Alternatively, the sheet of light can be generated by focusing a laser beam to a single line and by rapidly scanning it up and down during the exposure time. Pdf multidirectional digital scanned lightsheet microscopy. Embryonic development is one of the most complex processes encountered in biology. Nov 14, 2008 a longstanding goal of biology is to map the behavior of all cells during vertebrate embryogenesis. Alternatively, a digital scanned laser light sheetbased fluorescence microscope6 dslm generates the light sheet by moving a. The new system is equipped with one static and one scanned lightsheet and is called a scanning thinsheet laser imaging microscope stslim. Image formation by linear and nonlinear digital scanned light. It allows recording of long timelapse movies of live embryos in near physiological conditions. 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. Here we describe a new approach which will effectively mitigate the artifacts in digital scanned light sheet microscopy dslm and digital. For in vivo development of zebrafish embryos over a 24hr period. Pdf light sheet fluorescence microscopy lsfm functions as a nondestructive microtome and.
Stelzer cell biology and biophysics unit embl heidelberg meyerhofstrasse 1, 69117 heidelberg, germany email. Osa scanned light sheet microscopy with confocal slit detection. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Multidirectional digital scanned lightsheet microscopy. In digital scanned laser lightsheet fluorescence microscopy dslm, a specimen is rapidly scanned with a thin sheet of light while fluorescence is recorded perpendicular to the axis of illumination with a camera. In this device, light of a specific wavelength or set of wavelengths, often in the uv, is produced by passing. Structured light sheet fluorescence microscopy based on. Using light sheet fluorescence microscopy to image. Fundamentally, lsfm techniques are made possible by decoupling the illumination and detection optical pathways, allowing for novel illumination strategies that optimize the photon. This illumination scheme provides minimal photobleaching and phototoxicity. Light sheet fluorescence microscopy microscopyu enus. Oct 01, 2011 digital scanned laser lightsheet fluorescence microscopy dslm of zebrafish and drosophila embryonic development. Reconstruction of zebrafish early embryonic development by.
Sim in combination with lightsheetbased fluorescence microscopy csilsfm uses a pair of illumination objective lenses to. By combining twophoton fluorescence excitation with lsfm, truong et al. In digital scanned laser light sheet fluorescence microscopy dslm, a specimen is rapidly scanned with a thin sheet of light while fluorescence is recorded perpendicular to the axis of illumination with a camera. In digital scanned laser lightsheet fluorescence microscopy dslm, a specimen is rapidly scanned with a thin sheet of light while fluorescence is recorded perpendicular to the. The state key laboratory of biomembrane and membrane biotechnology, institute of molecular. A longstanding goal of biology is to map the behavior of all cells during vertebrate embryogenesis. Abstractin light sheet microscopy, optical sectioning by selective fluorescence excitation with a sheet of light is combined with fast fullframe acquisition. We demonstrate its application for imaging zebrafish eye development across wide spatiotemporal scales and present a pipeline for fusion and deconvolution of.
The light sheet itself can be static, formed by a cylindrical lens, the socalled selective plane illumination microscopy spim. Complemented with remote focusing and multiview acquisition, light sheet microscopy is the method of choice for acquisition of very fast biological processes. The light sheet is static, its intensity is easily controlled and it is well suited for massively parallel detectors such as very fast cameras. Segmentation is usually a timeconsuming, manual proce dure, but methods. 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. Carl friedrich gauss, 3, castelldefels barcelona, spain.
This device produces a linelike laser beam using an ftheta lens that is scanned across a small specimen, and the image is collected in the orthogonal plane. May 09, 2017 structured illumination microscopy sim is a superresolution technique that illuminates the specimen with a sinemodulated pattern. The state key laboratory of biomembrane and membrane biotechnology, institute of molecular medicine, peking university, beijing 100871. Olarte 1, jordi andilla 1, raphael jorand 2,3, bernard ducommun 2,3,4, corinne lorenzo 2,3 and pablo lozaalvarez 1 1icfothe institute of photonic sciences, av.
Schematic illustration of dslm, including the laser light source 1, the laser scanner 2, the illumination arm 3, the temperaturecontrolled specimen chamber 4 and two widefield fluorescence detection arms. Single plane illumination microscopy spim provides fast threedimensional recording of fluorescent biological specimens 1. Threecolor twophoton threeaxis digital scanned lightsheet microscopy 3c2p3adslm volume 21 supplement weijian zong, zuosheng liu, fuzeng niu, shaowa li, jia zhao, aimin wang, liangyi chen. Stelzer, quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy, curr. Ccd, chargecoupled device, crisprcas9, genome engineering technique crispr. Sim in combination with light sheet based fluorescence microscopy csilsfm uses a pair of illumination objective lenses to.
Digital scanned laser lightsheet fluorescence microscopy. The multidirectional digital scanned light sheet microscopy mdslm architecture utilizes an elliptical gaussian pencil beam with a higher na along one axis in the plane of the light sheet to. A digital scanned laser light sheet microscope dslm philipp. Light sheet microscopy, fluorescence microscopy, 3d imaging, structured illumination, spim, highthroughput. Single plane illumination microscopy and light sheet fluorescence microscopy main article. Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single lens or multiple lenses to allow a magnified view of the sample.
It is an optimized version of a lightsheet fluorescent microscope that is designed to image large specimens light sheet can be generated by rapidly scanning a laser beam up and down digital scanned light sheet microscopy, dslm, figure 3. The light sheet is aligned exactly with the focal plane of the detection lens. Threecolor twophoton threeaxis digital scanned light sheet microscopy 3c2p3adslm volume 21 supplement weijian zong, zuosheng liu, fuzeng niu, shaowa li, jia zhao, aimin wang, liangyi chen. Multilayer mounting enables longterm imaging of zebrafish. 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 is an excellent tool for imaging embryonic development. Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution. Fast, highcontrast imaging of animal development by scanned light sheet based structured illumination microscopy philipp j. Lightsheet 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. This is termed digital scanned laser light sheet microscopy dslm 8,9. Light sheet fluorescence microscopy lsfm is a fluorescence microscopy technique with an intermediatetohigh optical resolution, but good optical sectioning capabilities and high speed.
Pdf lightsheet fluorescence microscopy lsfm has emerged as a powerful method for rapid and optically efficient 3d microscopy. Dslm of zebrafish and drosophila embryonic development. Importantly, such recordings must be realized without compromising the physiological development of the specimen. Quantitative in vivo imaging of entire embryos with. Light sheet fluorescence microscopy using a plane of light formed by focusing light through a cylindrical lens at a narrow angle or by scanning a line of light in a plane perpendicular to the axis of objective, high resolution optical sections can be taken. Ploem is credited with the development of the vertical illuminator for reflected light fluorescence microscopy. Alternatively, a digital scanned laser light sheetbased fluorescence microscope6 dslm generates the light sheet by moving a focused that is, gaussian beam. Zebrafish embryo development using light sheet fluorescence microscopy duration. The combination of digital scanned laser light sheet microscopy and incoherent structured illumination allows intrinsic removal of scattered background fluorescence from the. Lightsheet fluorescence microscopy for quantitative biology. Modern applications in the life sciences are frequently based on in vivo imaging of biological specimens, a domain for which light microscopy approaches are. Light sheet microscopy is an important and widely used method for studying large and semiopaque biological specimens. Fundamentals of light microscopy and electronic imaging douglas b. Alternatively, a light sheet can be generated by rapidly scanning a laser beam up and down digital scanned light sheet microscopy, dslm, figure 3.
Digital scanned laser lightsheet fluorescence microscopy dslm. In contrast to epifluorescence microscopy only a thin slice usually a few hundred nanometers to a few micrometers of the sample is illuminated. Largefield highresolution twophoton digital scanned light. Sep 17, 2018 the multidirectional digital scanned light sheet microscopy mdslm architecture utilizes an elliptical gaussian pencil beam with a higher na along one axis in the plane of the light sheet to. The combination of digital scanned laser light sheet microscopy and incoherent structured illumination allows intrinsic removal of scattered background fluorescence from the desired fluorescent. Modern applications in the life sciences are frequently based on in vivo imaging of biological specimens, a domain for which light microscopy approaches are typically best suited. Here, we demonstrate that contrast and degree of confocality can greatly be increased by combining scanned light sheet. Digital scanned laser light sheet fluorescence microscopy. Here, we discuss digital scanned laser light sheet fluorescence microscopy dslm as a novel tool for quantitative in vivo imaging in the postgenomic era and show how this emerging technique relates to the currently most widely applied 3d microscopy techniques in biology. Largefield highresolution twophoton digital scanned.
Fast fluorescence microscopy with light sheets the. We develop a multidimensional fluorescence imaging technique by implementing a wide. Frontiers applications of lightsheet microscopy in. Pdf digital scanned laser light sheet fluorescence microscopy. Zeiss microscopy online campus light sheet microscopy. Quantitative in vivo imaging of entire embryos with digital. The authors propose that ocpi and opm can be fitted to existing microscopes for imaging small specimens. Microscopy tion are carried out through completely separate optical paths that are arranged perpendicular to one another. Digital scanned laser lightsheet microscopy structured illumination dslmsi duration. To achieve the imaging speed and quality for recording entire embryos, we developed digital scanned laser light sheet fluorescence microscopy dslm fig. Schematic illustration of dslm, including the laser light source 1, the laser scanner 2, the illumination arm 3, the temperaturecontrolled specimen chamber 4 and two widefield fluorescence detection arms with independently operated cameras 5. Threecolor twophoton threeaxis digital scanned lightsheet microscopy 3c2p3a dslm weijian zong1, 2, zuosheng liu3, fuzeng niu3, shaowa li1, jia zhao1, aimin wang3 and liangyi chen1 1. Scanning thinsheet laser imaging microscopy stslim with.
Osa scanned light sheet microscopy with confocal slit. The light sheet is generated by using either a cylindrical lens or a scanning laser beam. Image formation by linear and nonlinear digital scanned. A 2008 version of kellers lightsheet microscope, called digitally scanned laser lightsheet fluorescence microscopy dslm, gave him the speed he craved. The multidirectional digital scanned lightsheet microscopy mdslm architecture utilizes an elliptical gaussian pencil beam with a higher na along one axis in the plane of the light sheet to. Threecolor twophoton threeaxis digital scanned lightsheet microscopy 3c2p3a dslm weijian zong1, 2, zuosheng liu3, fuzeng niu3, shaowa li1, jia zhao1, aimin wang3 and liangyi chen1. The idea behind dslm is to generate a plane of light with a laser scanner that rapidly moves a micrometerthin beam of. Threecolor twophoton threeaxis digital scanned light. Lightsheet fluorescence microscopy lsfm has emerged as a powerful method for rapid and optically efficient 3d microscopy. Request pdf digital scanned laser lightsheet fluorescence microscopy dslm of zebrafish and drosophila embryonic development embryonic. This concept was recently introduced as digital scanned laser lightsheet fluorescence microscopy dslm keller et al. Schmidt 3, anthony santella 4, khaled khairy 1, zhirong bao 4, joachim wittbrodt 3,5 and ernst h.
The energy input into the sample is minimized, making light sheet microscopy ideally suited. Structured illumination microscopy sim is a superresolution technique that illuminates the specimen with a sinemodulated pattern. 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. However, light scattering within the sample often deteriorates the optical sectioning effect. A digital scanned laser light sheet microscope dslm philipp j. European molecular biology laboratory embl 5,388 views 0. Multidirectional digital scanned lightsheet microscopy enables. The resulting image can be detected directly by the eye, imaged on a photographic plate, or captured digitally. The single lens with its attachments, or the system of lenses and imaging equipment, along with.
648 1313 350 1044 246 857 1173 1474 856 535 100 1616 775 1643 639 1459 947 883 1351 722 1540 800 1102 1259 478 1060 1510 468 842 766 1222 835 559 903 1113 540 1030 843 171 894