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Basic Confocal Microscopy

Carolyn L. Smith¹

¹National Institute of Neurological Disorders and Stroke, Bethesda, Maryland

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 2.2

DOI:

10.1002/0471142301.ns0202s56

Online Posting Date:

July, 2011

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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 compared to other techniques for fluorescence imaging are described. There are also practical guidelines for sample preparation and optimization of imaging parameters, as well as examples of some of the applications of confocal microscopy. Curr. Protoc. Neurosci. 56:2.2.1‐2.2.18. © 2011 by John Wiley & Sons, Inc.

Keywords: confocal microscopy; fluorescence; imaging; resolution; three‐dimensional reconstruction

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Introduction
Basis of Optical Sectioning
Configuration of an LSCM
Practical Guidelines
Commentary
Literature Cited
Figures
Tables

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Figures

Figure 2.2.1

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, by permission of Cold Spring Harbor Laboratory Press). (A) A single optical section (∼2.5 µm) captured with a 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 an LSM410 (Carl Zeiss, Inc.).

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Figure 2.2.2

Three‐dimensional imaging in living specimens. Comparison of water‐ and oil‐immersion objectives. Living yeast cells expressing a GFP construct that targets the mitochondrial matrix were embedded in an aqueous solution with 0.2% agarose and visualized with (A,B) a C‐APO 63×, 1.2‐NA water‐immersion objective or (E,F) a Plan Apochromat 100×, 1.4‐NA oil‐immersion objective. The images show xy (A,E) and yz (B,F) 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. (C,G) yz projections of images of 0.19‐µm fluorescent beads embedded in an aqueous solution with 2% agarose and captured with a 63× water‐immersion (C) or 100× oil‐immersion (G) objective. (D,H) Intensity profiles along the horizontal and vertical axes of the beads. A 63×, 1.2‐NA water‐immersion objective (D) provides better axial resolution than a 100×, 1.4‐NA oil‐immersion objective (H) in specimens in an aqueous solution. Scale bars = 5 µm (A,B,E,F); 0.5 µm (C,G). Images were captured with an LSM510 laser scanning confocal microscope (Carl Zeiss, Inc.).

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Figure 2.2.3

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).

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Figure 2.2.4

The light path of a laser‐scanning confocal microscope (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.

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Figure 2.2.5

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.

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Literature Cited

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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, 1995, 2006. See above.
Two editions of comprehensive reference book on confocal microscopy.
	Russ, 2002. See above.
Guide to digital image processing.
Internet Resources
	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 crossover that will occur when imaging different combinations of fluorophores with specific laser lines and filter sets.
	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 list, go to the URL and type “confocal” in the box that asks which list one wishes to join.
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.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.invitrogen.com/site/us/en/home/support/Research‐Tools/Fluorescence‐SpectraViewer.html
Invitrogen Spectral Viewer.
	http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp
BD Fluorescence Spectral Viewer.