Light Sheet Microscopy
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Abstract:
This chapter focuses on the basics of the light sheet microscopy principle and construction, followed by an overview of its various implementations. It discusses some of the novel data handling solutions that light sheet microscopy gave rise to, as well as future prospects in this area. Two different ways of generating a light sheet are commonly used: in selective plane illumination microscopy (SPIM), the laser beam is expanded and focused using a cylindrical lens to form the sheet of light. Alternatively, a "virtual" light sheet can be generated by the digitally scanned light sheet microscopy (DSLM) technique, which uses a scanning mirror to rapidly sweep a beam across the field of view (FOV). The chapter discusses how to acquire 2D images of a sample using light sheet microscopy. However, most biological applications require 3D imaging. Given its excellent optical sectioning capability, speed, and low phototoxicity, light sheet microscopy is ideally suited for fast 3D imaging.Keywords:
Optical sectioning
Bright-field microscopy
Structured Light
We present a single-pixel microscope with optical sectioning by combining two structured illumination methods: structured illumination microscopy (SIM) and single-pixel imaging (SPI). Experimental results are shown for the application in 3D fluorescence microscopy by scanning different axial planes.
Optical sectioning
Photoactivated localization microscopy
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Optical sectioning
Micrometer
Bright-field microscopy
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Selective plane illumination microscopy (SPIM) offers an alternative way of optically sectioning the sample in fluorescence microscopy. By illuminating the sample with a sheet of light, a sectioning effect can be obtained that is similar to that in confocal microscopy. However, in combination with widefield detection, such an approach has several advantages over confocal scanning microscopy. SPIM offers reduced fluorophore1 bleaching, fast, highly efficient image recording, and high depth penetration, especially when multiple views are combined. SPIM performs especially well in large samples such as fish or fly embryos, which can be observed live for several days. The principle is universal and can also be applied to micrometre-sized samples.
Optical sectioning
Bright-field microscopy
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In light microscopy (or optical microscopy) visible light is either transmitted through or reflected from the specimen before it is observed or recorded. In its simplest imaging mode, bright field microscopy, the illumination light is modulated in intensity or color depending on the specimen's transmission or reflection properties before it enters the objective lens. The general drawbacks of this are limited resolution (which is constrained by the wavelength of visible light) and limited contrast. Several techniques exist for enhancing the contrast of light microscopes, such as dark field microscopy, phase contrast microscopy, (differential) interference contrast microscopy, or fluorescence microscopy. Most of them are applied to make otherwise invisible transparent objects, such a biological structures like cells, visible. Specimens that are too thick for transmitting light, however, require a reflected illumination - for which there are few alternatives for contrast enhancements.
Bright-field microscopy
Dark field microscopy
Polarized light microscopy
Visible spectrum
Interference microscopy
Digital Holographic Microscopy
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Significance: Currently, tissue biopsies are sectioned into 3- to 5-μm-thick slices that are used for conventional pathology analysis. Previous work by confocal microscopy and light-sheet microscopy has shown that analyzing biopsies intact in three-dimensions (3D) is possible and may lead to a better understanding of cancer growth patterns. Although accurate, these methods require fluorescent staining of the tissue, in addition to tissue clearing. If the 3D biopsy analysis could be done sufficiently swiftly, this approach may be used for on-site assessment of the adequacy of a biopsy taken. Aim: We aim to show that, by transmission microscopy of optically cleared tissue punches, the tissue architecture can be determined without the need for fluorescent staining. Approach: Transmission microscopy is used by combining bright field microscopy with dark field and epifluorescent microscopy to compare samples that have also been analyzed by fluorescent confocal microscopy. Results: With increasing distance to the focal plane, the higher-frequency part of the spatial frequency spectrum of transmitted light is attenuated increasingly. This property is exploited for tissue segmentation, detecting whether tissue is present at a certain position in the focal plane image. Using this approach, we show that a 3D rendering of the internal cavity or tubules structure of punch biopsies, which are up to 1-mm thick, can be acquired in ≈1 min scan time per imaging modality. The images of the overall tissue architecture that are obtained are similar to those from the confocal microscopy benchmark, without requiring fluorescent staining. Conclusions: Images of the overall tissue architecture can be obtained from transmission microcopy; they are similar to those from the confocal microscopy benchmark without requiring fluorescent staining. Tissue clearing is still needed. The total scan time of the present method is significantly shorter at a fraction of the device costs.
Bright-field microscopy
Optical sectioning
Autofluorescence
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We present a general summary of light sheet based microscopy for non-destructive optical sectioning of organisms and thick tissues. Optical sections are recorded using a thin sheet of light to induce a plane of fluorescence in transparent or fixed and cleared tissues. We explain the basic building units and compare our thin sheet laser imaging microscope (TSLIM) to similar microscope systems.
High resolution across the full width of a large specimen is achieved by moving the specimen through the thinnest region of the hyperbolically shaped light sheet, stitching together pixel columns with optimal resolution. We also show how to reduce absorption and scattering artefacts and explain optimizations to the optical system which help to produce thinner light sheets than achieved with cylindrical lenses alone.
Optical sectioning
Image stitching
Clearance
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Light-sheet microscopy, also named selective-plane illumination microscopy, enables optical sectioning with minimal light delivered to the sample. Therefore, it allows one to gather volumetric datasets of developing embryos and other light-sensitive samples over extended times. We have configured a light-sheet microscope that, unlike most previous designs, can image samples in formats compatible with high-content imaging. Our microscope can be used with multi-well plates or with microfluidic devices. In designing our optical system to accommodate these types of sample holders we encounter large optical aberrations. We counter these aberrations with both static optical components in the imaging path and with adaptive optics. Potential applications of this microscope include studying the development of a large number of embryos in parallel and over long times with subcellular resolution and doing high-throughput screens on organisms or cells where volumetric data is necessary.
Optical sectioning
Optical path
Sample (material)
Image plane
Bright-field microscopy
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A 3D structured light sheet microscope using a four-faceted symmetric pyramid is presented.The sample is illuminated by the resulting four beam interference field.This approach combines advantages of standing wave and structured illumination microscopy.Examples of micrographs of fluorescently labeled Chinese hamster ovary (CHO) cells as well as of the compound eyes of drosophila are shown and the optical sectioning ability of our system is demonstrated.The capabilities and the limitations of the scheme are discussed.
Optical sectioning
Bright-field microscopy
Interference microscopy
Structured Light
Pyramid (geometry)
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We report the development of a modular and optimized thin-sheet laser imaging microscope (TSLIM) for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear, and rat brain at a resolution that is comparable to wide-field fluorescence microscopy. TSLIM optically sections tissue using a thin sheet of light by inducing a plane of fluorescence in transparent or fixed and cleared tissues. Moving the specimen through the thinnest portion of the light sheet and stitching these image columns together results in optimal resolution and focus across the width of a large specimen. Dual light sheets and aberration-corrected objectives provide uniform section illumination and reduce absorption artifacts that are common in light-sheet microscopy. Construction details are provided for duplication of a TSLIM device by other investigators in order to encourage further use and development of this important technology.
Optical sectioning
Image stitching
Microtome
Clearance
Bright-field microscopy
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