Basic Confocal Microscopy

Carolyn L. Smith1

1 National Institute of Neurological Disorders and Stroke, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2C.1
DOI:  10.1002/9780471729259.mc02c01s01
Online Posting Date:  June, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit introduces the reader to the basic principles of confocal microscopy and the design and capabilities of current confocal microscopes. The advantages and disadvantages of confocal microscopy as compared to other techniques for fluorescence imaging are described. There are also practical guidelines for sample preparation and optimizing imaging parameters and examples of some of the applications of confocal microscopy.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basis of Optical Sectioning
  • Configuration of an LSCM
  • Practical Guidelines
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 2C.1.1 Applications of laser scanning microscopy. (A, B). Imaging in thick specimens. Neurons in a Drosophila embryo were immunolabeled with antibodies against three different transcription factors (images provided by Dr. Ward Odenwald of the National Institutes of Health, Bethesda, Md.; reproduced from Kamabadur et al., 1998). (A) A single optical section (~2.5-µm) captured with 25×, 0.8-NA objective. Labeled neurons in the plane of focus appear sharply defined, while those outside it are not visualized. (B) A maximum projection of 65 optical sections collected at 2-µm intervals in the z axis. (C) Imaging intracellular structures. Dissociated rat fibroblasts were immunolabeled with anti-tubulin antibodies to visualize microtubules (green) and stained with fluorescent probes for mitochondria (Mitotracker, red) and DNA (DAPI, blue). The image is a projection of 20 optical sections (0.3-µm intervals) captured with a 100×, 1.4-NA objective. (D) Measuring molecular mobility in living cells. In a living fibroblast expressing a Golgi membrane protein (galactosyltransferase) fused to GFP (S65T), GFP fluorescence (green) is localized in the Golgi complex, shown superimposed on a DIC image of the cell. After the first image was collected, the boxed region (yellow) was scanned with full laser power to photobleach the GFP in the boxed area. The second image was collected 2 sec later. Subsequent images (not illustrated) showed that the GFP-galactosyltransferase rapidly diffused back into the photobleached area. Images were captured with a LSM410 (Carl Zeiss, Inc.).
    View Image
  •  FigureFigure 2C.1.2 3-D Imaging in living specimens. Comparison of water- and oil-immersion objectives. (A,B,E,F) Living yeast cells expressing a GFP construct that targets to the mitochondrial matrix were visualized with C-APO 63× 1.2 NA (water) objective (A,B) or a Planapochromat 100 × 1.4 NA (oil) objective (E,F). The images show xy and yz projections of stacks of 40 images collected at 0.2-µm intervals along the optical axis. The xy projections appear sharper than the yz projections because the resolution is higher in the focal plane of the objective than along the optical axis. The yeast were embedded in an aqueous solution with 0.2% agarose. Panels C and G are yz projections of images of 0.19-µm fluorescent beads captured with a 63× (water; C) or 100× (oil; G) objective. The beads were embedded in an aqueous solution with 2% agarose. D and H are intensity profiles along the horizontal and vertical axes of the beads. A 63× 1.2 NA (water) objective provides better axial resolution than an 100× 1.4 NA objective (oil) in specimens in an aqueous solution. Scale bars = 5 µm (A,B,E,F); 0.5 µm (C,G). Images were captured with a LSM510 (Carl Zeiss, Inc.).
    View Image
  •  FigureFigure 2C.1.3 Applications of confocal microscopy in microbial research. (A, B). Human red blood cells (RBC) infected with malaria parasites (Plasmodium falciparum; 3D7 strain). Biotinylated human RBC were labeled with streptavidin-conjugated Quantum Dots 525 (CA; green color; http://www.qdots.com) and 0.5- to 4-µM FM-64 (Molecular Probes, red color). The cells were injected into chambers (HybriWell HBW20 from Grace Bio-Labs) and were examined with an LSM 510 confocal microscope (Carl Zeiss, Inc.) with a 100× 1.4-NA oil objective. Panel A shows two RBC, one containing a parasite at the trophozoite stage; panel B shows a parasite at the schizont stage. The schizont was extruded from the RBC for better imaging of individual parasites (red). The green signal in the center of the schizont represents autofluorescence from hemazoin in the digestive vacuole. Images were provided by Dr. Svetlana Glushakova (National Institutes of Health, Bethesda, Md.; methods described in Glushakova et al., 2005). (C, D) Biofilm composed of microcolonies of nitrifying bacteria (ammonia oxidizers; Nitrosomonas sp.) and nitrite oxidizers (Nitrospira sp.). Both populations were labeled by fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. Nitrosomonas colonies are green; Nitrospira colonies are red. Images are 3-D reconstructions created using the Daime imaging program (see Daims et al., 2006, and Internet Resources) from stacks of optical sections collected with an LSM 510 (Carl Zeiss, Inc). Panel C shows an overview of the projected image of the biofilm, while panel D shows a smaller region at higher zoom. Images were provided by Dr. Holger Daims (Universität Wien, Vienna, Austria).
    View Image
  •  FigureFigure 2C.1.4 The basis of optical sectioning. Illumination from a point light source is reflected by a dichroic mirror into the back aperture of a microscope objective. The objective lens focuses the light to a diffraction-limited spot within the specimen. Fluorophores at the focal spot and within the cones of illumination above and below it are excited, emitting fluorescence in all directions. The fluorescence captured by the objective passes through the dichroic mirror because the fluorescence is at a longer wavelength than the excitation. The confocal pinhole allows fluorescence from the focal spot to reach the photodetector and blocks fluorescence from out-of-focus areas. Redrawn from Shotton (1993).
    View Image
  •  FigureFigure 2C.1.5 The light path of a laser-scanning confocal microscope. The diagram illustrates the light path of a LSCM set up for simultaneous imaging of FITC and lissamine rhodamine. The 488- and 568-nm lines of a krypton-argon laser are reflected by dichroic beam splitter 1 into the optical axis of the microscope. The beam is reflected by a mirror into the microscope objective, which focuses the beam to a diffraction-limited spot in the specimen. The scanner consists of a pair of galvanometer mirrors that deflect the laser beams so as to scan the spot across the specimen in a raster pattern. Fluorescence emitted as each point is illuminated travels the reverse path through the scanning system. The FITC fluorescence (peak at 520 nm) and lissamine rhodamine fluorescence (peak at 590 nm) pass through dichroic beam splitter 1 to dichroic beam splitter 2, which transmits the lissamine rhodamine fluorescence to photomultiplier tube 1 and reflects the FITC fluorescence to photomultiplier tube 2. A variable pinhole in front of each photodetector blocks light from out-of-focus areas of the specimen while allowing light from the focal plane to reach the detector.
    View Image
  •  FigureFigure 2C.1.6 Excitation spectra of representative fluorophores and emission wavelengths of lasers for confocal microscopy. The graph at the top shows the excitation spectra of Marina Blue, Alexa 488, Alexa 555, and Alexa 647 (Molecular Probes). The emission wavelengths of lasers commonly used for confocal microscopy are shown below. Data for the excitation spectra are from Molecular Probes.
    View Image

Videos

Literature Cited

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. Proc. Natl. Acad. Sci. U.S.A. 99:12651-12656.
    Art, J. 1995. Photon detectors for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 183-196. Plenum, New York.
    Bacallao, R., Kiai, K., and Jesaitis, L. 1995. Guiding principles of specimen preservation for confocal fluorescence microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 311-326. Plenum, New York.
    Benham, G. 2002. Practical aspects of objective lens selection for confocal and multiphoton digital imaging techniques. In Cell Biological Applications of Confocal Microscopy, 2nd ed. (B. Matsumoto, ed.) pp. 247-300. Academic Press, San Diego, Calif.
    Boccacci, P. and Bertero, M. 2002. Image-restoration methods: Basics and algorithms. In Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (A. Diaspro, A., ed.), pp. 253-269. John Wiley & Sons, Hoboken, N.J.
    Brelje, T.C., Wessendorf, M.W., and Sorenson, R.L. 1993. Multicolor laser scanning confocal immunofluorescence microscopy: Practical applications and limitations. In Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.) pp. 98-182. Academic Press, San Diego, Calif.
    Brelje, T.C., Wessendorf, M.W., and Sorenson, R.L. 2002. Multicolor laser scanning confocal immunofluorescence microscopy: Practical applications and limitations. In Cell Biological Applications of Confocal Microscopy, 2nd ed. (B. Matsumoto, ed.), pp. 166-244. Academic Press, San Diego, Calif.
    Centonze, V. and Pawley, J. 1995. Tutorial on practical confocal microscopy and use of the confocal test specimen. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 549-570. Plenum, New York.
    Cole, N., Smith, C., Sciaky, N., Terasaki, M., Edidin, M., and Lippincott-Schwartz, J. 1996. Diffusional mobility of Golgi proteins in membranes of living cells. Science 237:797-801.
    Daims, H., Lucker, S., and Wagner, M. 2006. Daime, a novel image analysis program for microbial ecology and biofilm research. Environ. Microbiol. 8:200-213.
    Diaspro, A. (ed.) 2002. Confocal and Two Photon Microscopy: Foundations, Applications and Advances. Wiley-Liss, New York.
    Dickinson, M.E., Bearman, G., Tille, S., Lansford, R., and Fraser, S.E. 2001. Multi-spectral imaging and llinear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31:1274-1278.
    Drenkard, E. and Ausubel, F.M. 2002. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416:740-743.
    Elphick, G.F., Querbes, W., Jordan, J.A. Gee, G.V., Eash, S., Manley, K., Dugan, A., Stanifer, M., Bhatnagar, A., Koreze, W.K., Roth, B.L., and Atwood, J.W. 2004. The Human Polyomavirus, JCV, uses serotonin receptors to infect cells. Science 306:1380-1383.
    Ferrari, A., Pellegrini, V., Arcangeli, C., Fittipaldi, A., Giacca, M., and Beltram, F. 2003. Caveolae-mediated internalization of extracellular HIV-1 Tat fusion proteins visualized in real time. Mol. Ther. 8:284-294.
    Forest, T., Barnard, S., and Baines, J.D. 2005. Active intranuclear movement of herpes virus capsids. Nat. Cell Biol. 7:429-431.
    Giloh, H. and Sedat, J.W. 1982. Fluorescence microscopy: Reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217:1252-1255.
    Glushakova, S., Yin, D., Li, T., and Zimmerberg, J. 2005. Membrane transformation during malaria parasite release from human red blood cells. Curr. Biol. 15:1645-1650.
    Inoué, S. and Inoué, T. 2002. Direct-View High-Speed Confocal Scanner: The CSU-10. In Cell Biological Applications of Confocal Microscopy, 2nd ed. (B. Matsumoto, ed.) pp. 88-128. Academic Press, San Diego, Calif.
    Inoué, S. and Spring, K.R. 1997. Video Microscopy: The Fundamentals, 2nd ed. Plenum, New York.
    Jares-Erijman, E.A. and Jovin, T.M. 2003. FRET imaging. Nat. Biotechnol. 21:1387-1395.
    Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D., and Holborow, E.J. 1982. Fading of immunofluorescence during microscopy: A study of the phenomenon and its remedy. J. Immunol. Methods 55:231-242.
    Kamabadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S., and Odenwald, W. 1998. Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes Dev. 12:246-260.
    Karbowski, M., Arnoult, D., Chen, H., Chan, D.C., Smith, C.L., and Youle, R.J. 2004. Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis. J. Cell Biol. 164:493-499.
    Keller, E. 1995. Objective lenses for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 111-126. Plenum, New York.
    Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G.H. 2003. Photobleaching and photoactivation: Following protein dynamics in living cells. Nat. Cell Biol. Suppl:S7-S14.
    Majlof, L. and Forsgren, P. 2002. Confocal microscopy: Important considerations for accurate imaging. In Cell Biological Applications of Confocal Microscopy 2nd ed. (B. Matsumoto, ed.) pp. 149-164. Academic Press, San Diego, Calif.
    Matsumoto, B. (ed.) 2002. Cell Biological Applications of Confocal Microscopy. 2nd ed. Academic Press, London.
    McNally, J., Karpova, T., Cooper, J., and Conchello, J. 1999. Three-dimensional imaging by deconvolution microscopy. Methods 19:373-385.
    McNally, J.G. and Smith, C.L. 2002. Photobleaching by confocal microscopy. In Confocal and Two Photon Microscopy: Foundations, Applications and Advances (A. Diaspro, ed.) pp. 525-538. Wiley-Liss, New York.
    Nerbonne, J.M. 1996. Caged compounds: Tools for illuminating neuronal responses and connections. Curr. Opin. Neurobiol. 6:379-386.
    Patterson, G.H. and Lippincott-Schwartz, J. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873-1877.
    Perrin, A.J., Jiang, X., Birmingham, C.L., So, N.S., Burmell, J.H. 2004. Recognition of bacteria in the cytosol of mammalian cells by the ubiquitin system. Curr. Biol. 14:806-811.
    Rani, S.A., Pitts, B., and Stewart, P.S. 2005. Rapid diffusion of fluorescent tracers into Staphylococcus epidermis biofilms visualized by time lapse microscopy. Antimicrob. Agents Chemother. 49:728-732.
    Roux, P., Munter, S., Frischknecht, F., Herbomel, P., and Shorte, P. 2004. Focusing light on infection in four dimensions. Cell. Microbiol. 6:333-343.
    Sandison, D.R., Williams, R.M., Wells, K.S., Strickler, J., and Webb, W.W. 1995. Quantitative fluorescence confocal laser scanning microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 39-54. Plenum, New York.
    Satpute-Krishnan, P., DeGiorgis, J.A., and Bearer, E.L. 2003. Fast anterograde transport of Herpes Simplex Virus: Role for the amyloid precursor protein of Alzheimer's disease. Aging Cell 2:305-318.
    Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. Red fluorescent protein. Nat. Biotechnol. 22:1567-1572.
    Shotton, D.M. 1993. Electronic acquisition of light microscope images. In Electronic Light Microscopy (D.M. Shotton, ed.) pp. 1-38. Wiley-Liss, New York.
    Sprague, B.L. and McNally, J.G. 2005. FRAP analysis of binding: Proper and fitting. Trends Cell Biol. 15:84-91.
    Terasaki, M. and Dailey, M.E. 1995. Confocal microscopy of living cells. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 327-346. Plenum, New York.
    Tsien, R.Y. and Waggoner, A. 1995. Fluorophores for confocal microscopy. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 267-280. Plenum, New York.
    Viachou, D., Zimmermann, T., Cantera, R., Janse, C.J., Waters, A.P., and Kafatos, F.C. 2004. Real-time, in vivo analysis of malaria ookinete locomotion and mosquito midgut invasion. Cell Microbiol. 6:671-685.
    Waterman-Storer, C.M., Sanger, J.W., and Sanger, J.M. 1993. Dynamics of organelles in the mitotic spindles of living cells: Membrane and microtubule interactions. Cell Motil. Cytoskeleton 26:19-39.
    Webb, R.H. and Dorey, C.K. 1995. The pixilated image. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 55-68. Plenum, New York.
    Wilson, T. (ed.) 1990. Confocal Microscopy. Academic Press, London.
    Wilson, T. 1995. The role of the pinhole in confocal imaging system. In Handbook of Biological Confocal Microscopy, 2nd ed. (J. Pawley, ed.) pp. 167-182. Plenum, New York.
    Wouterlood, F.G. 2005. 3-D reconstruction of neurons from multichannel confocal laser scanning image series. In Current Protocols in Neuroscience (J.N. Crawley, C.R. Gerfen, M.A. Rogawski, D.R. Sibley, P. Skolnick, and S. Wray, eds) pp. 2.8.1-2.2.8.16. John Wiley & Sons, Hoboken, N.J.
    Wouters, F.S., and Bastiaens, P.I.H. 2000. Imaging protein-protein interactions by fluorescence resonance energy transfer (FRET) microscopy. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds.) pp. 17.1.1-17.1.15. John Wiley & Sons, Hoboken, N.J.
    Zhang, J., Campbell, R.E, Ting, A., and Tsien, R.Y. 2002. Creating new fluorescent probes for cell biology. Nature Rev. Mol. Cell Biol. 3:906-918.
 Key References
    Inoué and Spring, 1997. See above.

Covers the basics of light microscopy, video microscopy, and much more.

    Matsumoto, 2002. See above.

Good source of practical information about confocal imaging.

    Pawley, J. (ed.) 1995. See above.

Comprehensive reference book on confocal microscopy.

    Russ, J. 2002. The Image Processing Handbook. 4th edition. CRC Press, Boca Raton, Fla.

Guide to digital image processing.

 Internet Resources
    http://www.microbial-ecology.net/daime

Daime Web site, from which the Daime software application can be downloaded. Daime (digital image analysis in microbial ecology) is an open-source software program for 2-D and 3-D image analysis developed by Holger Daims, Sebastian Lücker, and Michael Wagner (Universität Wien, Vienna, Austria). The features of Daime and its application to analysis of biofilms are described in Daims et al. (2006).

    http://rsb.info.nih.gov/ij

ImageJ is a public domain image analysis program developed by W. Rasband (Research Services Branch, National Institute of Mental Health, NIH) for operating systems running Java (including Windows/PC and OSX/Macintosh). ImageJ has many useful tools for analysis of confocal images.

    http://www.uhnres.utoronto.ca/facilities/wcif/imagej/

A manual written by Tony Collins that describes the use of ImageJ to visualize and analyze confocal images.

    http://www.molecularexpressions.com

The Molecular Expressions Web site is a rich source of information about all aspects of light microscopy, including confocal microscopy. It includes sections on the basic principles of confocal imaging, instrumentation, sample preparation, and choices of fluorophores. An interactive tutorial “Choosing fluorophore combinations for confocal microscopy” allows the user to determine the extent of spectral cross-over that will occur when imaging different combinations of fluorophores with specific laser lines and filter sets.

 Web sites of vendors of confocal microscopes

These provide product descriptions, manuals, tutorials and literature.

    http://www.zeiss.com

Carl Zeiss, Inc.

    http://www.leica-microsystems.com/company

Leica Microsystems.

    http://www.nikonusa.com

Nikon, Inc.

    http://www.microscopyu.com

For information about light microscopy and confocal microscopy.

    http://www.olympusconfocal.com/

Olympus, Inc.

    http://www.perkinelmer.com/

PerkinElmer, Inc.

    http://www.solameretech.com/

Solamere Technology.

 Spectra of fluorophores
    http://fluorescence.nexus-solutions.net/frames6.htm

Biorad Microsciences fluorochrome database and charting application.

    http://home.earthlink.net/~fluorescentdyes/

George McNamara Multiprobe Microscopy.

    http://www.probes.com/

Molecular Probes.

    http://www.molecularexpressions.com

Molecular Expressions.

    http://www.olympusfluoview.com/resources/specimenchambers.html

Sources of chambers for maintaining living specimens during observation by microscopy.

    http://listserv.buffalo.edu/user/sub.html

Many topics of interest to confocal microscopists are discussed on the confocal listserver operated by the listserver at the University at Buffalo. To subscribe to the lis,t go to the URL and type “confocal” in the box that asks which list one wishes to join.

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library