessens the photodamage and stress generated on a living sample because only the region being studied is lit.
Moreover, the strong optical sectioning capacity lowers the background signal, increasing contrast and producing images similar to those obtained with confocal microscopy.
The illumination in this technique of microscopy is carried out perpendicularly to the direction of observation (see schematic image at the top of the article).
A cylindrical lens or a combination of a cylinder lens and a microscope objective, the latter of which is more readily available and has a higher numerical aperture than the former, concentrates the enlarged laser beam exclusively in one direction.
In this manner, a lightsheet or thin sheet of light is produced in the focal region that can be utilised to activate fluorescence only in a thin slice of the sample, typically one or two micrometres thick.
Following that, a normal microscope objective is used to gather the fluorescence light generated by the lightsheet perpendicularly, and it is then directed onto an imaging sensor.
An observation objective with a high working distance is utilised in order to leave ample room for the excitation optics and lightsheet.
The sample and the excitation/detection optics are typically embedded into a buffer-filled sample chamber, which can also be used to control the environmental conditions (temperature, carbon dioxide level) during the measurement.
This is the case for the majority of light sheet fluorescence microscopes. This is a more thorough explanation of sample mounting in light sheet fluorescence microscopy.
As the focal plane of the detection optics and the excitation lightsheet must coincide for an image to be formed, focusing different portions of the sample cannot be accomplished simply translating the detection objective; instead, the entire sample is often translated and rotated.
The Global light sheet microscopy market accounted for $XX Billion in 2023 and is anticipated to reach $XX Billion by 2030, registering a CAGR of XX% from 2024 to 2030.
The non-destructive microtome and microscope known as light sheet fluorescence microscopy (LSFM) employs a plane of light to optically segment and study tissues with subcellular resolution.
In contrast to wide-field fluorescence, confocal, or multiphoton microscopy, this technique is well suited for imaging deep within transparent tissues or within entire organisms.
Additionally, because tissues are only exposed to a thin plane of light, specimen photobleaching and phototoxicity are reduced.
For the three-dimensional reconstruction of tissue structures, LSFMs yield well-registered serial sections. Many variations of light sheet microscopes have been built by various researchers due to the unavailability of a commercial LSFM microscope.
This article discusses the history of the technique, examines current products, describes one LSFM device in detail, and includes examples of pictures and three-dimensional reconstructions of tissues made with LSFM.
Transparent tissues or complete organisms that have been fluorescently labelled can be optically sectioned using light sheet fluorescence microscopy (LSFM), which uses a thin plane of light.
demonstrates that when compared to other nondestructive tomographic techniques like magnetic resonance imaging (MRI) and computed tomography, LSFM delivers higher resolution (subcellular) and faster imaging speed (CT).
Because the specimen is only exposed to a narrow light sheet, LSFM can photograph thicker tissues with less photobleaching and phototoxicity than confocal and two-photon microscopy.
Moreover, LSFM is a nondestructive technique that yields optical sections with good registration that are appropriate for three-dimensional reconstruction and that can be processed by other histological techniques (such as mechanical sectioning) after imaging. In addition, LSFM devices are far less expensive to build than these other microscopes.
