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What is Light Sheet Microscopy?

Living specimens, particularly developing embryos, are often sensitive to light exposure. Light sheet microscopy – also referred to as single plane illumination microscopy or SPIM – is a gentle way of imaging sensitive samples or fast biological processes in vivo. The specimen is illuminated only in a single plane at a time and detected from the perpendicular direction. Since there is no out-of-focus excitation, phototoxic effects are reduced to the focal plane. Light sheet imaging has intrinsic optical sectioning. By moving the sample through the light sheet, 3D images of a specimen can be recorded.

  • Clarifying Tissue Clearing

    Biological specimens are intrinsically three dimensional; however because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by “clearing” the tissue have been ongoing for over a century, there have been a large number of recent innovations. This review introduces the physical basis for light-scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.
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  • Clearing of Fixed Tissue: A Review from a Microscopist’s Perspective

    Chemical clearing of fixed tissues is becoming a key instrument for the three-dimensional reconstruction of macroscopic tissue portions, including entire organs. Indeed, the growing interest in this field has both triggered and been stimulated by recent advances in high-throughput microscopy and data analysis methods, which allowed imaging and management of large samples.
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  • Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent

    Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations.
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  • Light Sheet Microscopy Turned Vertically

    Living cells and organisms often suffer from the high light intensities used for fluorescent imaging. Light sheet microscopy reduces phototoxic effects and bleaching by illuminating a specimen in only a single plane at a time. A new light sheet microscope combines light sheet and confocal microscopy in one system without compromising either functionality and allows the combination of the two methods, e.g. confocal photomanipulation with subsequent light sheet acquisition, for new applications.
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  • "The Leica Digital Light Sheet Module – a Clever Example of Thinking Out of the Box"

    Bram van den Broek is a postdoctoral fellow at the Netherlands cancer institute in Amsterdam where he supports the advanced microscopy techniques in the laboratory of Kees Jalink. Working with Leica Microsystems as a collaboration partner for beta-testing of microscopes he enjoys very much.
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  • Webinar: The Best of Both Worlds: Combining Light Sheet and Confocal Microscopy

    Living cells and organisms often suffer from high light intensities that are used in conventional imaging. Light sheet microscopy reduces phototoxic effects and bleaching, by only illuminating a specimen in a single plane at a time whilst the signal is detected in a perpendicular direction. In combination with high-speed cameras for image acquisition, light sheet microscopy is a very gentle method to observe fast biological processes in sensitive organisms over an extended time period.
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  • Confocal and Digital Light Sheet Imaging

    Optical imaging instrumentation can magnify tiny objects, zoom in on distant stars and reveal details that are invisible to the naked eye. But it notoriously suffers from an annoying problem: the limited depth of field. Our eye-lens (an optical imaging instrument) has the same trouble, but our brain smartly removes all not-in-focus information before the signal reaches conscious cognition.
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  • Webinar: Light Sheet Imaging – New Solutions and Their Applications in Zebrafish Embryogenesis

    Living cells and organisms often suffer from high light intensities that are used in conventional imaging. Light sheet microscopy reduces phototoxic effects and bleaching, by only illuminating a specimen in a single plane at a time whilst the signal is detected in a perpendicular direction. In combination with high-speed cameras for image acquisition, light sheet microscopy is a very gentle method to observe fast biological processes in sensitive organisms over an extended time period.
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  • Webinar: Light Sheet Imaging for Fast 3D Live Cell and Tissue Imaging

    In recent years, light sheet microscopy has emerged as the biological imaging modality of choice to achieve high performance in imaging speed, resolution, and penetration depth – all with minimal photo-induced damage.
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  • Nature Methods: Light-sheet Fluorescence Microscopy - Method of the Year 2014

    Just about everyone who has examined fluorescent samples under the microscope is aware of the constant struggle to have enough signal to see the labeled structures while also avoiding fluorophore bleaching. What may be less apparent, at least to those who image bright, robust or fixed samples, is how stressful and potentially toxic to living cells and tissues it is to illuminate them with high-intensity light.
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  • Video Talk by Ernst Stelzer: Light Sheet Sectioning

    This talk discusses the technique of light sheet microscopy, also known as selective plane illumination (SPIM). This uses two objectives, one to illuminate the sample and a second to image it and allows long-term 3D imaging of thick specimens like developing embryos with minimal photobleaching and phototoxicity.
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  • Light Sheet Fluorescence Microscopy: Beyond the Flatlands

    Light Sheet Fluorescence Microscopy (LISH-M) is a true fluorescence optical sectioning technique, first described by Heinrich Siedentopf in 1902 under the name of Ultramicroscopy. Light sheet microscopy utilises a plane of light to optically section samples. This allows deep imaging within transparent tissues and whole organisms. This book chapter will provide the reader with a comprehensive view on this emerging technology.
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  • Quantitative Imaging in Cell Biology: Light Sheet Microscopy

    This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation.
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  • Light Sheet Fluorescence Microscopy - A Review

    Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy.
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  • Selective Plane Illumination Microscopy Techniques in Developmental Biology

    Selective plane illumination microscopy (SPIM) and other fluorescence microscopy techniques in which a focused sheet of light serves to illuminate the sample have become increasingly popular in developmental studies. Fluorescence light-sheet microscopy bridges the gap in image quality between fluorescence stereomicroscopy and high-resolution imaging of fixed tissue sections.
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Useful Links

Publications on Light Sheet Microscopy

Visualization and targeting of LGR5+ human colon cancer stem cells
Shimokawa, M. et al., 2017

Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent
Ke, M.-T. et al., 2016

Light Sheet Microscopy to Measure Protein Dynamics.
Rieckher, M., 2016

Further reading on Light Sheet Microscopy

www.sciencemag.org/content/305/5686/1007.long
Huisken, J. et al., 2004

www.ncbi.nlm.nih.gov/pubmed/8371260?dopt=Abstract&holding=npg
Voie, A. H. et al., 1993

Light Sheet Fluorescence Microscopy International Conference

www.lsfm2017.org/
Singapore 2017

www.lsfm2016.org/
Sheffield 2016

www.lsfm2015.org/
Genoa 2015

www.lsfm2014.com/
Barcelona 2014

Communities and Web Sources

www.researchgate.net/
Social network for scientists

www.ibiology.org/
Teaching tools, video lectures on biology and microscopy

bitesizebio.com
Online magazine and community for molecular and cell biology researchers

www.somersault1824.com
Resource for high-end scientific illustrations, images and animations

Search Engines and Data Bases

www.cellimagelibrary.org
Public resource database of images, videos, and animations of cells

harvester.fzk.de/harvester
Bioinformatic meta search engine for genes and proteins

www.gopubmed.com
Search interface for pubmed

en.wikipedia.org/wiki/List_of_academic_databases_and_search_engines
List of academic databases and search engines

scholar.google.com
Beta of Google's search engine for scientific article abstracts

Journals

www.doaj.org/
Directory of open access journals

emboj.embopress.org/
The EMBO Journal

www.lifescied.org
CBE-Life Sciences Education – an ASCB online journal

www.sciencemag.org/
Science

www.nature.com/
Nature

www.cell.com/
Biweekly publication of exceptional research articles

jcs.biologists.org/
Journal of Cell Science

dev.biologists.org/
Development

jeb.biologists.org/
The Journal of Experimental Biology

dmm.biologists.org/
DMM Disease Models & Mechanisms

www.biotechniques.com/
International Journal of Life Science Methods

www.opticsinfobase.org/
Collection of Journals and Proceedings in Optics and Photonics

spie.org/x576.xml
SPIE - peer-reviewed journals on applied research in optics and photonics

onlinelibrary.wiley.com/journal/10.1002/(ISSN)1864-0648
Journal of Biophotonics

www.plosone.org/home.action
International, peer-reviewed, open-access, online publication

rspb.royalsocietypublishing.org/
Proceedings B - the Royal Society's biological research journal

www.microscopy-analysis.com/
International Journal for microscopists

Organizations / Institutes

www.microscopy.org/
Microscopy Society of America

www.eurmicsoc.org/
European Microscopy Society

www.rms.org.uk/
Royal Microscopical Society

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