The principle of light sheet based fluorescence microscopy. We hereby present an imaging technique that allows volumetric reconstruction of the fluorescent. Because the thickness of the light sheet is smaller than the depth of field of the detection lens for low numerical apertures, and. 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. Nov 14, 2008 a longstanding goal of biology is to map the behavior of all cells during vertebrate embryogenesis. 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.
Lsfm can be operated with laser cutters and for fluorescence correlation spectroscopy. This is basically a reflected light microscopy mode in which the wavelength of the reflected light is longer than that of the excitation. State key laboratory of transient optics and photonics, xian institute of optics and. Light sheet based fluorescence microscopy lsfm takes advantage of modern camera technologies. Lightsheetbased fluorescence microscopy lsfm is a fluorescence technique that combines optical sectioning, the key capability of confocal and twophoton. Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution. 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. 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. 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 based fluorescence microscopy lsfm is a fluorescence technique that combines optical sectioning, the key capability of confocal and twophoton fluorescence. Illumination device for light sheet based fluorescence. 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. 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. Lightsheetbased fluorescence microscopy for threedimensional imaging of biological samples jim swoger, francesco pampaloni, and ernst h. Light sheetbased fluorescence microscopy lsfm reduces. 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. Deep penetration into the specimen, high imaging speed and ultralow intensity of the excitation light are key requirements. More dimensions, more photons, and less photodamage. Multipleview microscopy with lightsheet based fluorescence. Highresolution deep imaging of live cellular spheroids. Threedimensional live imaging of filamentous fungi with light sheet based fluorescence microscopy lsfm francesco pampaloni, laura knuppertz, andrea hamann, heinz d. Light sheet fluorescence microscopy by zeiss adapted by dirk pacholsky, biovis facility, uppsala, sweden for methods for cell analysis, 2014.
Similarly, the light sheet microscope has origins that are over 100 yr old and are based on the ultramicroscope siedentopf and zsigmondy, 1903. A natural extension of confocal microscopy is twophoton excitation 2pe fluorescence microscopy, which has also found applications in light sheet microscopy. To achieve optical sectioning, the sample is illuminated with a sheet of light. Jan 25, 2019 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. Using light sheet fluorescence microscopy to image. Fundamentally, lsfm techniques are made possible by decoupling the illumination and detection optical pathways, allowing for novel. Comparison of optical projection tomography and light.
Light sheet imaging comes of age journal of cell biology. Light sheet fluorescence microscopy microscopyu enus. 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. Exciting the fluorescent sample perpendicular to the detection by a focused light sheet offers two major advantages. Fast highcontrast imaging of animal development with scanned. Light sheet based fluorescence microscopy lsfm allows the imaging of large threedimensional specimens over periods of several days 7.
Further improvements in 3d microscopy require limiting the light exposure of the sample and increasing the volumetric acquisition rate. Stelzerhighresolution deep imaging of live cellular spheroids with light sheet based fluorescence microscopy cell tissue res. We present an overview of recent technological advances in light. Light sheet fluorescence microscopy lsfm has proven to be the technique of choice to accomplish such challenging objectives 2. Lightsheet fluorescence microscopy for quantitative. Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. 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.
Light sheet based fluorescence microscopy lsfm for the quantitative imaging of cells and. 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 fluorescence microscopy lsfm uses a thin plane of light to optically section transparent tissues or whole organisms that have been labeled with a fluorophore. Lightsheet fluorescence microscopy for quantitative biology.
Light sheet fluorescence microscopy an overview sciencedirect. To achieve optical sectioning, the sample is illuminated with a sheet of light, an approach. 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. Howbeit, fluorophore excitation is performed in a thin sheet that can. Light sheet fluorescence microscopy was performed using a homebuilt instrument similar in design to that of keller et al. Light sheet fluorescence microscopy, to the same extent as laser scanning confocal microscopy, now refers to a wide range of microscope systems figure 8. Light sheetbased fluorescence microscopes lsfm rely on two main optical paths1.
First, it allows 3d microscopy utilizing a wide field microscope. 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. Fast, highcontrast imaging of animal development with. A bright fluorescent probe for h2s enables analyteresponsive. We demonstrate its application for imaging zebrafish eye development across wide spatiotemporal scales and present a pipeline for fusion and deconvolution of. A bright fluorescent probe for h2s enables analyte. 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. Here we discuss lsfm fundamentals and outline the basic principles of several major lightsheetbased imaging modalities spim, inverted spim, multiview spim. The protocol is also suitable for both canonic implementations, i. Stelzer in modern biology, most optical imaging technologies are applied to twodimensional cell culture systems. In this device, light of a specific wavelength or set. 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. Laboratory embls single plane illumination fluorescence. Please use one of the following formats to cite this article in your essay, paper or report.
Using light sheet fluorescence microscopy to image zebrafish. Conventional and confocal light sheet microscopy light sheet microscopy singleplane acquisition 3d data acquisition figure 1. Photon budget is how many photons a dye can produce before it goes dark. The new imaging properties are first highlighted in terms of optical parameters. Light sheetbased fluorescence microscopy of living or fixed. 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. Light sheet fluorescence microscopy has become one of the most widely used techniques for threedimensional imaging due to its high speed and low phototoxicity. Pdf light sheet fluorescence microscopy lsfm functions as a nondestructive microtome and. This was first introduced by richard zsigmondy and henry siedentopf in 1903 using a slit based system when they were working for carl zeiss.
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 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. Light sheet based microscopy, such as spim is a new method for imaging large fluorescent samples 1. The authors describe optical sectioning, photobleaching, and phototoxicity differences between the. In contrast to epifluorescence microscopy only a thin slice usually a few hundred nanometers to a few micrometers of the sample is illuminated. 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. Light sheet fluorescence microscopy versus confocal. Light sheetbased fluorescence microscopy of living or. Both techniques have made a great progress in recent decades and have been widely applied in life science, medical research and so on. 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. Deeplearning lightsheet fluorescence microscopy for high.
Light sheet fluorescence microscopy enables relatively gentle imaging of biological samples with high resolution in three dimensions 3d and over long periods of time. Lightsheet fluorescence microscopy, to the same extent as laser scanning confocal. Light sheet fluorescence microscopy by zeiss adapted by dirk pacholsky, biovis facility, uppsala. 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. Frontiers applications of lightsheet microscopy in. We propose an algorithm for 3d multiview deblurring using spatially variant point spread functions psfs.
Light sheet fluorescence microscopy lsfm is gaining more and more popularity as a method to image embryonic development. The excitation path is perpendicular to the detection path. Structured light sheet fluorescence microscopy based on. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled. Fundamentally, lsfm techniques are made possible by decoupling the illumination and detection optical pathways, allowing for novel illumination strategies that. How to define and optimize axial resolution in lightsheet. 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. Reconstruction of zebrafish early embryonic development by. In lsfm, illumination and detection are split into at least two optical paths. Request pdf multipleview microscopy with lightsheet based fluorescence microscope the axial resolution of any standard singlelens light microscope is lower than its lateral resolution.
The new player is light sheet fluorescence microscopy, an illumination approach orthogonal to the detection that allows for a single plane illumination. The light sheet and the focal plane of the detection microscope objective overlap fig. Light sheet optics by zeiss the first microscope built around your. Lightsheetbased fluorescence microscopes lsfms, such as the european molecular biology. Light sheetbased microscopy, such as spim is a new method for imaging large fluorescent samples 1. In its technical implementation, a light sheet microscope differs fundamentally from a conventional microscope stand. Ploem is credited with the development of the vertical illuminator for reflected light fluorescence microscopy. Nov 27, 2018 please use one of the following formats to cite this article in your essay, paper or report.
The dynamic horizontal light sheet focus guarantees excellent zresolution covering the entire field of view. Another way to look at it is that a light sheetbased fluorescence. Such microscopes distinguish themselves for having very low phototoxicity levels and high. 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. The detection path consists of the detection microscope objective lens, a spectral filter, which discriminates the excitation light, a tube lens and a camera. The horizontal microscope beam path for lightsheet z. 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. Zeiss microscopy online campus light sheet microscopy. Multiview deblurring for 3d images from lightsheetbased. Based on a rectangular slit combined with white light, it enabled the. 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. 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. Light sheet microscopy as confocal and multiphoton microscopy, light sheet microscopy is based on fluorescence imaging 72. State key laboratory of transient optics and photonics, xian institute of optics and precision mechanics, chinese academy of sciences, xian 710119, china.
The principle of light sheetbased fluorescence microscopy. Special attention goes to designs that allow integration of lsfm on standard microscope bodies. In this device, light of a specific wavelength or set of wavelengths, often in the uv, is produced by passing. 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. It allows recording of long timelapse movies of live embryos in near physiological conditions. A comparison of digital light sheet microscopy with conventional widefield and confocal fluorescence techniques. Light sheet light illumination in a horizontal microscope.
Osa spatially modulated illumination allows for light sheet. Digital scanned laser lightsheet fluorescence microscopy dslm. Light sheet fluorescence microscopy lsfm is a fluorescence microscopy technique with an intermediatetohigh optical resolution, but good optical sectioning capabilities and high speed. 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. Highresolution threedimensional imaging of large specimens. Spatially modulated illumination allows for light sheet. A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. Julien colombelli, corinne lorenzo, in fluorescence microscopy, 2014. It directs a light sheet into the side of the specimen.