General and Historical

Memoir on Inventing the Confocal Scanning Microscope

Marvin Minsky's recollections on the invention and patenting of the principles of the confocal microscope. (Text is from an article originally published in Scanning 10:128-138, 1988). A short biographical sketch of Dr. Minsky is available (Molecular Expressions, Florida State University).

How the Confocal Laser Scanning Microscope entered Biological Research

A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge. From: Biology of the Cell 95(6):335-342 (2003). Please note, your institution may need to have a subscription to access this journal on-line.

Collections of Microscopic images (including confocal)

This link will take you to a different page on this site that contains links to images from a variety of microscopic techniques.

Confocal Principles, Optics and Related techniques

Confocal - theory and operation:

Laser Scanning Confocal Microscopy - The Molecular Expressions website (Florida State University) has a number of excellent tutorials and information about confocal microscopy. These web pages are written by some of the most prominent names in the field. They include many diagrams and interactive tutorials that make understanding the concepts much easier. Other similar sites can be found at the Nikon MicroscopyU, the Olympus FluoView Resource Center and the Olympus Microscopy Resource Center.

Confocal Microscopy tutorial - An introductory site from the Advanced Microscopy unit (Department of Pathology, Haartman Institute, University of Helsinki, Finland)

Suggested reading/reference materials (PDF)
The Confocal Laser Scanning Microscope - Overview (1 page PDF) (Carl Zeiss Inc)
Confocal Laser Scanning Microscopy (37 page PDF) (Carl Zeiss Inc)
Laser Scanning Confocal Microscopy (37 page PDF) (hosted by Olympus America, written by Nathan S. Claxton, Thomas J. Fellers, and Michael W. Davidson)

 

Information about Confocal Optics and Hardware:

Laser Fundamentals - The Molecular Expressions website (Florida State University) has a number of excellent tutorials and information about lasers used in microscopy. The web pages include many diagrams and interactive tutorials. Note: most visible light wavelength lasers for confocal microscopes use a shielded fiber optic cable to safely transmit the light from the laser to the microscope. More powerful lasers (e.g., pulsed infrared lasers used for multiphoton imaging) may have an exposed beam path and their use is often regulated by government occupational safety agencies. For general information on laser safety, see: Laser Safety (The Laser Institute) and Laser Safety Guide (Michigan Technical University)

Photomultiplier Tubes - PMTs and Side-on PMTs are used to detect the fluorescent light in laser scanning confocal microscopes (Molecular Expressions, Florida State University).

AOTF (Acusto-Optical Tunable Filter) - used by many confocals to adjust the intensity of the laser beam before it reaches the sample (Molecular Expressions, Florida State University).

Optical Filters: Chroma Technology has a free booklet entitled Handbook of Optical Filters for Fluorescence Microscopy (40 page PDF) that is available by mail or download.

Microscope Objectives - High numerical aperture objectives typically give the highest resolution images for confocal microscopy (Molecular Expressions, Florida State University). In some instances water immersion objectives and/or objectives with a correction collar may be need to be used (Nikon MicroscopyU).

Suggested reading/reference materials (PDF)

The 39 Steps: A Cautionary Tale of Quantitative 3-D Fluorescence Microscopy, James Pawley, BioTechniques 28:884-888 (2000).
Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. Allison J. North, Journal of Cell Biology 172(1): 9-18 (2006). Please note, your institution may need to have a subscription to access this journal on-line.
A guided tour into subcellular colocalization analysis in light microscopy, Susanne Bolte and Fabrice Cordelières, Journal of Microscopy 224:213–232 (2006). Please note, your institution may need to have a subscription to access this journal on-line.
Multicolor Imaging: The Important Question of Co-Localization, Anna Smallcombe, BioTechniques 30: 1240-1247 (2001). Biotechniques requires a free subscription to access it's archives.
Colocalization (20 page PDF), Tony Collins, Biophotonics Facility, McMaster University, Canada.
Basic Principles of Microscope Objectives, Mortimer Abramowitz, Kenneth R. Spring, H. Ernst Keller, and Michael W. Davidson, BioTechniques 33:772-781 (2002). Biotechniques requires a free subscription to access it's archives.
Optical Aberrations and Objective Choice in Multicolor Confocal Microscopy, Dunn, K.W. and E. Wang, BioTechniques 28:542-550 (2000) (reproduced at Nikon MicroscopyU)
The Good, the Bad and the Ugly, Helen Pearson, Nature 447:138-140 (2007). See also: Under the Microscope Please note, your institution may need to have a subscription to access this journal on-line.

 

2-Photon or Multi-photon Confocal Microscopy

This non-linear optical technique offers several advantages over confocal microscopy.  An overview of Multiphoton Fluorescence Microscopy can be found at the always excellent Molecular Expressions site (Florida State University). Information about the Ti:sapphire pulsed infrared laser can be found at the Molecular Expressions site or at Wikipedia.

Fluorescent molecules respond to 2-photon excitation somewhat differently than the standard excitation and emission curves provided by vendors for routine confocal microscopy (UV through visible light wavelengths). Some molecules that work well with routine confocal excite very poorly in 2-photon microscopy. At this point in time there does not appear to be a definative source of suggested infrared wavelengths for 2-photon excitation. While there are values for specific fluorescent molecules in the literature, some people have opined that many of these published values are incorrect. Consider published values and the following references as starting points.
     Fluorescent dyes and their multiphoton characteristics (University of Georgia)
    Commercially available fluorophores and the GFP's -- 2P cross sections (Cornell University)
    Multiphoton Excitation Cross-Sections of Molecular Fluorophores (University of Milano-Bicocca, Italy)

Suggested reading/reference materials (PDF)

Microscopy Research and Technique has published three special issues covering the topic of two-photon microscopy. They are: Volume 47, Issue 3 (1999), Volume 63, Issue 1 (2004) and Volume 70, Issue 5 (May 2007). Please note that your institution will need to have a subscription to access these journals on-line.
Nonlinear magic: multiphoton microscopy in the biosciences. Warren R. Zipfel, Rebecca M. Williams and Watt W. Webb. Nature Biotechnology 21:1369-1377 (2003). Please note that your institution will need to have a subscription to access this journal on-line.
Deep tissue two-photon microscopy. Helmchen F. and Denk W. Nature Methods2:932-40 (2005). Please note that your institution will need to have a subscription to access this journal on-line.
When Two Is Better Than One: Elements of Intravital Microscopy. David W. Piston. PLoS Biol 3(6): e207 (2005)
Multiphoton Excitation Microscopy - tutorial. Coherent Laser Group (2000).

Specialty imaging techniques

FRET - Flourescence (sometimes "Förster") resonance energy transfer (Molecular Expressions, Florida State University)
FLIP / FRAP - fluorescence loss in photobleaching (FLIP) / fluorescence recovery after photobleaching (FRAP) (Olympus Fluoview Resource Center)
Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms, Paul J Campagnola & Leslie M Loew, Nature Biotechnology 21: 1356 - 1360 (2003). Please note that your institution will need to have a subscription to access this journal on-line.

Deconvolution Microscopy

Uses the point spread function and sophisticated mathematical algorithms to create images that are comparable to confocal images (using a standard epi-fluorescence microscope and some serious computing power).  An introduction to Deconvolution Microscopy can be found at the Molecular Expressions web site (Florida State University) .

Further information on the applications of this technique can be found at the AutoQuant, VayTech, and Applied Precision WWW sites.

Suggested reading/reference materials (PDF)
Workingperson's Guide to Deconvolution in Light Microscopy, Wes Wallace, Lutz H. Schaefer, and Jason R. Swedlow, BioTechniques 31:1076-1097 (2001). Biotechniques requires a free subscription to access it's archives.

Spinning Disk (Nipkow Disk) Confocal Microscopy

Instead of scanning a diffraction-limited laser spot across the sample, a spinning disk confocal uses multiple pinholes to obtain confocality with an arc lamp light source (Hg or Xe). This type of confocal is better suited to high-speed imaging, with the limitation being that the disk's pinholes are not adjustable, so optimum confocal images can only be acquired with a specific objective lens. For a brief description of how this technique works, see The Principle of Confocal Microscopy (Atto Bioscience).

Further information on spinning disk confocals is available from Atto Bioscience, PerkinElemer LifeSciences and VisiTech. Note, other microscope vendors have attempted to bypass the shortcomings of the spinning disk by using a variation on line scanning to perform high speed confocal imaging (e.g., Nikon, Zeiss).

Differential Interference Contrast (DIC or Nomarski)

Many confocal microscopes are able to image unstained cells and tissues with DIC as a compliment to the fluorescence imaging. Molecular Expressions (Florida State University) has an excellent series of tutorials on Differential Interference Contrast.

Flow Cytometry

While not a confocal technique, cytometry is a useful compliment to the confocal microscope. Flow cytometry used in combinatiuon with a cell sorting instrument can be used to separate large numbers of cells and provide population data such as specific fluorescent labeling, size, cytoplasmic granularity, DNA content and apoptosis. A new tool, called a Laser Scanning Cytometer, scans populations of cells attached to a microscope slide.

For additional cytometry information and resources, see the Purdue University Cytometry Labs web site.

Additional Information and Resources

Fluorescence Techniques - Links to information about fluorescent dyes, antibodies & related techniques, and sample preparation.

 

Confocal Listserver (searchable archive)

On-line discussion group for problems and and idea related to confocal microscopy.

Multiphoton Confocal Listserver

This list is more for individuals that are interested in developing the latest techniques and technologies related to multiphoton microscopy.

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