Threedimensional live imaging of filamentous fungi with light sheet based fluorescence microscopy lsfm francesco pampaloni, laura knuppertz, andrea hamann, heinz d. Lightsheetbased fluorescence microscopes lsfms, such as the european molecular biology. Light sheet based microscopy, such as spim is a new method for imaging large fluorescent samples 1. Light sheetbased fluorescence microscopy of living or fixed. Lightsheetbased fluorescence microscopy lsfm offers a favorable combination of these requirements and is therefore currently established as the technique of choice for the study of threedimensional cell cultures. Light sheet fluorescence microscopy was performed using a homebuilt instrument similar in design to that of keller et al. More dimensions, more photons, and less photodamage. Light sheetbased microscopy, such as spim is a new method for imaging large fluorescent samples 1. The excitation path is perpendicular to the detection path. Similarly, the light sheet microscope has origins that are over 100 yr old and are based on the ultramicroscope siedentopf and zsigmondy, 1903. Light sheet fluorescence microscopy lsfm is gaining more and more popularity as a method to image embryonic development. In light sheetbased fluorescence microscopy lsfm, optical sectioning in the excitation process minimizes fluorophore bleaching and phototoxic effects.
Colombelli 2009 and for fluorescence correlation spectroscopy fcs, wohland 2010. Recently, we developed digital scanned laser light sheet fluorescence microscopy dslm, in which a single plane in a specimen is quickly scanned with a thin laser beam while a camera based. Light sheet fluorescence microscopy versus confocal. Another way to look at it is that a light sheetbased fluorescence. Deeplearning lightsheet fluorescence microscopy for high. The protocol is written for light sheet based fluorescence microscopes that are equipped with a sample chamber and a rotatable clamp mechanism for standardized sample holders54,59,60, which are typically cylindershaped elements made of metal, plastic or glass with a diameter in the millimeter range. The principle of light sheetbased fluorescence microscopy. Fundamentally, lsfm techniques are made possible by decoupling the illumination and detection optical pathways, allowing for novel. We demonstrate its application for imaging zebrafish eye development across wide spatiotemporal scales and present a pipeline for fusion and deconvolution of.
Reconstruction of zebrafish early embryonic development by. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled. Photon budget is how many photons a dye can produce before it goes dark. Frontiers applications of lightsheet microscopy in. Light sheet based fluorescence microscopy lsfm takes advantage of modern camera technologies. Lightsheet fluorescence microscopy for quantitative. The protocol is also suitable for both canonic implementations, i.
Light sheet fluorescence microscopy by zeiss adapted by dirk pacholsky, biovis facility, uppsala, sweden for methods for cell analysis, 2014. During the last few years, lsfm was used to record zebrafish development from the early 32cell stage until late neurulation with subcellular resolution and short. Fast, highcontrast imaging of animal development with. Fundamentally, lsfm techniques are made possible by decoupling the illumination and detection optical pathways, allowing for novel illumination strategies that. In contrast to epifluorescence microscopy only a thin slice usually a few hundred nanometers to a few micrometers of the sample is illuminated perpendicularly to the direction of observation. Fast highcontrast imaging of animal development with scanned. Please use one of the following formats to cite this article in your essay, paper or report. Light sheet based fluorescence microscopy lsfm allows the imaging of large threedimensional specimens over periods of several days 7. The light sheet and the focal plane of the detection microscope objective overlap fig. Further improvements in 3d microscopy require limiting the light exposure of the sample and increasing the volumetric acquisition rate. The new player is light sheet fluorescence microscopy, an illumination approach orthogonal to the detection that allows for a single plane illumination. Lightsheet fluorescence microscopy with structured light. Lsfm can be operated with laser cutters and for fluorescence correlation spectroscopy.
In september 2009 the first light sheet based fluorescence microscopy workshop in dresden gathered, for the very first time, all researchers developing, using or simply interested in the various flavors of light sheet microscopy. The authors describe optical sectioning, photobleaching, and phototoxicity differences between the. Light sheet fluorescence microscopy lsfm uses a thin plane of light to optically section transparent tissues or whole organisms that have been labeled with a fluorophore. Lightsheetbased fluorescence microscopy lsfm has emerged as one of the most valuable novel tools in developmental biology 15, plant biology 6, and 3d cell biology 7. Such microscopes distinguish themselves for having very low phototoxicity levels and high. Conventional and confocal light sheet microscopy light sheet microscopy singleplane acquisition 3d data acquisition figure 1. This was first introduced by richard zsigmondy and henry siedentopf in 1903 using a slit based system when they were working for carl zeiss. Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. Using light sheet fluorescence microscopy to image. Light sheet based fluorescence microscopy lsfm for the quantitative imaging of cells and. The dynamic horizontal light sheet focus guarantees excellent zresolution covering the entire field of view. Professor kubitscheck develops single molecule and light sheet imaging techniques, has authored over 80 scientific publications and has extensive experience in teaching courses on physical chemistry, biophysics and quantitative microscopy.
Nov 27, 2018 please use one of the following formats to cite this article in your essay, paper or report. The new imaging properties are first highlighted in terms of optical parameters. At least one illumination arm is used to generate the light sheet cyan, while at least one detection arm is equipped with an appropriate filter and a camera for the parallel detection of the. However, both technologies lay fallow until the development of fluorescence microscopy, the advent of lasers, and the ability to digitally encode and rebuild images using computers. Light sheet optics by zeiss the first microscope built around your. Lightsheetbased fluorescence microscopy for threedimensional imaging of biological samples jim swoger, francesco pampaloni, and ernst h. State key laboratory of transient optics and photonics, xian institute of optics and precision mechanics, chinese academy of sciences, xian 710119, china. Here we discuss lsfm fundamentals and outline the basic principles of several major lightsheetbased imaging modalities spim, inverted spim, multiview spim. In contrast to epifluorescence microscopy only a thin slice usually a few hundred nanometers to a few micrometers of the sample is illuminated. Light sheet imaging comes of age journal of cell biology. Osa spatially modulated illumination allows for light sheet. Light sheet based fluorescence microscopy lsfm overcomes many drawbacks of conventional fluorescence microscopy by using an orthogonalazimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The principle of light sheet based fluorescence microscopy.
In its technical implementation, a light sheet microscope differs fundamentally from a conventional microscope stand. In this device, light of a specific wavelength or set of wavelengths, often in the uv, is produced by passing. To achieve optical sectioning, the sample is illuminated with a sheet of light. Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution. In this device, light of a specific wavelength or set. Lightsheetbased fluorescence microscopy lsfm is a fluorescence technique that combines optical sectioning, the key capability of confocal and twophoton. Multipleview microscopy with lightsheet based fluorescence. Pdf light sheet fluorescence microscopy lsfm functions as a nondestructive microtome and. Light sheet fluorescence microscopy lsfm is a general name for a constantly growing family of planar illumination techniques that have revolutionized how optical imaging of biological specimens can be performed. Light sheet fluorescence microscopy an overview sciencedirect. Both techniques have made a great progress in recent decades and have been widely applied in life science, medical research and so on.
Structured light sheet fluorescence microscopy based on four. Spatially modulated illumination allows for light sheet. We present an overview of recent technological advances in light. Deep penetration into the specimen, high imaging speed and ultralow intensity of the excitation light are key requirements. As such, emphasis is placed on the advantages that ls microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. To achieve optical sectioning, the sample is illuminated with a sheet of light, an approach. Multiview deblurring for 3d images from lightsheetbased. A bright fluorescent probe for h2s enables analyte. Nov 14, 2008 a longstanding goal of biology is to map the behavior of all cells during vertebrate embryogenesis. First, it allows 3d microscopy utilizing a wide field microscope.
A bright fluorescent probe for h2s enables analyteresponsive. We propose an algorithm for 3d multiview deblurring using spatially variant point spread functions psfs. Julien colombelli, corinne lorenzo, in fluorescence microscopy, 2014. State key laboratory of transient optics and photonics, xian institute of optics and. Lightsheetbased fluorescence microscopy lsfm is a fluorescence technique that combines optical sectioning, the key capability of confocal and twophoton fluorescence microscopes with multipleview imaging, which is used in optical tomography. Light sheetbased fluorescence microscopy lsfm reduces. Highresolution threedimensional imaging of large specimens. Light sheet fluorescence microscopy, to the same extent as laser scanning confocal microscopy, now refers to a wide range of microscope systems figure 8. Special attention goes to designs that allow integration of lsfm on standard microscope bodies. Request pdf multipleview microscopy with lightsheet based fluorescence microscope the axial resolution of any standard singlelens light microscope is lower than its lateral resolution.
Howbeit, fluorophore excitation is performed in a thin sheet that can. How to define and optimize axial resolution in lightsheet. It directs a light sheet into the side of the specimen. Fahrbach 1, 1 leica microsystems cms gmbh, am friedensplatz 3, 68165 mannheim, germany 2 institute of genetics and molecular and cellular biology igbmc, 67404 illkirch. Light sheetbased fluorescence microscopes lsfm rely on two main optical paths1. Most modern microscopy is based on fluorescence fluorescent dyes are useful because the intensity of emissions can be quantified unlike colorimetric stains and sensitivity is increases since background is dark unlike for transmitted light. Light sheet microscopy as confocal and multiphoton microscopy, light sheet microscopy is based on fluorescence imaging 72. Light sheet based fluorescence microscopy lsfm is a fluorescence technique that combines optical sectioning, the key capability of confocal and twophoton fluorescence microscopes with multiple. The detection path consists of the detection microscope objective lens, a spectral filter, which discriminates the excitation light, a tube lens and a camera. Sep 15, 2008 light sheet based fluorescence microscopy lsfm is a fluorescence technique that combines optical sectioning, the key capability of confocal and twophoton fluorescence microscopes with multipleview imaging, which is used in optical tomography. Although it has great advantages over conventional widefield microscopy because of its strong 3d capacity, high speed and low phototoxicity, the imaging resolution of light sheet fluorescence microscopy. Light sheet based fluorescence microscopy lsfm has emerged as one of the most valuable novel tools in developmental biology 15, plant biology 6, and 3d cell biology 7.
Light sheet fluorescence microscopy lsfm is a fluorescence microscopy technique with an intermediatetohigh optical resolution, but good optical sectioning capabilities and high speed. Laboratory embls single plane illumination fluorescence. Based on a rectangular slit combined with white light, it enabled the. Light sheet fluorescence microscopy has become one of the most widely used techniques for threedimensional imaging due to its high speed and low phototoxicity. Light sheet fluorescence microscopy lsfm has been present in cell biology laboratories for quite some time, mainly as custommade systems, with imaging applications ranging from single cells in the micrometer scale to small organisms in the millimeter scale. Stelzerhighresolution deep imaging of live cellular spheroids with light sheet based fluorescence microscopy cell tissue res. Pdf lightsheetbased fluorescence microscopy lsfm is a fluorescence technique that combines optical sectioning, the key capability of confocal and. It allows recording of long timelapse movies of live embryos in near physiological conditions. Light sheet fluorescence microscopy microscopyu enus. Light sheet based fluorescence microscopes lsfm rely on two main optical paths1. Highresolution deep imaging of live cellular spheroids. Recently, we developed digital scanned laser light sheet fluorescence microscopy dslm, in which a single plane in a specimen is quickly scanned with a thin laser beam, while a camera based detection arm records a fluorescence image at a right angle to the illumination axis.
The main advantages of lsfm compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles. Zeiss microscopy online campus light sheet microscopy. Development of light sheet fluorescence microscopy the first published account of a very simple version of an lsfm called ultramicroscopy was described by siedentopf and zsigmondy 1903 in which sunlight was projected through a slit aperture to observe gold particles fig. Ploem is credited with the development of the vertical illuminator for reflected light fluorescence microscopy. Illumination device for light sheet based fluorescence microscopy of living cells sarah schickinger1, thomas bruns 1, rainer wittig2, and herbert schneckenburger1,2 1hochschule aalen, institut fur angewandte forschung, beethovenstr. Illumination device for light sheet based fluorescence. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wildtype and mutant zebrafish embryos over the first 24 hours of development. Digital scanned laser lightsheet fluorescence microscopy dslm. This is basically a reflected light microscopy mode in which the wavelength of the reflected light is longer than that of the excitation. Table 1 shows that lsfm offers higher resolution subcellular and faster imaging speed compared with other nondestructive tomographic devices such as magnetic resonance. Light sheet fluorescence microscopy by zeiss adapted by dirk pacholsky, biovis facility, uppsala. Light sheet fluorescence microscopy enables relatively gentle imaging of biological samples with high resolution in three dimensions 3d and over long periods of time. Light sheet based fluorescence microscopy lsfm is a fluorescence technique that combines optical sectioning, the key capability of confocal and twophoton fluorescence.
Lightsheet fluorescence microscopy for quantitative biology. The horizontal microscope beam path for lightsheet z. Comparison of optical projection tomography and light. Structured light sheet fluorescence microscopy based on.
Fluorescence microscope refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope. Structured light sheet fluorescence microscopy based on four beam interference ming lei, 1, 2 andreas zumbusch1, 1 department of chemie, university of konstanz, d78457 konstanz, germany 2 permanent address. Stelzer in modern biology, most optical imaging technologies are applied to twodimensional cell culture systems. Light sheet light illumination in a horizontal microscope.
Light sheet fluorescence microscopy lsfm has proven to be the technique of choice to accomplish such challenging objectives 2. Light sheetbased fluorescence microscopy of living or. In lsfm, illumination and detection are split into at least two optical paths. Polarized light microscopy only works with birefringent samples those that alter polarity of light some polymers such as microtubules depends on orientation, so rotating stage desirable compatible with fluorescence microscopy good way to read out orientation of certain chromophores requires strainfree optics. Using light sheet fluorescence microscopy to image zebrafish. A natural extension of confocal microscopy is twophoton excitation 2pe fluorescence microscopy, which has also found applications in light sheet microscopy. A comparison of digital light sheet microscopy with conventional widefield and confocal fluorescence techniques. We hereby present an imaging technique that allows volumetric reconstruction of the fluorescent.
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