- In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded in light from a light source.
- Due to the conservation of light intensity transportation, all parts of the specimen throughout the optical path will be excited and the fluorescence detected by a photo-detector or a camera.
- In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information.
- Only the light within the focal plane can be detected, so the image quality is much better than that of wide-field images.
- As only one point is illuminated at a time in confocal microscopy, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen.
- The thickness of the focal plane is defined mostly by the square of the numerical aperture of the objective lens, and also by the optical properties of the specimen and the ambient index of refraction.
- Three types of confocal microscopes are commercially available: Confocal laser scanning microscopes, spinning-disk (Nipkow disk) confocal microscopes and Programmable Array Microscopes (PAM).
- Confocal laser scanning microscopy yields better image quality than Nipkow and PAM, but the imaging frame rate was very slow (less than 3 frames/second) until recently; spinning-disk confocal microscopes can achieve video rate imaging—a desirable feature for dynamic observations such as live cell imaging.
- Confocal laser scanning microscopy has now been improved to provide better than video rate (60 frames/second) imaging by using MEMS based scanning mirrors
- Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens.
- The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus.
- There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional optical microscopy, and in its great number of applications in many areas of current research interest
Basic Concepts –
- Current instruments are highly evolved from the earliest versions, but the principle of confocal imaging advanced by Marvin Minsky, and patented in 1957, is employed in all modern confocal microscopes.
- In a conventional wide field microscope, the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected onto an image capture device or photographic film.
- In contrast, the method of image formation in a confocal microscope is fundamentally different.
- Illumination is achieved by scanning one or more focused beams of light, usually from a laser or arc-discharge source, across the specimen.
- This point of illumination is brought to focus in the specimen by the objective lens, and laterally scanned using some form of scanning device under computer control.
- The sequences of points of light from the specimen are detected by a photomultiplier tube (PMT) through a pinhole (or in some cases, a slit), and the output from the PMT is built into an image and displayed by the computer.
- Although unstained specimens can be viewed using light reflected back from the specimen, they usually are labeled with one or more fluorescent probes.
Imaging Modes –
- A number of different imaging modes are used in the application of confocal microscopy to a vast variety of specimen types.
- They all rely on the ability of the technique to produce high-resolution images, termed optical sections, in sequence through relatively thick sections or whole-mount specimens.
- Based on the optical section as the basic image unit, data can be collected from fixed and stained specimens in single, double, triple,or multiple-wavelength illumination modes, and the images collected with the various illumination and labeling strategies will be in register with each other.
- Live cell imaging and time-lapse sequences are possible, and digital image processing methods applied to sequences of images allow z-series and three-dimensional representation of specimens, as well as the time-sequence presentation of 3D data as four-dimensional imaging.
- Reflected light imaging was the mode used in early confocal instruments, but any of the transmitted light imaging modes commonly employed in microscopy can be utilized in the laser scanning confocal microscope.
- Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology.
- However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components.
- In fact, optical aberrations that cause subtle defects in image quality in wide field microscopy can have devastating effects in confocal microscopy.
- Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly.
Three-Color Imaging for Confocal Microscopy –
- The laser scanning confocal microscope (LSCM) is routinely used to produce digital images of single-, double-, and triple-labeled fluorescent samples.
- The use of red, green and blue (RGB) color is most informative for displaying the distribution of up to three fluorescent probes labeling a cell, where any co-localization is observed as a different additive color when the images are colorized and merged into a single three-color image.