Light sheet fluorescence microscope (LSFM; sometimes also called SPIM - Selective Plane Illumination Microscopy) has become a key method for fast and gentle imaging of living specimens in life science and biomedical research. Its potential has been proven with an array of diverse specimens ranging from single cells to organoids and multicellular organisms in developmental biology or in plant science. Unlike other fluorescence imaging techniques such as widefield, spinning disk or confocal microscopy that illuminate and detect the specimen along the same direction and typically through one objective lens, a light sheet fluorescence microscope illuminates and detects the specimen along two orthogonal, typically horizontal directions. This allows the microscope to illuminate only the part of the specimen that is in the focus of the detection objective lens, and thus record a well-focused, high-contrast optical section of the fluorescent specimen.
LSFM illuminates only the section of the specimen that is currently imaged, while most of the specimen remains unexposed. In case of thick specimens that require many sections, planar illumination leads to a massive reduction in photobleaching and phototoxicity. Moreover, as an entire section is recorded in one shot with a high-speed digital camera without the need to scan through every pixel in the image as in confocal laser scanning microscopy, LSFM is also considerably faster. It records a section consisting of millions of pixels in a few milliseconds, and a stack of thousands of such images in a few seconds. The unique combination of speed and gentleness have made LSFM the tool of choice for imaging of large, delicate and dynamic specimens. Examples include cleared tissues, organs and organisms, developing living specimens and cultured organoids - the list is practically endless.
Although recent years have given rise to a number of specialized implementations for diverse research applications, the basic concept and essential building blocks remain similar to those described in early LSFM publications. The illumination optics of shape a laser beam into a thin sheet of light that extends over the microscope’s entire field of view. Instead of using a static light sheet, modern LSFM implementations generate a “virtual” light sheet by digitally scanning a single laser beam sideways through the imaging plane of the specimen.
Due to light diffraction, a light sheet is only thin in the middle of the field of view. Moreover, the thinner the light sheet is in the middle, the quicker its thickness increases towards the sides. Thickness of the light sheet must therefore be fitted to the size of the field of view, which a modern light sheet fluorescence microscope manages with automated zoom optics in the illumination beam path. Additionally, opaque and scattering specimens will deform the light sheet as it travels through the matter. Once the light sheet reaches the far end of the specimen, it is often considerably thicker than optimal, which can be avoided by illuminating the specimen from the opposite direction. For example, the dual illumination option of Lightsheet 7 employs two opposing illumination objective lenses to illuminate the specimen from two opposite directions, and thus efficiently alleviates the problem of light sheet degradation in the sample.
Sidewise illumination of a light sheet microscope can produce stripe-like image artefacts, which are shadows of absorbing particles in the specimen that stretch sideways through an image. These stripes can be reduced by quickly pivoting the light sheet around the specimen so that the specimen is illuminated along a continuously changing direction. Pivoting varies the direction of the shadows in the image and, if the direction changes rapidly, disperse the shadows until they virtually disappear.
When the light sheet is passing through the sample, some structures of the specimen, e.g. nuclei, will absorb or scatter the excitation light. This will cast shadows along the illumination axis, as you see in the left figure. This effect occurs in all fluorescence microscopes, but the illumination axis in light sheet fluorescence microscopy is perpendicular to the observation axis and so this effect is more obvious.
In Lightsheet 7, a patented Pivot Scanner alters the angle of the light sheet upwards and downwards during image acquisition. By altering the illumination angle the shadows will be cast in different directions and excitation light will also reach regions behind opaque structures, as you see in right figure. This patented Pivot Scanner is a perfect way to acquire artifact-free images and to improve downstream processing and analysis steps. It is always better to tackle artifacts right at their origin.
The detection beam path of an LSFM resembles a fluorescence widefield microscope: light is collected through a detection objective lens, illumination light is blocked using an emission filter, and the image is recorded using a CCD or CMOS camera. Additionally, advanced LSFM microscopes like Lightsheet 7 feature zoom optics that allow the microscope to quickly zoom into interesting areas of the specimen. To further increase the imaging speed, advanced LSFM implementations employ two cameras to collect two fluorescence channels in parallel.
Finally, the specimen positioning unit of a light sheet fluorescence microscope translates and rotates the specimen. Most LSFM implementations assemble a three-dimensional image by moving the specimen through the light sheet while recording a series of sections. In case of opaque specimens, sample rotation allows the microscope to image parts of the specimen that are obscured in any single view. The Multiview mode for Lightsheet 7 uses specimen rotation to record multiple three-dimensional images from different directions relative to the specimen and computationally assemble them into a single, high-quality image, typically unattainable by other microscopy methods.
During the experiment, the specimen is submerged in a solution that is contained in an experimental chamber. The solution is chosen to provide near-physiological conditions for a living specimen and optimal imaging conditions for cleared and non-cleared specimens.
Light sheet microscopy has been successfully applied in almost all fields of biological and medical research that typically rely on fluorescence microscopy, but it has proven especially potent for imaging of large, delicate and dynamic specimens. For example, LSFM in neuroscience allows scientists to record entire animal brains with cellular resolution, in morphogenesis and physiology research LSFM enables imaging of whole organs. Developmental biology was furthered by recordings of the formation of entire organs and embryos with a spatial and temporal resolution that allows individual cells in embryogenesis and morphogenesis to be tracked over many cell divisions. In embryos and small organisms, cellular dynamics such as cell migration, cardiac development, blood flow, vascular development, neuronal development or calcium imaging can be recorded and visualized over time and in three dimensions. Plant biologists can follow biological processes in organogenesis over hours and days with exceptional light efficiency and next to no photo damage, and cell biologists gently image three-dimensional cell cultures, spheroids and organoids, for example in tumor and cancer research.
Finally, emerging tissue clearing methods generate specimens that are clear and well suited for optical microscopy, but often too large to be imaged with traditional fluorescence microscopy techniques. LSFM with its speed and the possibility to have large, intact specimens such as tissue sections, brains, embryos, organs, spheroids or biopsies fully immersed in a medium during imaging is a perfect companion for these clearing methods.
Imagine you had access to an imaging system that could deliver optical sections of large samples, with virtually no phototoxicity or bleaching and with high temporal resolution. That is exactly what Lightsheet 7 from ZEISS does. The unique Multiview light sheet fluorescence microscope allows you to record the development of large, living samples and gently image them to deliver exceptionally high information content. It is also fast: Lightsheet 7 is your microscope for optical sections at high speed. Acquire images of your whole sample volume at sub-cellular resolution – in a fraction of the time it takes using other techniques.
Enhance your Lightsheet Z.1
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Adjusting Refractive Index for Clearing Applications
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Application Note: Fast Imaging of Cellular Spheroids with Light Sheet Fluorescence Microscopy
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