Confocal Microscopy

David Carter1

1 University of California at Riverside, Riverside, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 9.4
DOI:  10.1002/9780470089941.et0904s07
Online Posting Date:  October, 2013
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This overview covers the two main types of confocal scanner. Point scanners are more flexible and give the best resolution, while camera‐based systems can be more sensitive, faster, and more automated. The procedure for setting up and using these instruments is described in sufficient depth for basic operation. Beyond collecting single images, a Z‐series, which captures a volume of data, a kinetics series, collecting a sequence of images, and generation of a large montage image are all described. Confocal is an extension of regular fluorescence microscopy, with the ability to handle thicker and more heavily stained specimens and to work more precisely with physiological markers in live samples and organelle stains. Such techniques are highly divergent, depending on specimen type, so they cannot be covered in any detail here, but a general idea will give the user confidence to explore the literature and collect good data on their own systems. Curr. Protoc. Essential Lab. Tech. 7:9.4.1‐9.4.36. © 2013 by John Wiley & Sons, Inc.

Keywords: confocal; imaging; laser scanning; Z‐series; kinetics; montage

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Table of Contents

  • Overview and Principles
  • Sample Preparation
  • Types of Fluorophore
  • Lasers
  • Safety Considerations
  • Objective Lenses
  • Protocols
  • Basic Protocol 1: Use of a Confocal Microscope
  • Basic Protocol 2: Raster Scanning
  • Basic Protocol 3: Careful Collection of Quality Data
  • Basic Protocol 4: Imaging with a Camera
  • Basic Protocol 5: Collecting a Z‐Stack
  • Basic Protocol 6: Kinetics Imaging
  • Basic Protocol 7: Montage Imaging
  • Concluding Remarks
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

  Ando, R., Hama, H., Yamamoto‐Hino, M., Mizuno, H., and Miyawaki, A. 2002. An optical marker based on the UV‐induced green‐to‐red photoconversion of a fluorescent protein. PNAS99:12651‐12656.
  Avila, E., Zouhar, J., Agee, A., Carter, D., Chary S.N., and Raikhel, N.V. 2003. Tools to study organelle biogenesis: Point mutation lines with disrupted vacuoles and high speed confocal screening of green fluorescent protein tagged organelles. Plant Physiol.133:1‐4.
  Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E., and Webb, W.W. 1976. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biopys. J.16:1055‐1069.
  Deerinck, T.J. 2008. The application of fluorescent quantum dots to confocal, multiphoton, and electron microscopic imaging. Toxicol. Pathol.36:112‐116.
  Hama, H., Kurokawa, H., Kawano, H., Ando, R., Shimogori, T., Noda, H., Fukami, K., Sakaue‐Sawano, A., and Miyawak, A. 2011. Scale: A chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci.14:1481‐1488.
  Kwaaitaal, M., Keinath, N.F., Pajonk, S., Biskup, C., and Panstruga, R. 2010. Combined bimolecular fluorescence complementation and Förster resonance energy transfer reveals ternary SNARE complex formation in living plant cells. Plant Physiol.152:1135‐1147.
  Matsumoto, B. (ed.) 2002. Cell Biological Applications of Confocal Microscopy. In Methods in Cell Biology, Volume 70, 2nd ed. (70) Academic Press, San Diego.
  Rizzo, M.A., Davidson, M.W., and Piston, D.W. 2010. Fluorescent protein tracking and detection: Fluorescent protein structure and color variants. Cold Spring Harb Protoc. 2009 Dec;2009(12):pdb.top63. doi: 10.1101/pdb.top63.
Internet Resources
  A free calendar reservation system which is widely used for core resources such as seminar rooms and shared instrumentation.
  The Electron Microscopy Sciences online catalog contains most supplies needed for optical microscopy too, including all types of mounting media, slides, and coverslips.
  Describes a well designed objective lens heater for use with a heated stage insert.
  Non‐setting mounting media with DAPI for nuclear counter‐staining. Vector labs sells many kits and reagents for all kinds of staining protocols, including secondary antibodies for a dozen species for immunostaining, and kits for fluorescent in‐situ hybridization (FISH).
  An air‐filtration system that reduces the dust burden in a microscopy lab and optionally can remove chemical contaminants, such as DMSO, from a robotics room.
  A version of ImageJ that is a good starting point for microscopy analysis of images. ImageJ is free and has a large user base who constantly add to its functionality.
  This school supply house has a large collection of histological slides, some of which are fluorescent (from the eosin in H & E), and can be used as practice pieces.
  Another online school supply house with an extensive collection of histological slides.
  Fine science tools supplier with fixatives, stains and equipment for sample preparation.‐Probes.html
  The most comprehensive source for fluorescent reagents is Molecular Probes, now part of Invitrogen.
  Titled “Molecular Expressions,” this Florida State University site is an exhaustive resource for all things microscopy and includes many interactive Java applets for hands‐on demonstration of various aspects of instrument performance.
  This Nikon‐sponsored extension to “Molecular Expressions” has many tutorials on various aspects of confocal imaging and sample preparation.
  This camera company is pioneering scientific CMOS cameras and offers integrated spinning disc confocal systems.
  Olympus makes relatively affordable confocals with the most extensive analysis and quantitation software. Their FluoView 1000 can be configured with a whole second scanner for photobleaching and photoconversion applications.
  Leica makes premium confocal systems with excellent sensitivity and ease of use. They were the first to market with a real‐time super‐resolution system.‐Frame/3d7a40584d71593841256a71004cb2d4
  Zeiss makes premium confocal systems with excellent image quality and high performance objectives.
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