Laser Scanning Confocal MicroscopyREVIEW 127 Principles and Practices of Laser Scanning Confocal Microscopy Stephen W. Paddock* Abstract The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging appli- cations at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today’s sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstruc- tions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice. Index Entries: Confocal microscopy; laser scanning; fluorescence light microscopy. 1. Introduction field light microscope (theoretical maximum The major application of confocal microscopy resolution of 0.2 µm), but not as great as that in in the biomedical sciences is for imaging either the transmission electron microscope (0.1 nm), it fixed or living tissues that have been labeled with has bridged the gap between these two commonly one or more fluorescent probes. When these used techniques. samples are imaged using a conventional light The method of image formation in a confocal microscope, fluorescence in the specimen in focal microscope is fundamentally different from that planes away from the region of interest interferes in a conventional wide-field microscope where with resolution of structures in focus, especially the entire specimen is bathed in light from a mer- for those specimens that are thicker than 2 µm or cury or xenon source, and the image can be so (Fig. 1). The confocal approach provides a viewed directly by eye. In contrast, the illumina- slight increase in both lateral and axial resolution. tion in a confocal microscope is achieved by scan- It is the ability of the instrument to eliminate the ning one or more focused beams of light, usually “out-of-focus” flare from thick fluorescently from a laser, across the specimen. An image pro- labeled specimens that has caused the explosion duced by scanning the specimen in this way is in its popularity recently (1). Most modern con- called an optical section. This term refers to the focal microscopes are now relatively easy to noninvasive method of image collection by the operate and have become integral parts of instrument, which uses light rather than physical many multiuser imaging facilities. Since the means to section the specimen. The confocal resolution achieved by the LSCM is a little bet- approach has thus facilitated the imaging of liv- ter than that achieved in a conventional wide- ing specimens, enabled the automated collection *Author to whom all correspondence and reprint requests should be addressed. Department of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, Wisconsin 53706, USA, E-mail: [email protected] Molecular Biotechnology 2000 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2000/16:2/127–149/$15.75 MOLECULAR BIOTECHNOLOGY 127 Volume 16, 2000 128 Paddock Fig. 1. Conventional epifluorescence image (A) compared with a confocal image (B) of a similar region of a whole mount of a butterfly (Precis coenia) pupal wing stained with propidium iodide. Note the improved resolu- tion of the nuclear detail (B). of three-dimensional data in the form of Z-series, by Minsky, and all of the modern confocal imag- and improved the images of multiple labeled ing systems employ the principle of confocal specimens (1). imaging that he patented in 1957 (5). Emphasis has been placed on the laser scan- In Minsky’s original confocal microscope the ning confocal microscope (LSCM) throughout point source of light is produced by a pinhole this article because it is currently the instrument placed in front of a zirconium arc source. The of choice for most biomedical research applica- point of light is focused by an objective lens into tions, and it is therefore most likely to be the the specimen, and light that passes through it is instrument first encountered by the novice user. focused by a second objective lens at a second Several alternative designs of confocal instru- pinhole, which has the same focus as the first pin- ments occupy specific niches within the biologi- hole, i.e., it is confocal with it. Any light that cal imaging field (2). Optical sections can be passes through the second pinhole strikes a low- produced using other, nonconfocal, methods. For noise photomultiplier, which produces a signal example, using deconvolution, which calculates that is directly proportional to the brightness of the out-of-focus information in an image and the light. The second pinhole prevents light from removes it digitally (3), and multiple photon above or below the plane of focus from striking imaging (4), which uses the same method of scan- the photomultiplier. This is the key to the confo- ning as the LSCM but uses a laser that only cal approach, namely the elimination of out-of- excites the fluorochromes that are imaged in the focus light or “flare” in the specimen by spatial optical section itself. filtering. Minsky also described a reflected light version of the microscope that uses a single objec- 2. Evolution of the Confocal Approach tive lens and a dichromatic mirror arrangement. The development of confocal microscopes This is the basic configuration of most modern was driven largely by a desire to image biologi- confocal systems used for fluorescence imaging cal events as they occur in vivo. The invention (Fig. 2). of the confocal microscope is usually attributed In order to build an image, the focused spot of to Marvin Minsky, who built a working micro- light must be scanned across the specimen in scope in 1955 with the goal of imaging neural some way. In Minsky’s original microscope the networks in unstained preparations of living beam was stationary and the specimen itself was brains. Details of the microscope, and of its devel- moved on a vibrating stage. This optical arrange- opment can be found in an informative memoir ment has the advantage of always scanning on the MOLECULAR BIOTECHNOLOGY Volume 16, 2000 Laser Scanning Confocal Microscopy 129 image quality in his microscope was not very impressive because of the quality of the oscillo- scope display and not because of lack of resolu- tion achieved with the microscope itself. It is clear that the technology was not available to Minsky in 1955 to fully demonstrate the poten- tial of the confocal approach especially for imag- ing biological structures. According to Minsky, this is perhaps a reason why confocal microscopy did not immediately catch on with the biological community, who were, (as they are now) a highly demanding and fickle group when it came to the quality of their images. After all, at the time they could quite easily view and photograph their brightly stained and colorful histological tissue sec- tions using light microscopes with excellent optics. In modern confocal microscopes the image is either built up from the output of a photo- multiplier tube or captured using a digital charge coupled device (CCD) camera, directly pro- cessed in a computer imaging system, and then displayed on a high-resolution video monitor and recorded on modern hard copy devices, with Fig. 2. The light path in a typical LSCM is based upon that of a conventional reflected-light wide-field spectacular results. epifluorescence microscope, but with pinholes placed The optics of the light microscope have not in front of the light source (the point source is now a changed drastically in decades since the final laser) and in front of the photodetector. The pinhole at resolution achieved by the instrument is governed the light source, the focused point in the specimen, by the wavelength of light, the objective lens and and the pinhole in front of the detector are all confocal properties of the specimen itself. However, the with one another. associated technology and the dyes used to add contrast to the specimens have been improved sig- optical axis, which can eliminate any lens defects. nificantly over the past 20 yr. The confocal However, for many biological specimens, move- approach is a direct result of a renaissance in ment of the specimen can cause them to wobble light microscopy that has been fueled largely by and distort, which results in a loss of resolution in advancements in modern technology. Several the image. Moreover, it is impossible to perform major technological advances that would have various manipulations such as microinjection of benefited Minsky’s confocal design are now fluorescently labeled probes when the specimen available (and affordable) to biologists. These is moving. advances include: Finally an image of the specimen has to be pro- 1. Stable multiwavelength lasers for brighter duced. A real image is not formed in Minsky’s point sources of light. original microscope but rather the output from 2. More efficiently reflecting mirrors. the photodetector is translated into an image of 3. Sensitive low-noise photodetectors. the region-of-interest. In Minsky’s original design 4. Fast microcomputers with image processing the image was built up on the screen of a military capabilities. surplus oscilloscope with no facility for hard 5. Elegant software solutions for analyzing the copy. Minsky admitted at a later date that the images. MOLECULAR BIOTECHNOLOGY Volume 16, 2000 130 Paddock 6. High-resolution video displays and digital of confocal instrumentation have been covered printers. elsewhere (2), but briefly there are two funda- 7. Brighter and more stable fluorescent probes. mentally different methods of beam scanning: multiple-beam scanning or single-beam scan- These technologies have been developed inde- ning. The most popular way is currently single- pendently, and since 1955, they have slowly been beam scanning, which is typified by the LSCM.